Preparation and / or formulation of proteins cross-linked by polysaccharides

Abstract

Therapeutic compositions and / or formulations and methods of making the same are provided.SOLUTION: The composition and / or formulation comprises at least one cross-linked protein matrix, wherein at least one of the cross-linked protein matrices comprises at least one protein residue and at least one residue having a sugar. The cross-linked protein matrix may be prepared by cross-linking a full-length or substantially full-length protein with a saccharide-containing cross-linker, such as a polysaccharide cross-linker Wherein the protein is for example tropoelastin, elastin, albumin, collagen, collagen monomers, immunoglobulins, insulin and / or derivatives or combinations thereof, and wherein the polysaccharide cross-linker is for example derived from hyaluronic acid or cellulose derivatives. The therapeutic composition may be administered topically or by injection.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 61 / 3444,940 (filed November 23, 2010), which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to and may be applied to preparations and / or formulations of proteins that are cross-linked by polysaccharides. [Background technology]

[0003] Implants are currently used in medical applications to augment or augment tissue. Medical applications for the use of such implants range from vocal cord reconstruction to the treatment of fecal and urinary incontinence and cosmetic treatment of wrinkles. Current implants are composed of a variety of materials, including hyaluronic acid, proteins (collagen), polymers (polylactic acid), and biocompatible materials (hydroxyapaptite).

[0004] For example, hyaluronic acid ("HA"), also known as hyaluronan or hyaluronate, is a naturally occurring mucopolysaccharide present in synovial fluid, vitreous humor, blood vessel walls, the umbilical cord, and other connective tissues. This polysaccharide is composed of alternating N-acetyl-D-glucosamine and D-glucuronic acid residues linked to each other by alternating β-1-3 glucuronide and β-1-3 glucosaminide bonds. Hyaluronic acid-based products are crosslinked using various techniques, including chemicals such as BDDE and divinylsulfane. The crosslinked hyaluronic acid is then micronized for injectability (e.g., Restylane® and Juvederm®). The effect of hyaluronic acid implants is achieved by tissue augmentation and water retention within the hyaluronic acid implant. Hyaluronic acid implants are gradually degraded and absorbed by the body.

[0005] Another example is a collagen-based implant extracted from animal or human tissue, which is further crosslinked (e.g., glutaraldehyde-based crosslinking (Zyplast®) or ribose-based crosslinking (Evolence®)), homogenized, and then resuspended in saline to prepare it for implantation. The effect of collagen implants is similar to that of hyaluronic acid implants, as they augment tissue. On the other hand, the use of collagen implants allows for increased cell infiltration into the implant and the production of new collagen material.

[0006] Techniques using polylactic acid (e.g., Sculptra®) and biomaterials (e.g., Radiesse®) have been based on the preparation of a suspension of particulate material (typically a polysaccharide such as hyaluronic acid or carboxymethylcellulose) in an injectable gel. The action of particulate implants is achieved by inducing a foreign body reaction in the body, which results in the fibroblasts enveloping the particles, leading to collagen production and subsequent tissue development and growth.

[0007] One problem with current approaches to tissue augmentation is that they are unable to deliver biomaterials based on or incorporating full-length (or substantially full-length) proteins. Formulations based on or incorporating full-length (or substantially full-length) protein materials that resemble proteins naturally occurring in the body are more likely to maintain the levels of biocompatibility and self-recognition desired for many intended applications. Chemical conjugation methods, on the other hand, typically result in substantial intramolecular crosslinking, which can disrupt the natural structure of the molecules. Furthermore, the micronization or homogenization techniques used to enable the injection of the product do not contribute to the preservation of the structure of full-length (or substantially full-length) protein molecules. Additionally, chemical crosslinkers used to crosslink hyaluronic acid and proteins have known toxicities and can be irritating, inflammatory, or pose a risk of cancer.

[0008] The present disclosure relates, in part, to providing injectable formulations of protein-based or protein-derived coherent biomaterials, enabling protein residues incorporated into the formulations to maintain full-length (or substantially full-length) structure, and protecting the protein residues to prevent rapid resorption and / or degradation due to, for example, proteolysis. The present disclosure also relates, in part, to full-length (or substantially full-length) protein-based or full-length (or substantially full-length) protein-derived biomaterials that are suitable for injection using a needle, maintain a coherent structure, or are sufficiently crosslinked to slow resorption in vivo, or a combination of these characteristics. The present disclosure also relates, in part, to biomaterials that are substantially free of toxic chemical crosslinkers. The present disclosure also provides, in part, methods, systems, and / or kits for preparing and / or formulating at least one crosslinked protein matrix, comprising at least one protein residue and at least one biomolecular crosslinker residue, wherein the at least one protein molecule is crosslinked by the at least one biomolecular crosslinker to form the crosslinked protein matrix. The present disclosure also provides, in part, systems and / or kits for preparing and / or formulating at least one crosslinked protein matrix, comprising at least one protein residue and at least one polysaccharide residue, wherein the protein molecules (substantially full-length protein molecules or full-length protein molecules) are crosslinked by the polysaccharide crosslinker to form at least one such crosslinked protein matrix. There is a need for the compositions, methods, systems, and / or kits disclosed herein. Summary of the Invention

[0009] In some embodiments, the injectable composition can be at least one cross-linked protein matrix, the at least one cross-linked protein matrix comprising at least one protein residue and at least one cross-linking residue having a sugar.

[0010] In some embodiments, the injectable composition can be a composition that is substantially soluble in aqueous and / or physiological media, hi some embodiments, the injectable composition can be a composition that is substantially soluble, partially soluble, or substantially insoluble in aqueous and / or physiological media.

[0011] In some embodiments, the injectable composition may comprise at least one sugar-bearing residue derived from at least one sugar-bearing crosslinking molecule, which may be substantially bioavailable, substantially biodegradable, substantially bioabsorbable, and / or substantially bioabsorbable. In some aspects, the at least one sugar-bearing residue may comprise at least one polysaccharide residue or at least one oligosaccharide residue, or a combination thereof. In some aspects, the injectable composition may comprise at least one polysaccharide, wherein the at least one polysaccharide residue comprises a low, medium, and / or high molecular weight polysaccharide residue. In some aspects, the injectable composition may comprise at least one polysaccharide residue having a molecular weight of about 500 to about 500,000 daltons. In some aspects, the injectable composition may comprise at least one sugar-bearing residue, wherein the at least one sugar-bearing residue comprises at least one oligosaccharide residue or at least one polysaccharide residue comprising one or more positively charged functional groups and / or one or more negatively charged functional groups. In some aspects, the injectable composition may comprise at least one polyanionic polysaccharide residue or at least one polyanionic oligosaccharide residue. In some aspects, the injectable composition may comprise at least one polysaccharide residue derived from or containing a residue of hyaluronic acid, cellulose derivatives, carboxycellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylamylose, xanthan gum, guar gum, α-glucan, β-glucan, β-1,4-glucan, β-1,3-glucan, alginate, carboxymethyldextran, glycosaminoglycan derivatives, chondroitin-6-sulfate, dermatin sulfate, heparin, heparin sulfate, or a biomaterial (e.g., polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), tricalcium phosphate, 1-hydroxyapatite), and / or pharmaceutically acceptable salts, derivatives, and / or combinations thereof.In some aspects, the injectable composition can include at least one cross-linked protein matrix containing at least one sugar-bearing residue at a concentration of about 0.01% to about 30%.

[0012] In some embodiments, the injectable composition may comprise at least one protein residue derived from or containing a residue of a full-length protein. In some aspects, the injectable composition may comprise at least one protein residue containing an amine-bearing side chain residue, the side chain residue comprising at least one lysine residue and / or at least one arginine residue. In some aspects, the injectable composition may comprise at least one protein residue derived from or containing a residue of tropoelastin, elastin, albumin, collagen, collagen monomer, immunoglobulin, insulin, and / or derivatives or combinations thereof.

[0013] In some embodiments, the injectable composition may comprise at least one crosslinked protein matrix extrudable to a length of at least 10 cm or about 10 cm. In some embodiments, the injectable composition is an extrudable composition. In some embodiments, the injectable composition is an extrudable composition to a length of about 5 cm to about 30 cm. In some embodiments, the injectable composition may comprise at least one crosslinked protein matrix comprising about 25 mg / ml or at least about 25 mg / ml of protein residue. In some embodiments, the injectable composition may comprise at least one crosslinked protein matrix comprising about 1 mg / ml to about 250 mg / ml of protein residue.

[0014] In some embodiments, the injectable composition may comprise at least one cross-linked protein matrix that has been prepared by forming one or more linkages and / or cross-links using (i) an activating agent and / or a coupling agent; and (ii) a modifying agent and / or an auxiliary coupling agent.

[0015] In some embodiments, the injectable compositions may be used therapeutically, including surgical procedures, cosmetic procedures, tissue augmentation, incontinence treatment, skin replacement products, dermatology, dermatological surgery, eye surgery, rheumatology, pharmacology, and / or cosmetics.

[0016] In some embodiments, methods of preparing the composition are disclosed, the methods comprising crosslinking at least one protein molecule with at least one sugar-bearing crosslinking molecule. In some embodiments, the methods of preparing the composition comprise (i) modifying at least one sugar-bearing molecule to include at least one reactive chemical group complementary to a reactive chemical group of at least one protein molecule, (ii) combining the modified at least one sugar-bearing molecule with at least one protein molecule, and (iii) forming at least one bond between the modified at least one sugar-bearing molecule and the at least one protein molecule.

[0017] In some embodiments, the method of preparing the composition comprises: (i) modifying at least one sugar-bearing molecule to include at least one reactive chemical group; (ii) combining the at least one modified sugar-bearing molecule with at least one protein molecule that includes at least one reactive chemical group complementary to the reactive group on the at least one sugar-bearing molecule; and (iii) forming at least one covalent bond between the at least one sugar-bearing molecule and the at least one protein molecule.

[0018] In some aspects, the at least one modified sugar-bearing molecule can include a modified polysaccharide molecule prepared by attaching at least one moiety containing a reactive linker. The linker can be attached to a protein molecule or modified protein molecule during solid-phase polysaccharide synthesis. In some aspects, the at least one moiety can be attached by a covalent bond. Furthermore, the at least one moiety can include a spacer group. Furthermore, the spacer group can include polymerized ethylene oxide. The spacer group can also be PEG or PEO.

[0019] In some embodiments, the bond may be formed by a covalent bond. Furthermore, in some embodiments, the covalent bond may be selected from the group consisting of amide, oxime, hydrazone, sulfide, ether, amine (e.g., secondary amine or tertiary amine), enol ether, thiol ether, ester, triazole, and disulfide. In some embodiments, the covalent bond may include an amide or hydrazone.

[0020] In some embodiments, the disclosed methods may be robust, more efficient, cost-effective, convenient, and / or a combination thereof.

[0021] In some embodiments, the cross-linked protein matrix may comprise one or more protein residues or modified protein residues. In some embodiments, the cross-linked protein matrix may comprise two different protein residues or modified protein residues.

[0022] In some embodiments, the cross-linked protein matrix may comprise one or more polysaccharide residues or modified polysaccharide residues. In some embodiments, the cross-linked protein matrix may comprise two different polysaccharide residues or modified polysaccharide residues.

[0023] In some embodiments, the cross-linked protein matrix can be an injectable composition.

[0024] The accompanying drawings facilitate an understanding of various embodiments of the present disclosure. Exemplary embodiments of processes, systems, kits, preparations, purifications, or combinations thereof will be described in further detail, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0025] [Figure 1] According to some embodiments, a typical repeating structure of hyaluronic acid is shown: Hyaluronic acid is a polysaccharide consisting of disaccharide units of β-D-glucuronic acid-[1,3]-β-DN-acetyl-glucosamine, each of which contains a carboxyl group that can be activated and crosslinked. [Figure 2] 1 shows the EDC- and NHS-mediated reaction of a carboxylate-containing molecule (1) with an amine (2), according to some embodiments. The suitable intermediate structures shown include O-acylisourea ester and NHS ester intermediates formed from a modifying agent (e.g., N-hydroxysuccinimide (NHS)) to form an NHS-activated carboxylate intermediate capable of reacting with a primary amino group (1) to form an amide bond. [Figure 3] 1 shows an SDS PAGE gel of proteins extracted from the formulation of Example 5 soaked in PBS, according to some embodiments. The SDS PAGE gel includes the following lanes: marker (lane M), supernatant (lanes A and B), and pure TE (lane TE). DETAILED DESCRIPTION OF THE INVENTION

[0026] The following description is provided in terms of various embodiments that may share representative characteristics and features. It will be understood that one or more features of one embodiment may be combined with features of other embodiments. Furthermore, a single feature or combination of features in some embodiments may constitute an additional embodiment.

[0027] In this specification, the word "comprising" is to be understood in its "open" sense (i.e., the sense of "including") and is therefore not limited to its "closed" sense (i.e., the sense of "consisting only of"). Where clear, a corresponding intention applies to the corresponding words "comprise", "included" and "comprises".

[0028] The headings used in the detailed description are included solely for ease of reference by the reader and should not be used to limit the subject matter found in the disclosure or claims as a whole. The headings should not be used to interpret the scope of or restrict the scope of the claims.

[0029] Unless otherwise specified, technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0030] The term "activated" can encompass intermediate forms of a molecule that can be sensitive and / or strongly susceptible to nucleophilic attack and / or nucleophilic substitution by a nucleophilic compound. For example, in some embodiments, a molecule containing a carboxyl group (e.g., a sugar-bearing molecule containing a carboxyl group (e.g., a polysaccharide containing a carboxyl group)) can be activated when treated with an activating agent to form an activated intermediate (e.g., an activated ester), where the activated intermediate is sensitive and / or weakly susceptible to nucleophilic attack and / or nucleophilic substitution by a nucleophilic compound, such as an amine, and can form a bond (e.g., an amide bond) between the molecule containing a carboxyl group and the nucleophilic compound. In some embodiments, a hydroxyl-containing molecule (e.g., a saccharide-bearing molecule (e.g., a hydroxyl-bearing polysaccharide) containing a hydroxyl group) can be activated when treated with an activating agent (e.g., a reactive epoxy group or halohydrin group) to form an activated intermediate, which can react with a compound (e.g., an amine) to form a bond (e.g., a secondary amine bond or a tertiary amine bond) between the hydroxyl-containing molecule and the compound.

[0031] The term "amino acid" can refer to α-amino acids that are racemic or in either the D or L configuration. In some embodiments, an amino acid can be a naturally occurring amino acid or a non-naturally occurring amino acid (e.g., a synthetically produced non-naturally occurring amino acid). The designation "d" before an amino acid designation (e.g., dAla, dSer, dVal) refers to the D-isomer of that amino acid. The designation "dl" before an amino acid designation (e.g., dlSer) refers to a mixture of the L- and D-isomers of that amino acid.

[0032] The term "biocompatible" can encompass the ability of a material to function with an appropriate host response in a particular situation (e.g., the absence of medically unacceptable toxicity or impairing effects on biological function).

[0033] The term "bioconjugate" can refer to a conjugate derived from at least two biomolecules, at least two biopolymers, or at least one biomolecule and at least one biopolymer. Bioconjugates also encompass conjugates derived from three or more biomolecules, biopolymers, or combinations thereof, where at least one biomolecule and / or biopolymer is conjugated to two or more biomolecules and / or biopolymers, resulting in intermolecular crosslinks. Bioconjugates can also include one or more linkages (e.g., intramolecular crosslinks) between the individual conjugated components. In some embodiments, bioconjugates can have one or more intermolecular crosslinks (e.g., bioconjugates can be exclusively intermolecularly crosslinked, or primarily or substantially intermolecularly crosslinked). In some embodiments, bioconjugates can have one or more intramolecular crosslinks (e.g., bioconjugates can be exclusively intramolecularly crosslinked, or primarily or substantially intramolecularly crosslinked). In some embodiments, bioconjugates can have both intermolecular and intramolecular crosslinks. The bioconjugate may also include one or more spacer groups between one or more links connecting one or more individual elements, or the spacer groups may be between the individual elements and the links. For example, spacer groups may include, but are not limited to, ethylene oxide moieties, polymers based on repeating ethylene oxide moieties, -(-CH2-CH2-O-)- moieties, polyethylene glycol (PEG), polyethylene oxide (PEO), and / or derivatives thereof.

[0034] The term "biomolecule" refers to a naturally occurring compound, a derivative of a naturally occurring compound (i.e., a naturally occurring molecule), a synthetically modified analog of a naturally occurring compound, a genetically modified analog of a naturally occurring compound, or a genetically modified modified analog of a naturally occurring compound. For example, biomolecules include, but are not limited to, amino acids, peptides, bioactive peptides, genetically modified peptides, proteins, glycoproteins, bioactive proteins, partially digested proteins, pre-active proteins, genetically modified proteins, enzymes, antibodies, genetically modified antibodies, sugars, disaccharides, trisaccharides, oligosaccharides, polysaccharides, oligonucleotides, RNA, DNA, peptide nucleic acids (PNAs), antigens, oligosaccharides, substrates for enzymes, substrates for nuclear receptors, and / or derivatives or combinations thereof.

[0035] The term "biopolymer" can refer to naturally occurring compounds, derivatives of naturally occurring compounds, synthetically modified analogs of naturally occurring compounds, genetically modified analogs of naturally occurring compounds, and genetically modified modified analogs of naturally occurring compounds, where the biopolymer can be made up of multiple monomeric units. For example, biopolymers include, but are not limited to, peptides, peptide nucleic acids (PNAs), oligonucleotides, RNA, DNA, proteins, enzymes, antibodies, glycoproteins, trisaccharides, oligosaccharides, polysaccharides, and / or derivatives thereof. In some embodiments, biopolymers can be linear or branched, or can have a specific three-dimensional design (e.g., a star-shaped or matrix-like structure). Examples of monomeric units include, but are not limited to, amino acids, amino acid derivatives, monosaccharides, disaccharides, trisaccharides, sugar derivatives, PNA monomers, nucleotides, nucleosides, and / or derivatives or combinations thereof.

[0036] In some embodiments, the compounds described herein may contain chiral centers. Such chiral centers may be in either the R or L configuration, or a mixture thereof. For example, the compounds described herein may be enantiomerically homogeneous, diastereomeric, or stereoisomerically homogeneous. In some embodiments, the compounds described herein may be a mixture of stereoisomers or a mixture of diastereoisomers. For example, in the case of amino acid residues, each residue may be in either the L or D configuration. For example, the preferred configuration for naturally occurring acid residues is L.

[0037] The term "complementary reactive groups" refers to those groups that form a covalent bond when reacted together. For example, a reactive amino group refers to a moiety that can react directly with a moiety containing amine reactivity to form an amide or amine bond. For example, a reactive thiol functional group refers to a moiety that can react directly with a group containing sulfhydryl reactivity to form a highly stable sulfide bond. For example, an amino group can be a complementary group to a carboxyl derivative. For example, an amino group can be a complementary group to a hydroxyl derivative. For example, a hydrazino group can be a complementary group to a carbonyl derivative. And, for example, an oxyamino group can be a complementary group to a carbonyl derivative.

[0038] The term "conjugate" refers to a compound comprising at least two or more elements (e.g., at least two or more biomolecules and / or biopolymers) linked to each other. The individual elements are directly bound to each other by one or more covalent bonds, one or more ionic bonds, chelation, and / or a mixture or combination of these bonds. In some embodiments, a conjugate can include multiple direct linkages (e.g., multiple ionic or covalent bonds (e.g., amide bonds)) between the individual elements that directly link the at least two or more biomolecules and / or biopolymers to each other. For example, a conjugate can include a first element (e.g., a protein) that can be directly linked to a second element (e.g., a polysaccharide) via one or more covalent bonds to form a conjugate (e.g., a protein-polysaccharide conjugate). In some embodiments, a conjugate can include spacer groups between the individual elements, where the conjugate includes at least two linkages via the spacer groups to connect the two elements to each other. For example, a first biomolecule can form a first linkage with a spacer group, and a second biomolecule can form a second linkage with the spacer group. A conjugate can include one or more spacer groups between one or more bonds connecting two or more individual elements, or between individual elements and links. For example, the spacer group can include, but is not limited to, glycol moieties, ethylene oxide moieties, polymers formed from repeating "-(-CH2-CH2-O-)-" moieties (e.g., polyethylene glycol (PEG) or polyethylene oxide (PEO)), polyamines, polyols, and / or derivatives or combinations thereof.

[0039] The term "crosslinked protein matrix" can refer to one or more protein residues containing at least one or more crosslinks to at least one or more molecular residues, e.g., one or more biomolecule residues and / or one or more biopolymer residues, or derivatives or combinations thereof.

[0040] The term "residue" can refer to a portion of molecular material or residual molecular material remaining in a reaction product. For example, a portion of a protein molecular material remaining in a reaction product (e.g., a cross-linked product resulting from the reaction of a protein molecule and a cross-linking agent) is referred to as a protein residue. For example, a portion of a sugar-bearing molecular material remaining in a reaction product (e.g., a cross-linked product resulting from the reaction of a sugar-bearing molecule and a protein molecule) is referred to as a sugar-bearing residue.

[0041] The terms "fine needle," "fine gauge needle," or "fine needle injection" may refer to, but are not limited to, the use of needles sized about 25 G or less. Larger needles may be used for some applications as further described herein.

[0042] The term "hyaluronic acid" or "HA" can include hyaluronic acid and any of hyaluronic acid salts (for example, sodium hyaluronate (sodium salt), potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate).In this specification, hyaluronic acid obtained from various sources can be used.For example, hyaluronic acid can be extracted from animal tissue, recovered as a product of bacterial fermentation, or produced in industrial quantities by bioprocessing.

[0043] The term "linkage" refers to the connection or bond between two individual molecular elements that are linked together. In some embodiments, the individual molecular elements that are linked together include, but are not limited to, biopolymers, modified biopolymers (biologically and / or synthetically modified biopolymers), biomolecules, and modified biomolecules (biologically and / or synthetically modified biomolecules). For example, a connection or bond between two biomolecules, a connection or bond between a biomolecule and a spacer group, a connection or bond between two biopolymers, a connection or bond between a biopolymer and a spacer group, a connection or bond between two modified molecules, and / or derivatives or combinations thereof. In some embodiments, the linkage may be stable against thermal degradation, hydrolysis, or both. In some embodiments, the linkage may be biocompatible. In some embodiments, the linkage may be formed by the formation of a covalent bond, an ionic bond, and / or a combination thereof. For example, the linkage may be formed by the formation of a combination of one or more covalent bonds and / or one or more ionic bonds. In some embodiments, the covalent linkage may include, but is not limited to, an amide bond, an oxime bond, a hydrazone bond, a triazole bond, a sulfide bond, an ether bond, an amine bond (e.g., a secondary amine bond or a tertiary amine bond), an enol ether bond, an ester bond, a disulfide bond, or a combination thereof. In some embodiments, an amide bond may be formed, for example, between a carboxylic acid group or an activated carboxylic acid group of a sugar-bearing biomolecule and an amino acid-containing biomolecule (e.g., a protein (e.g., a protein containing a lysine residue)). For example, in some embodiments, an amide bond may be formed between, for example, a biomolecule containing a modified sugar moiety (e.g., a sugar moiety modified with a spacer group) and a biomolecule (e.g., a protein) containing an amino acid moiety. In some embodiments, an amide bond may be formed between, for example, a sugar moiety and a biomolecule containing a modified amino acid moiety (e.g., a protein modified with a spacer group).

[0044] The term "modified" refers to the modification of a molecule (e.g., a biomolecule or biopolymer) and / or a portion of the molecule by naturally occurring processes, chemical synthetic modification, bioengineering, etc., and / or a combination or variation thereof. In some embodiments, the molecule and / or a portion of the molecule can be modified by converting a moiety already present on the molecule (e.g., by a chemical synthetic conversion process, and / or a naturally occurring process, by adding an additional moiety, and / or a combination or variation thereof). For example, in some embodiments, the addition of a moiety to the molecule can be based on the formation of a covalent bond. In some embodiments, for example, a modified molecule containing the converted moiety can be or is more capable of reacting with a complementary reactive group to form a linkage, crosslink, and / or a combination or derivative thereof. In some embodiments, for example, a molecule containing the added moiety can be or is more capable of reacting with a complementary reactive group to form a linkage, crosslink, and / or a combination or derivative thereof. In some embodiments, molecules comprising the converted and / or attached moieties can include, for example, reactive groups, linkable groups, spacer groups, complementary reactive groups, and / or combinations or derivatives thereof. In some embodiments, molecules comprising the converted and / or attached moieties can include moieties (e.g., reactive groups) that can be formed and / or deprotected by chemical synthetic modification or naturally occurring processes, and that are available for reaction, e.g., to form linkages or crosslinks by reacting with complementary reactive groups. For example, in some embodiments, modified molecules can be obtained by activating a chemical group (e.g., a carboxyl group), adding a spacer group, deprotecting a reactive moiety, and / or combinations or modifications thereof.

[0045] As used herein, the term "moles" or "molarity (M)" of a polysaccharide refers to the moles of repeating monomer units contained in the polymer.

[0046] The term "polysaccharide" can encompass, for example, a sugar-bearing molecule that includes at least three sugar residues (e.g., at least three sugar monomer repeat units), e.g., at least three monosaccharide repeat units, at least three disaccharide repeat units, at least three trisaccharide repeat units, at least three oligosaccharide repeat units, and / or combinations or derivatives thereof. In some embodiments, a polysaccharide can include multiple sugar residues that are the same and / or different (e.g., one or more of the same and / or different sugar residues, two or more of the same and / or different sugar residues, three or more of the same and / or different sugar residues, and / or combinations or derivatives thereof).

[0047] The term "sugar-bearing molecule" can encompass, for example, monosaccharide-, disaccharide-, trisaccharide-, oligosaccharide-, and / or polysaccharide-bearing molecules. In some embodiments, for example, a sugar-bearing molecule can include a monosaccharide-, disaccharide-, trisaccharide-, oligosaccharide-, or polysaccharide-bearing monomer repeat unit. In some embodiments, a sugar-bearing molecule can include one or more sugar monomer repeat units, which may be the same or different. For example, it can include one or more disaccharide-, trisaccharide-, and / or polysaccharide monomer repeat units, which may be the same or different.

[0048] In some embodiments, the sugar-bearing residue can be (1) a sugar-bearing residue derived from an oligosaccharide, a modified oligosaccharide, a polysaccharide, a modified polysaccharide, and / or a derivative thereof, or (2) a sugar-bearing residue derived from a sugar-bearing crosslinking molecule (e.g., an oligosaccharide crosslinker, a modified oligosaccharide crosslinker, a polysaccharide crosslinker, a modified polysaccharide crosslinker, and / or a derivative thereof).

[0049] The terms "protein," "protein unit," or "protein monomer" can include, for example, a full-length protein, a substantially full-length protein, a protein fragment, a biologically active protein, a biologically active protein fragment, a pre-active form of a protein, an inactive protein, a protein having an active site, a protein having a binding site, a protein having a proteolytic cleavage site, a partially digested protein, a partially hydrolyzed protein, a protein having one or more point mutations, a protein comprising about 50% to about 99.99% of the full-length protein, or a protein comprising about 50% to about 99.99% conservation of the amino acids in the full-length protein. In some embodiments, proteins can include, for example, peptides comprising the sequence of at least one biologically active peptide, peptides comprising at least one receptor binding site, peptides comprising at least one proteolytic cleavage site, oligopeptides, polypeptides, and / or combinations or derivatives thereof.

[0050] In some embodiments, the protein may include, for example, a protein containing at least one lysine residue, at least one arginine residue, at least one cysteine ​​residue, at least one serine residue, at least one threonine residue, at least one tyrosine residue, at least one glutamic acid residue, at least one aspartic acid residue, at least one proline residue, and / or combinations or derivatives thereof. In some embodiments, the protein may include, for example, a protein containing at least one dimerization residue (e.g., at least one cystine residue).

[0051] In some embodiments, the protein may include, for example, a protein containing at least one amine group, a protein containing at least one amine-bearing side chain, a protein containing at least one amine-bearing amino acid residue (e.g., a protein containing at least one lysine residue), a protein containing at least one arginine residue, and / or a combination or derivative thereof. For example, in some embodiments, the protein may include a protein containing an amine-rich region (lysine-rich region or arginine-rich region) and / or a combination or derivative thereof. In some embodiments, the protein may include, for example, a poly(amine residue) protein (e.g., polylysine, polyarginine, and / or a combination or derivative thereof).

[0052] In some embodiments, the protein may include a homopolymer or copolymer (e.g., a homopolymer or copolymer of amino acid residues). For example, in some embodiments, the protein may include a homopolymer or copolymer of lysine, arginine, and / or histidine residues (e.g., a protein having a lysine-rich region). In some embodiments, for example, a protein including a lysine-rich region includes at least two lysine units (e.g., a polylysine region (e.g., at least five lysine units)). In some embodiments, for example, a protein including an arginine-rich region includes at least two arginine units (e.g., a polyarginine region (e.g., at least five arginine units)). In some embodiments, the protein may include regions rich in at least two different residues. For example, the protein may include a protein including at least one lysine-rich region and at least one arginine-rich region, and / or a combination or derivative thereof. In some embodiments, the protein may include, but is not limited to, tropoelastin, elastin, albumin, collagen, multiple collagen monomers, immunoglobulin, insulin, and / or a derivative or combination thereof.

[0053] In some embodiments, the protein may include a modified protein or protein derivative. In some embodiments, for example, the modified protein or protein derivative may be a protein prepared and / or obtained by a naturally occurring process, chemical synthetic modification, and / or a combination thereof. In some embodiments, for example, the modified protein or protein derivative may be a protein prepared and / or obtained from a naturally occurring process (e.g., a protein present in a eukaryotic cell, a prokaryotic cell, and / or a combination thereof). For example, in some embodiments, the naturally occurring process may include protein synthesis, proteolysis, hydrolysis, enzymatic treatment and / or conjugation, oxidation, reduction, glycosylation, amination, carboxylation, incorporation of amino acid residues or spacer groups (also referred to as linker groups), modification and / or derivatization of amino acid residues or spacer groups, and / or a combination or variation thereof. In some embodiments, for example, the modified protein or protein derivative may be a protein prepared and / or obtained from a chemical synthetic modification. For example, in some embodiments, chemical synthetic modifications may include oxidation, reduction, conjugation, hydrolysis, amination, esterification, amidation, reductive amination, carboxyl group activation, carboxyl group modification, incorporation of amino acid residues or spacer groups (also called linker groups), modification and / or derivatization of amino acid residues or spacer groups, and / or combinations thereof. In some embodiments, modified proteins or protein derivatives may be prepared by solid-phase synthesis, solution-phase synthesis, and / or combinations thereof. In some embodiments, modified proteins or modified protein derivatives may be prepared from proteins containing regions rich in amine-bearing amino acid residues (lysine-rich regions).

[0054] In some embodiments, the protein residue may be derived from a protein and / or a derivative thereof as disclosed herein.

[0055] The term "spacer group" can include, for example, a moiety that links one or more individual elements (eg, links a protein and a polysaccharide).

[0056] The term "synthetic molecule" can refer to a small molecule or polymer that is not naturally occurring. For example, synthetic molecules can be prepared by chemical modification via solid phase synthesis, liquid phase synthesis, or a combination thereof.

[0057] Some embodiments provide methods for modifying or derivatizing sugar-bearing molecules (e.g., polysaccharides) with chemical groups capable of forming covalent bonds when combined with proteins. The polysaccharides can be modified to remain soluble or sufficiently soluble in water and / or saline. Most of the reactants remaining after polysaccharide modification are removed, for example, by precipitation or filtration. The modified sugar-bearing molecules (polysaccharides) can then be used as cross-linking agents. The modified polysaccharide solution can then be mixed with and reacted with the desired protein(s). The chemical modification groups on the modified polysaccharide react with the protein to form a biomaterial. The biomaterials produced in this manner have several unique properties compared to biomaterials purified using conventional methods. Formulations produced from proteins cross-linked using chemicals that result in intramolecular cross-links are typically opaque and often colored yellowish-white or brownish-white. Formulations obtained using some of the disclosed methods are clear and colorless. Furthermore, proteins cross-linked using chemicals (e.g., glutaraldehyde) have residual chemicals remaining in the formulation that can cause inflammation in vivo and / or reduce the biocompatibility of the product. Formulations obtained using some of the disclosed methods are substantially free of any such residual chemicals.

[0058] Formulations made with chemical crosslinkers or short crosslinking molecules often result in biocompatible materials that require micronization or homogenization to enable delivery using a syringe or needle; or that require a minimum level of crosslinking to enable extrusion through a needle. Proteins that are overly crosslinked, such as those crosslinked with chemicals (e.g., glutaraldehyde), cannot be extruded through a fine-gauge needle.

[0059] Formulations produced by some of the disclosed methods can be extruded through a fine-gauge needle without or substantially without further processing. Some of the disclosed formulations retain sufficient cohesion even after extrusion through a needle so that sufficiently long (e.g., >10 cm, >12 cm, >15 cm, >18 cm, >20 cm) strands of material can be extruded through the needle without shearing the material. Furthermore, using some embodiments described herein, formulations based on full-length proteins using modified polysaccharides as crosslinkers result in flexible matrix structures that can produce highly stable biomaterials that still retain sufficient flexibility to be removed through a fine-gauge needle.

[0060] (cross-linked protein matrix) The cross-linked protein matrix can vary in the disclosed embodiments.

[0061] For example, a cross-linked protein matrix can be obtained from the cross-linking of one or more protein molecules (one or more full-length proteins) with one or more sugar-bearing molecules (e.g., one or more sugar-bearing modified molecules).

[0062] For example, the cross-linked protein matrix can include one or more protein residues cross-linked with one or more sugar-bearing residues (glyco-, di-, tri-, oligo-, or polysaccharide-cross-linked proteins).

[0063] A crosslinked protein matrix can include linkages between one or more protein residues, e.g., via one or more linkages to one or more polysaccharide residues (e.g., one or more protein residues can be connected or linked to one another via one or more linkages to one or more polysaccharide residues). The crosslinked protein matrix can include, e.g., at least one protein residue linked to at least one polysaccharide-bearing residue (e.g., an oligosaccharide or polysaccharide residue) by at least one covalent bond and / or at least one ionic bond, or a combination thereof.

[0064] A crosslinked protein matrix can include, for example, linkages (i.e., one or more crosslinks (e.g., one or more intermolecular crosslinks and / or one or more intramolecular crosslinks, or a mixture or combination thereof)). A crosslinked protein matrix can be intermolecularly crosslinked, substantially intermolecularly crosslinked, intramolecularly crosslinked, substantially intramolecularly crosslinked, and / or both intermolecularly and intramolecularly crosslinked. The crosslinked protein matrix can be derived from one or more crosslinkers (e.g., a crosslinker bearing a sugar (e.g., a polysaccharide or modified polysaccharide, such as hyaluronic acid or modified hyaluronic acid)). For example, the crosslinked protein matrix can be derived from one or more crosslinkers, which can link to and / or crosslink one or more protein molecules and / or form one or more linkages to the same single protein molecule. For example, the crosslinked protein matrix can comprise a matrix structure (e.g., a matrix of protein residues linked to and / or crosslinked by one or more sugar-bearing crosslinker residues). The matrix structure of the crosslinked protein matrix can provide flexibility, where the degree of crosslinking within the crosslinked protein matrix can alter the provided flexibility.

[0065] In some embodiments, the use of crosslinked protein matrices as disclosed herein, derived from full-length proteins substantially lacking intramolecular crosslinks, can result in formulations that are more tissue compatible, promote tissue ingrowth, promote tissue regeneration, or a combination thereof, and that can be remodeled into a more standard desired structure and / or incorporated into new tissue.

[0066] In some embodiments, the use of crosslinked protein matrices as disclosed herein, derived from substantially full-length proteins substantially lacking intramolecular crosslinks, can result in formulations that are more tissue compatible, promote tissue ingrowth, promote tissue regeneration, or a combination thereof, and that can be remodeled into a more standard desired structure and / or incorporated into new tissue.

[0067] Other embodiments disclosed herein may have a degree of intramolecular cross-linking in the protein residues of the cross-linked protein matrix and still provide sufficient properties to be acceptable for use.

[0068] In some embodiments, the use of crosslinked protein matrices in which the structure of the protein residues is substantially uncovered by crosslinking processes and derived from substantially full-length proteins may result in formulations that are more tissue compatible, promote tissue ingrowth, promote tissue regeneration, or a combination thereof, and such formulations may be remodeled into a more standard desired structure and / or incorporated into new tissue.

[0069] (degree of crosslinking) In some embodiments, the solubility of sugar-bearing crosslinkers (e.g., polysaccharide crosslinkers) can be maintained by using specific ratios of chemical reagents during polysaccharide modification, derivatization, and / or processing. In some embodiments, useful precautions during post-derivatization processing may be required to prevent the derivatized polysaccharide from self-crosslinking. For example, the derivatized HA may need to be processed reasonably quickly after precipitation of the derivatized HA to remove residual reactants. In some embodiments, precipitation of the derivatized HA, removal of the reactants, and resuspension in aqueous solution can be performed within approximately 30 minutes. Other time periods may be used depending on the specific circumstances. For example, precipitation of the derivatized HA, removal of the reactants, and resuspension in aqueous solution can be performed within at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, or 2 hours.

[0070] Also, in some embodiments, dividing the derivatized HA precipitate into smaller portions before dissolution may be useful to improve the dissolution rate, which may require several hours (e.g., at least 1, 2, or 3 hours). Once dissolved, it may be desirable in certain applications to use the derivatized polysaccharide within a certain period of time (e.g., at least 1, 2, 3, 4, or 24 hours). However, this is not required and will depend on the particular formulation and / or application.

[0071] In some embodiments, the protein molecules utilized to form the cross-linked protein matrix in the formulation may be limited by the requirement for suitable reactive groups to render the protein molecules cross-linkable by the polysaccharide cross-linker.

[0072] In some embodiments, at least or about 50% of the protein monomers may be cross-linked with biomolecules and / or biopolymers (e.g., sugar-bearing molecules (e.g., oligosaccharides, polysaccharides, or derivatives thereof)). In other embodiments, at least or about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% of the protein monomers may be cross-linked with biomolecules and / or biopolymers or derivatives thereof. In some embodiments, the protein monomers may be substantially or completely cross-linked with biomolecules and / or biopolymers.

[0073] In some embodiments, the number of crosslinks per possible crosslinking site per polysaccharide may be at least 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 50%.

[0074] In some embodiments, the number of protein units or protein monomers remaining unbound and not incorporated into the crosslinked protein matrix or complex may be at least 1, 3, 5, 7, 9, 10, 15, or 20%. Minimizing the percentage of protein units remaining unbound after formation of the crosslinked protein matrix or complex is desirable in certain applications. For example, having less than 20%, 15%, 10%, 7%, 5%, 3%, or 1% unbound protein units in the crosslinked formulation is desirable in certain applications. A lack of unbound protein units or protein monomers is one of the advantages of certain applications of the present disclosure.

[0075] The percentage can depend on many considerations, including but not limited to, the protein and chemical species selected for a particular application. For example, the number of available sites for tropoelastin and lysine binding in some applications is typically about 30 to about 35, with the ratio being 1 to 35 (i.e., about 3% to 100%). For this combination, a preferred percentage for a crosslinked protein matrix can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the number of available sites on one or more protein molecules crosslinked with one or more biomolecules and / or biopolymers (e.g., sugar-bearing molecules or their derivatives).

[0076] Other considerations are the length of the sugar-bearing molecule (e.g., polysaccharide) and the components used in the activation, derivatization, or modification reaction. For example, in certain applications, 1%-30%, 1%-40%, 3%-30%, or 5%-30% of one or more carboxylic acid groups on a single carboxyl group on an oligosaccharide or polysaccharide may be activated with an activating agent (e.g., NHS) to make 1%-30%, 1%-40%, 3%-30%, or 5%-30% of activated ester sites available for cross-linking with proteins. For example, in certain applications, 1%-30%, 1%-50%, 3%-30%, or 5%-30% of one or more hydroxyl groups on an oligosaccharide or polysaccharide may be activated with an activator (e.g., allyl glycidyl ether) and further modified with a halogen compound (e.g., bromine) to form 1%-30%, 1%-40%, 3%-30%, 5%-30%, or 1%-50% of activated epoxy or halohydrin sites available for protein crosslinking. Other approximately effective components may also be used. Another consideration is keeping the percentage of protein units that may remain monomeric (i.e., unbound) low (e.g., 5% or less).

[0077] In some embodiments, protein monomers may be crosslinked with biomolecules and / or biopolymers to the extent that about 40% to about 90% of the protein monomers can be incorporated into the formulation, while in other embodiments, protein monomers may be crosslinked to the extent that about 30% to about 99%, about 40% to about 99%, about 50% to about 100%, about 60% to about 100%, about 70% to about 99%, about 80% to about 100%, or about 90% to about 100% of the protein monomers can be incorporated into the formulation.

[0078] In some embodiments, the crosslinked protein matrix can be acceptably resistant to biodegradation, degradation, hydrolysis, and / or combinations thereof over a period of time. In some embodiments, a formulation including the crosslinked protein matrix can be acceptably resistant to biodegradation, degradation, thermal degradation, hydrolysis, and / or combinations thereof over a period of time. Depending on the crosslinked protein matrix, the period of time can be at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months. Also, depending on the formulation including the crosslinked protein matrix, the period of time can be at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months.

[0079] In some embodiments, a formulation comprising a crosslinked protein matrix may remain acceptably intact or persist in vivo for between one week and one year. Depending on the particular formulation, the duration may vary. For example, a formulation comprising the crosslinked protein matrix may persist for at least one week to four weeks, two weeks to eight weeks, one month to three months, one month to six months, three months to nine months, or six months to twelve months, one week to 24 months, or 12 months to 24 months. In some embodiments, a formulation comprising the crosslinked protein matrix may persist in vivo for at least one week, two weeks, four weeks, two months, three months, six months, nine months, 12 months, 15 months, or 24 months.

[0080] For example, in some embodiments, a formulation (0.05 ml, 0.1 ml, 0.2 ml, or 0.5 ml, 1 ml, 2 ml, or 3 ml implant) containing the crosslinked protein may persist in vivo for about 1 week to about 2 years.

[0081] In some embodiments, the cross-linked protein matrix component within the formulation may be thermally stable, thermally resistant, hydrolytically stable, hydrolytically resistant, or a combination thereof during storage of the formulation. For example, the formulation may be stable for at least 6, 12, or 24 months when stored at a temperature of about 2°C to 8°C. The cross-linked protein matrix component within the formulation may be thermally stable, thermally resistant, hydrolytically stable, hydrolytically resistant, or a combination thereof during storage of the formulation when the formulation is lyophilized and stored at an appropriate temperature (e.g., below about -10°C), and thus may be stable for several years. The cross-linked protein matrix component within the formulation may be thermally and / or hydrolytically stable and / or resistant during storage of the formulation at room temperature for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 6 months, or 12 months. In some embodiments, the cross-linked protein matrix component within the formulation may be stable and / or resistant to thermal and / or hydrolytic degradation during storage of the formulation at room temperature for at least 1 week to 12 months, 2 weeks to 8 months, 1 week to 5 weeks, or 1 month to 6 months.

[0082] (homogeneous and heterogeneous cross-linked protein matrices) The structure and composition of the crosslinked protein matrix can vary. The crosslinked protein matrix can include, for example, one or more homologous or homogeneous protein residues (e.g., the same biopolymer) crosslinked with one or more homologous or homogeneous biomolecules, such as a homogeneous sugar-bearing molecule. The crosslinked protein matrix can include one or more homologous protein residues (e.g., two or more different biomolecule residues (e.g., heterogeneous biopolymer residues)) crosslinked with one or more heterogeneous or heterologous biomolecule residues, such as heterogeneous sugar-bearing residues. The crosslinked protein matrix can include one or more protein residues crosslinked with one or more different sugar-bearing residues (one or more different oligosaccharide or polysaccharide residues, and / or combinations thereof). The crosslinked protein matrix can include one or more protein residues crosslinked with a mixture of one or more different polysaccharide residues (e.g., a blend or mixture of hyaluronic acid and carboxymethylcellulose residues). A cross-linked protein matrix can be prepared by cross-linking one or more protein molecules with a mixture of one or more different activated polysaccharides (a blend or mixture of activated hyaluronic acid and activated carboxymethylcellulose).

[0083] Cross-linked protein matrices can include, for example, cross-linked protein matrices comprising heterologous protein residues (e.g., two or more different protein residues) cross-linked using a homogeneous biomolecular residue (e.g., a homogeneous sugar-bearing molecular residue (e.g., a homogeneous biopolymer residue)). Cross-linked protein matrices can include, for example, cross-linked protein matrices comprising heterologous protein residues (e.g., two or more different protein residues) cross-linked using a heterogeneous biopolymer residue (e.g., two or more different biomolecular residues (e.g., heterogeneous sugar-bearing residues)). A cross-linked protein matrix can comprise one or more (e.g., two or more) different protein residues cross-linked using a molecular residue (e.g., a biomolecular residue, a biopolymer residue, and / or derivatives or combinations thereof). A cross-linked protein can comprise one or more (e.g., two or more) different protein residues cross-linked using a sugar-bearing residue (e.g., an oligosaccharide-bearing residue or a polysaccharide-bearing residue).

[0084] A cross-linked protein matrix can include one or more (e.g., two or more) different protein residues that are cross-linked using one or more different molecular residues (e.g., one or more different biomolecule residues, one or more different biopolymer residues, and / or derivatives or combinations thereof). For example, a cross-linked protein matrix can include one or more (e.g., two or more) different protein residues that are cross-linked using one or more different sugar-bearing residues (e.g., one or more different oligosaccharide residues and / or one or more different polysaccharide residues (e.g., two or more different oligosaccharide residues and / or two or more different polysaccharide residues), or mixtures or combinations thereof).

[0085] The cross-linked protein matrix can include protein residues that are cross-linked with one or more different molecular residues (e.g., one or more different biomolecule residues, one or more different biopolymer residues, and / or derivatives or combinations thereof). For example, in some embodiments, the cross-linked protein matrix may include protein residues that are cross-linked using one or more (e.g., two or more) different sugar-bearing residues (e.g., one or more (two or more) different oligosaccharide residues and / or one or more (two or more) different polysaccharide residues, or mixtures or combinations thereof).

[0086] The cross-linked protein matrix may, for example, contain one or more protein residues for each polysaccharide residue (e.g., two or more protein residues for each polysaccharide residue). The cross-linked protein matrix may, for example, contain one or more polysaccharide residues for each protein residue (e.g., two or more polysaccharide residues for each protein residue). In some embodiments, the cross-linked protein matrix may, for example, contain protein residues and polysaccharide residues in a ratio of about 2.5% to 10% protein residues to about 0.1% to 1.5% polysaccharide residues. Other example ratios include 0.75% to 1.5% polysaccharide residues for 3% to 6% protein residues, 0.1% to 1.5% polysaccharide residues for 0.1% to 6% protein residues, 0.25% to 0.85% polysaccharide residues for 1% to 4% protein residues, 0.1% to 3% polysaccharide residues for 0.5% to 15% protein residues, 3% or less polysaccharide residues for at least 0.5% protein residues, at least 0.25% polysaccharide residues for 15% or less protein residues, at least 0.01% polysaccharide residues for 12% or less protein residues, or at least 1% polysaccharide residues for 8% or less protein residues. Other ratios may be used, depending on the desired properties and structure of the protein molecules used to induce the crosslinked protein matrix. For example, in softer formulations, the amount of polysaccharide molecules used may be reduced. For example, in a particular formulation, using longer chain sugars may allow for the use of fewer polysaccharide molecules and still allow for an acceptable formulation.

[0087] In some embodiments, the crosslinked protein matrix can include a biocompatible and / or bioavailable material. For example, the crosslinked protein matrix can be or be derived from a biocompatible and / or bioavailable material; i.e., the crosslinked protein matrix can be biocompatible and / or bioavailable.

[0088] In some embodiments, the crosslinked protein matrix can be a crosslinked protein matrix comprising and / or derived from a water-soluble crosslinker. For example, the crosslinked protein matrix can comprise or be derived from a water-soluble crosslinker bearing a sugar (e.g., a water-soluble oligosaccharide crosslinker) or a water-soluble modified crosslinker bearing a sugar (e.g., a water-soluble oligosaccharide crosslinker). Alternatively, the crosslinked protein matrix can comprise or be derived from a water-soluble crosslinker bearing a polysaccharide or a water-soluble modified crosslinker bearing a polysaccharide.

[0089] In some embodiments, the crosslinked protein matrix can include, for example, biomolecule-protein conjugates, biopolymer-protein conjugates, or combinations thereof. For example, the crosslinked protein matrix can include sugar-bearing molecule-protein conjugates (sugar-protein conjugates, disaccharide-protein conjugates, trisaccharide-protein conjugates, oligosaccharide-protein conjugates, polysaccharide-protein conjugates, and / or combinations thereof).

[0090] (molecule containing sugar) In some embodiments, sugar-bearing molecules (e.g., oligosaccharides) can contain one or more disaccharides, one or more trisaccharides, two or more disaccharides, two or more trisaccharides, three or more disaccharides, three or more trisaccharides, and / or combinations or derivatives thereof. For example, oligosaccharides can contain at least or about 3, 4, 5, 6, 7, 8, or 11 sugar residues or units. Additionally, oligosaccharides used to obtain the crosslinked protein matrix can contain, in certain formulations, from about 3 to about 15, from about 3 to about 14, from about 3 to about 12, from about 3 to about 11, from about 3 to about 10, from about 4 to about 15, from about 5 to about 15, or from about 5 to about 10 polysaccharide residues or units.

[0091] In some embodiments, a polysaccharide may comprise one or more disaccharide units or residues, one or more trisaccharide units or residues, one or more oligosaccharides, two or more disaccharide units or residues, two or more trisaccharide units or residues, two or more oligosaccharides, three or more disaccharide units or residues, three or more trisaccharide units or residues, three or more oligosaccharides, or a combination thereof. For example, a polysaccharide may comprise at least or about 25, 50, 100, 200, 500, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000, or 20,000 saccharide units or residues. Additionally, the polysaccharide used to obtain the crosslinked protein matrix may contain, in a particular formulation, from about 25 to about 5,000, from about 500 to about 2,000, from about 3,000 to about 5,000, from about 150 to about 250, from about 175 to about 225, from about 100 to about 175, from about 150 to about 200, or from about 100 to about 200 sugar residues or units.

[0092] In some embodiments, HA may be used in the range of about 100 to about 300 saccharide units or residues, e.g., about 200 saccharide units or residues. In other embodiments, HA may be used in the range of 200 to 20,000 saccharide units or residues. In other embodiments, HA may be used in the range of 500 to 2,000 saccharide units or residues. In other embodiments, HA may be used in the range of 3,000 to 5,000 saccharide units or residues. In other formulations, the HA used may contain at least or about 25, 50, 75, 100, 125, 150, 175, 200, 500, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000, or 20,000 saccharide units or residues. Additionally, the HA used to obtain the crosslinked protein matrix may contain, in certain formulations, from about 25 to about 5,000, from about 500 to about 2,000, from about 3,000 to about 5,000, from about 150 to about 250, from about 175 to about 225, from about 100 to about 175, from about 150 to about 200, or from about 100 to about 200 sugar residues or units.

[0093] The sugar-bearing molecules (e.g., polysaccharides) can be low, medium, or high molecular weight. For example, the compositions or formulations can be derived from low, medium, or high molecular weight polysaccharides or polysaccharide cross-linkers.

[0094] Low molecular weight molecules bearing sugars can have a molecular weight of about 25,000 to about 300,000 daltons (e.g., about 50,000 to about 275,000 daltons, about 100,000 to about 250,000 daltons, or about 50,000 to about 300,000 daltons). Medium molecular weight molecules bearing sugars can have a molecular weight of about 300,000 to about 900,000 daltons, about 600,000 to about 800,000 daltons, about 500,000 to about 900,000 daltons, or about 500,000 to about 750,000 daltons. High molecular weight sugar-bearing molecules can have molecular weights of about 900,000 daltons to about 4,000,000 daltons, about 1,000,000 daltons to about 3,500,000 daltons, about 900,000 daltons to about 3,500,000 daltons, about 1,500,000 daltons to about 3,700,000 daltons, or about 1,250,000 daltons to about 3,000,000 daltons. Polysaccharides having molecular weights ranging between any combination of the ranges listed herein are also contemplated. For example, polysaccharides having molecular weights of about 25,000 daltons to about 750,000 daltons, about 50,000 daltons to about 900,000 daltons, about 100,000 daltons to about 750,000 daltons, or about 250,000 daltons to about 500,000 daltons can be used, and other ranges can also be selected.

[0095] In some embodiments, the ability to use low- to medium-molecular-weight polysaccharides facilitates these approaches based on anticipated manufacturing / processing. For example, using low-molecular-weight HA allows the HA to be modified, precipitated, and washed, remaining in a reasonably low-viscosity solution that can be easily used as a crosslinker. While using higher molecular weight polysaccharides can introduce additional handling issues (e.g., problems with mixing, aeration, etc., viscous solutions), in some embodiments, a wide range of molecular weights can be used to achieve the desired results. One approach to handling higher molecular weight polysaccharides can be by using more dilute solutions, for example (e.g., using 1,500,000 dalton HA but using a 0.1% solution to keep the viscosity down).

[0096] The crosslinked protein matrix can include sugar-bearing residues (e.g., polysaccharide residues) at a concentration of about 0.1% to about 15%. In some embodiments, the crosslinked protein matrix can include sugar-bearing residues at a concentration of about 0.1% to about 10%, about 0.2% to about 5%, about 0.25% to about 5%, about 0.1% to about 3.5%, about 0.20% to about 3%, about 0.25% to about 3%, about 0.5% to about 4%, between about 0.5% and about 3%, about 0.75% to about 3.5%, about 1% to about 3%, a concentration range of about 1.5% to about 3.5%, or about 0.2% to about 4%.

[0097] The sugar-bearing molecule (e.g., a polysaccharide) can have a molecular weight of at least or about 500 daltons (e.g., at least or about 5,000 daltons, 10,000 daltons, 25,000 daltons, 50,000 daltons, 100,000 daltons, 150,000 daltons, 200,000 daltons, 250,000 daltons, 300,000 daltons, 500,000 daltons, 750,000 daltons, or 1,500,000 daltons).

[0098] In some embodiments, a molecule, such as a biomolecule or biopolymer, can include at least one linkable moiety (e.g., at least one cross-linkable moiety, such as a carboxyl group, a hydroxyl group, an amine, a thiol, an alcohol, an alkene, an alkyne, a cyano group, or an azide, and / or modifications, derivatives, or combinations thereof). For example, in some embodiments, a biomolecule or biopolymer (e.g., a protein or a sugar-bearing molecule, such as an oligosaccharide or polysaccharide) can include at least one cross-linkable moiety, such as a carboxyl group, a hydroxyl group, an amine, a thiol, an alcohol, an alkene, an alkyne, a cyano group, or an azide, and / or modifications, derivatives, or combinations thereof).

[0099] In some embodiments, the linkable moiety (e.g., cross-linkable moiety) can be an activatable moiety (e.g., a carboxyl or hydroxyl moiety) such that activation of the linkable moiety enables and / or facilitates reaction to form a linkage with a complementary reactive group on the same and / or a second molecule, e.g., a covalent bond with the same and / or a second molecule, and the reaction is, e.g., to form a cross-link to the second molecule (e.g., a second biomolecule or biopolymer).

[0100] In some embodiments, a molecule, such as a biomolecule or biopolymer (e.g., a sugar-bearing molecule or a protein), can include a spacer group that can link to the same and / or a second molecule (e.g., a second biomolecule or biopolymer). For example, in some embodiments, a spacer group can include at least one or more linkable moieties, thereby enabling the spacer group to link to the same and / or a second molecule. In some embodiments, a molecule, such as a sugar-bearing molecule or a protein, can include a spacer group that includes at least one or more linkable moieties, thereby enabling the molecule to form a linkage (e.g., a crosslink) with a second molecule (e.g., a second biomolecule or biopolymer (e.g., a protein or sugar-bearing molecule)) via a linkage formed by the linkable moieties on the spacer group. For example, in some embodiments, a sugar-bearing molecule (e.g., an oligosaccharide molecule, a polysaccharide molecule, or a modified sugar molecule) can include a spacer group containing at least one linkable moiety (a carboxyl group, an activated carboxyl group, or a modified carboxyl group) that allows the polysaccharide to form a linkage (e.g., a cross-link) with a second molecule (e.g., a protein (e.g., an amine-containing protein)) via an amide bond formed by the linkable moiety on the oligosaccharide, polysaccharide, or modified polysaccharide.

[0101] In some embodiments, the sugar-bearing molecule (e.g., an oligosaccharide or polysaccharide) can have a negatively or positively charged functional group (e.g., an oligosaccharide containing a negatively or positively charged functional group; or a polysaccharide containing a negatively or positively charged functional group; and / or derivatives or combinations thereof). For example, in some embodiments, the sugar-bearing molecule (e.g., an oligosaccharide or polysaccharide) can include iduronic acid, glucuronic acid, or N-acetylglucosamine residues. In some embodiments, for example, the sugar-bearing molecule can include a carboxyl-containing oligosaccharide or polysaccharide (e.g., a polycarboxylic acid-containing polysaccharide (e.g., hyaluronic acid or carboxymethylcellulose)), an amine-containing oligosaccharide or polysaccharide, and / or derivatives thereof.

[0102] (Structural characteristics - linear or branched chain) In some embodiments, the sugar-bearing molecule may include linear oligosaccharides, branched oligosaccharides, linear polysaccharides, and / or branched polysaccharides. Examples of sugar-bearing molecules include oligosaccharides and / or polysaccharides such as hyaluronic acid (HA), cellulose derivatives (e.g., carboxycellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose, carboxymethylamylose (CMA), xanthan gum, guar gum, α-glucan, β-glucan, β-1,4-glucan, β-1,3-glucan, alginate, carboxymethyldextran, glycosaminoglycans, and the like. Examples of suitable sugar-bearing molecules include, but are not limited to, aminoglycan derivatives, chondroitin-6-sulfate, dermatin sulfate, heparin, heparin sulfate, polylactic acid (PLA) or biocompatible materials such as polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), tricalcium phosphate (TCP), 1-hydroxyapatite (PAH), and / or pharmaceutically acceptable salts, derivatives, or combinations thereof. Sugar-bearing molecules may include pectin and / or its derivatives bearing linear and branched chain oligosaccharides and / or polysaccharides.

[0103] The sugar-bearing molecule can be a sugar-bearing molecule prepared and / or obtained from naturally occurring processes, chemical synthetic modifications, and / or combinations thereof.

[0104] For example, sugar-bearing molecules can include sugar-bearing molecules prepared and / or obtained from eukaryotic or prokaryotic cells (e.g., naturally occurring processes occurring via eukaryotic or prokaryotic cells, or combinations thereof).

[0105] For example, sugar-bearing molecules can include sugar-bearing molecules prepared and / or obtained from chemical synthetic modification (solid phase synthesis) and / or sugar-bearing molecules derived from chemical synthetic modification (solid phase synthesis), which incorporate a linking agent during solid phase synthesis of the polysaccharide.

[0106] Sugar-bearing molecules can include substantially soluble sugar-bearing molecules, for example, fully or partially soluble sugar-bearing molecules (oligosaccharides or polysaccharides that are substantially soluble in aqueous and / or physiological solutions).

[0107] Sugar-bearing molecules can include, for example, polyanionic sugars, polycationic sugars, biocompatible sugar molecules, bioavailable sugars, biodegradable sugars, bioabsorbable sugars, bioresorbable sugars, or combinations thereof.

[0108] (proteins and polysaccharides) In some embodiments, the crosslinked protein matrix can include, for example, sugar-bearing residue elements having electronic charge properties complementary to those of the protein residue elements of the crosslinked protein matrix. The charge-complementary properties of each element can aid or facilitate the integration of the elements. The charge-complementary properties of each element can add to the general overall properties of the composition. The crosslinked protein matrix can further include pharmaceutically and / or physiologically acceptable counterions that can complement the sugar-bearing residue elements having electronic charge properties, pharmaceutically and / or physiologically acceptable counterions that can complement the electronic charge properties of the protein residue elements, or both. For example, the crosslinked protein matrix can include sugar-bearing polyanionic residue elements (polyanionic polysaccharide residues crosslinked with positively charged protein residues). For example, the crosslinked protein matrix can include sugar-bearing polycationic residue elements (polycationic polysaccharide residues crosslinked with negatively charged protein residues).

[0109] (Protein and polysaccharide selection) In some embodiments, the selection of protein components (e.g., tropoelastin) included in the crosslinked protein matrix may be based on the functional end-use requirements of the resulting biomaterial product. For example, the protein residue components included in the crosslinked protein matrix may include protein residues (e.g., albumin residues or collagen residues). In some embodiments, the selection of protein residue components included in the crosslinked protein matrix may be based on the end-use requirements for the bioactivity of the resulting biomaterial product. ...

[0110] In some embodiments, the protein residue component included in the crosslinked protein matrix formulation can vary in composition. For example, in some embodiments, the formulation can have 25 mg / ml to 50 mg / ml of protein residue and 1 mg / ml to 30 mg / ml of polysaccharide crosslinker residue. In some formulations, the protein residue component included in the crosslinked protein matrix formulation can be 1 mg / ml to 200 mg / ml, 5 mg / ml to 30 mg / ml, 20 mg / ml to 100 mg / ml, 50 mg / ml to 200 mg / ml, 20 mg / ml to 100 mg / ml, 25 mg / ml to 80 mg / ml, 30 mg / ml to 60 mg / ml, 40 mg / ml to 70 mg / ml, or 25 mg / ml to 65 mg / ml. In some embodiments, the preferred ranges for the amount of protein residue component and the preferred ranges for the amount of polysaccharide residue component included in the crosslinked protein matrix formulation can vary based on the requirements of a particular application.

[0111] (Coupling / Conjugation / Crosslinking) In some embodiments, crosslinked protein matrices can be prepared by linking (e.g., coupling and / or crosslinking) proteins (amine-containing proteins) to sugar-bearing molecules to form amide or amine bonds, where the sugar-bearing molecules are molecules containing carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, modified carboxyl, or modified hydroxyl groups (e.g., oligosaccharides, polysaccharides, and / or derivatives thereof containing carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, modified carboxyl, or modified hydroxyl groups). For example, crosslinked protein matrices can be prepared by coupling and / or crosslinking proteins (amine-bearing proteins) to oligosaccharides and / or modified oligosaccharides to form amide or amine bonds between the protein and the oligosaccharide, where the oligosaccharides and / or modified oligosaccharides contain carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, modified carboxyl, or modified hydroxyl groups. For example, crosslinked protein matrices can be prepared by coupling and / or crosslinking proteins (amine residue-bearing proteins) to oligosaccharides and / or modified oligosaccharides, forming amide or amine bonds between the protein and the oligosaccharide, wherein the polysaccharides and / or modified polysaccharides contain carboxyl groups, hydroxyl groups, activated carboxyl groups, activated hydroxyl groups, modified carboxyl groups, or modified hydroxyl groups.

[0112] In some embodiments, the formation of a crosslinked protein matrix can be facilitated by using either (1) an activating agent and / or coupling agent, and (2) both an activating agent and / or coupling agent and a modifying agent and / or auxiliary coupling agent to form linkages and / or crosslinks between a protein component of the crosslinked protein and a second molecule of the crosslinked protein (e.g., a biomolecule, a biopolymer, a spacer group, or a derivative or combination thereof). The crosslinked protein matrix can be prepared by forming linkages and / or crosslinks using an activating agent and / or coupling agent. For example, the crosslinked protein matrix can be prepared by activating one or more carboxylic acid groups of a carboxyl-bearing oligosaccharide or polysaccharide (e.g., hyaluronic acid) with an activating agent and / or coupling agent, and coupling and / or crosslinking the activated oligosaccharide or polysaccharide to a protein (e.g., an amine-bearing protein) to form an amide bond between the oligosaccharide or polysaccharide and the protein. For example, a crosslinked protein matrix can be prepared by activating one or more hydroxyl groups of a hydroxyl-bearing oligosaccharide or polysaccharide (e.g., hyaluronic acid) with an activating and / or coupling agent, and linking and / or crosslinking the activated oligosaccharide or polysaccharide to a protein (e.g., an amine residue-bearing protein) to form an amine bond between the oligosaccharide or polysaccharide and the protein.

[0113] (Activator / Coupling Agent / Modifier) In some embodiments, the activating agent (also referred to as a coupling agent) may include, but is not limited to, a diimide (e.g., a carbodiimide or a water-soluble carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ("EDC"), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide ("ETC"), 1-cyclohexyl-3-(2-)morpholinoethyl)-carbodiimide ("CMC"), and / or a corresponding salt or mixture thereof). The activating agent may also include, but is not limited to, benzotriazol-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate ("Bop-reagent"), O-benzotriazol-1-yl-N,N,N',N'-tetramethylyluronium hexafluorophosphate, bromo-tris-(dimethylamino)-phosphonium hexafluorophosphate, and / or a corresponding halide salt or mixture thereof. In some embodiments, activating agents (also called coupling agents) may include, but are not limited to, epoxides (e.g., allyl glycidyl ether) or haloalkenes (e.g., allyl chloride), and / or their corresponding salts or mixtures.

[0114] A crosslinked protein matrix can be prepared by linking and / or crosslinking using both an activating agent and / or coupling agent and a modifying agent and / or auxiliary coupling agent. For example, a crosslinked protein matrix can be prepared by (1) activating one or more carboxylic acid groups of a carboxyl-containing oligosaccharide or polysaccharide (e.g., hyaluronic acid) using an activating agent and / or coupling agent, (2) modifying one or more activated carboxylic acid groups of the activated oligosaccharide or activated polysaccharide using a modifying agent and / or auxiliary coupling agent, and (3) linking or crosslinking the modified oligosaccharide or modified polysaccharide to a protein (e.g., an amine residue-bearing protein) to form an amide bond between the oligosaccharide or polysaccharide and the protein.

[0115] In some embodiments, modifying agents (also called auxiliary coupling agents) can include, but are not limited to, reagents that react in the presence of activated carboxyl and / or activated hydroxyl moieties (e.g., activated carboxyl and / or activated hydroxyl moieties on a polysaccharide) to form modified species that may be more stable and / or more reactive with nucleophiles. For example, modifying or auxiliary coupling agents may include, but are not limited to, N-hydroxy-succinimide (NHS), N-hydroxysulfosuccinimide ("sulf-NHS"), 1-hydroxy-benzotriazole hydrate ("HOBt"), 1-hydroxybenzotriazole monohydrate, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAT), 4-nitrophenol, 2-nitrophenol, 4-nitrothiophenol, 2-nitrothiophenol, pentachlorophenol, pentafluorophenyl, imidazole, tetrazole, 4-dimethylaminopyridine, halogen compounds, and / or other related compounds.

[0116] In some embodiments, a crosslinked protein matrix can be prepared by activating and / or modifying a sugar-bearing molecule (e.g., an oligosaccharide or polysaccharide, such as hyaluronic acid) having one or more carboxyl and / or hydroxyl groups with an activating and / or modifying agent, and combining it with a protein to form one or more linkages and / or crosslinks (one or more amide or amine bonds) between the sugar-bearing molecule and the protein.

[0117] Methods for preparing crosslinked protein matrices can include mixing and / or combining a sugar-bearing molecule (e.g., hyaluronic acid) containing one or more carboxyl and / or hydroxyl groups with an activating agent and / or modifying agent to form an activated sugar-bearing molecule and / or a modified sugar-bearing molecule, and mixing and / or combining the activated sugar-bearing molecule and / or the modified sugar-bearing molecule with a protein to form one or more linkages and / or crosslinks (one or more amide or amine bonds) between the sugar-bearing molecule and the protein. For example, the sugar-bearing molecule (e.g., hyaluronic acid) containing one or more carboxyl and / or hydroxyl groups can be activated and / or modified using an activating agent (EDC or allyl glycidyl ether) and / or a modifying agent (NHS, HOBt, or bromine). For example, the activated and / or modified sugar-bearing molecules can contain one or more carboxyl and / or hydroxyl groups that have been activated and / or modified as activated and / or modified esters (e.g., activated and / or modified triazole esters, or activated and / or modified N-hydroxysuccinimide esters), activated and / or modified epoxides, or activated and / or modified halohydrins. Molecules (e.g., hyaluronic acid) bearing sugars with one or more carboxyl and / or hydroxyl groups can be activated and / or modified using activating agents (EDC or allyl glycidyl ether) and / or modifying agents (NHS, HOBt, or bromine) and combined, mixed, and / or reacted with compounds bearing one or more amine moieties (proteins bearing one or more amine-bearing side chains) to form one or more linkages and / or crosslinks (amide or amine bonds) between the sugar-bearing molecule and the compound bearing one or more amine moieties.

[0118] In some embodiments, a molecule having one or more amine moieties (e.g., a protein, peptide, or spacer group having one or more amine moieties) can be coupled to one or more carboxyl and / or hydroxyl groups on a sugar-bearing molecule. For example, in an aqueous environment, the molecule containing one or more amine moieties can be coupled to an oligosaccharide or polysaccharide having one or more carboxyl and / or hydroxyl groups (e.g., an oligosaccharide or polysaccharide having one or more carboxyl and / or hydroxyl groups that have been activated and / or modified using an activating and / or modifying agent).

[0119] In some embodiments, the method used to modify the polysaccharide may depend on the protein being cross-linked and / or the polysaccharide used as the cross-linking agent. For example, methods used to modify polysaccharides may include the use of periodate oxidation. Methods used to modify polysaccharides may include the use of an activating agent, such as a carbodiimide (e.g., EDC). Methods used to modify the polysaccharides may include the use of an activating agent, such as an epoxide (e.g., allyl glycidyl ether). Methods used to modify the polysaccharides may further include the use of a modifying agent, such as a halogen compound (e.g., N-hydroxysuccinimide (NHS) or bromine). For example, hyaluronic acid and / or carboxymethylcellulose may be activated with an activating agent, such as a carbodiimide, and further modified with a modifying agent, such as N-hydroxysuccinimide. For example, hyaluronic acid and / or carboxymethylcellulose may be activated with an activating agent, such as an epoxide, and further modified with a modifying agent, such as bromine.

[0120] (Bifunctional molecular reagents) In some embodiments, crosslinked protein matrices can be prepared by reacting a protein with a multifunctional reagent (a sugar-bearing molecule containing two or more reactive moieties or a spacer group containing two or more reactive moieties) to form two or more linkages or crosslinks. For example, the multifunctional reagent can contain two or more reactive moieties, which can be the same or different (e.g., a mixture of carboxyl and / or hydroxyl groups, a mixture of activated carboxyl and / or activated hydroxyl groups, a mixture of modified carboxyl and / or modified hydroxyl groups, and / or combinations or derivatives thereof). For example, each of the reactive moieties on the multifunctional reagent can be reactive with complementary reactive groups on the same or different molecules. For example, one or more reactive moieties of the multifunctional reagent may require deprotection (e.g., removal of a protecting group) to be able to react with complementary reactive groups on the same or different molecules.

[0121] (spacer group) A spacer group may include a moiety that connects one or more individual elements. The spacer group may be linked to one or more molecules (e.g., covalently linked to a protein, covalently linked to a polysaccharide, covalently linked to both a protein and a polysaccharide, and / or combinations thereof). Spacer groups may include, but are not limited to, glycol moieties, ethylene oxide moieties, polymers formed from repeating -(-CH2-CH2-O-)- moieties (e.g., polyethylene glycol (PEG) or polyethylene oxide (PEO)), polyamines, or polyols. The spacer may be stable to thermal degradation, hydrolysis, or both. The spacer may be biocompatible, bioavailable, soluble and / or substantially soluble in aqueous and / or physiological media, or a combination thereof. The biomolecule or biopolymer may include one or more spacer group residues (e.g., polyethylene glycol (PEG) or polyethylene oxide (PEO) groups). The crosslinked protein matrix may include one or more spacer group residues (e.g., polyethylene glycol (PEG) or polyethylene oxide (PEO) groups). The sugar-bearing molecule may include one or more spacer group residues (e.g., polyethylene glycol (PEG) or polyethylene oxide (PEO) groups). The sugar-bearing molecule may include one or more moieties that include a reactive group (e.g., a reactive group that can form a covalent bond when reacted with a complementary reactive group that may be part of a protein or modified protein). The protein or modified protein may include one or more moieties that include a reactive group (e.g., a reactive group that can form a covalent bond when reacted with a complementary reactive group that may be part of a sugar-bearing molecule).

[0122] (Degree of polysaccharide modification) In some embodiments, sugar-bearing crosslinkers (e.g., hyaluronic acid) containing one or more carboxyl and / or hydroxyl groups can be activated and / or modified to include various activated and / or modified carboxyl and / or hydroxyl groups, as well as various unactivated and / or unmodified carboxyl and / or hydroxyl groups. For example, sugar-bearing crosslinkers can be activated and / or modified to include at least or about 2% (e.g., at least or about 0.5%, 1%, 3%, 5%, 10%, 20%, 25%, 30%, or 35%) of activated and / or modified carboxyl and / or hydroxyl groups. In certain applications, sugar-bearing crosslinkers can include substantially or completely activated and / or modified carboxyl and / or hydroxyl groups. In some embodiments, the sugar-bearing activated and / or modified crosslinker can be activated and / or modified to contain from about 0.5% to about 40%, from about 1% to about 30%, from about 1% to about 25%, from about 3% to about 30%, or from about 5% to about 25% activated and / or modified carboxyl groups and / or activated and / or modified hydroxyl groups.

[0123] In some embodiments, varying the level of activated and / or modified functional groups on a sugar-bearing crosslinker can increase and / or decrease the ability of the sugar-bearing crosslinker to crosslink with a biomolecule. For example, the level of activated and / or modified functional groups on a sugar-bearing crosslinker can result in the formation of one or more bonds between the sugar-bearing crosslinker and a biomolecule or biopolymer. Varying the level of activated and / or modified functional groups on a sugar-bearing crosslinker used to prepare a crosslinked protein can control or substantially control the number of activated and / or modified functional groups available to react with a biomolecule or biopolymer (capable of linking and / or crosslinking with the protein), stabilizing or substantially stabilizing the protein.

[0124] (Extrusion) In some embodiments, the crosslinked protein matrix can be extruded through a needle. For example, the crosslinked protein matrix can be extruded through a fine-gauge needle. The crosslinked protein matrix can maintain sufficient cohesion (e.g., maintain sufficient cohesion even after extrusion through a needle, such that long strands of material can be extruded through the needle without breaking). For example, strands of material at least about 15 cm in length can be extruded through a needle without breaking. The crosslinked protein matrix can include a flexible matrix structure. For example, the flexible matrix structure of the crosslinked protein matrix can facilitate the production of a stable biomaterial that maintains sufficient flexibility to be ejected through a thin needle. In some embodiments, a formulation including the crosslinked protein matrix can be extruded through a needle (e.g., extruded through a fine-gauge needle without further processing). In some embodiments, a formulation including the crosslinked protein matrix can maintain sufficient cohesion even after extrusion through a needle, such that long strands of material (strands of material having a length of at least about 15 cm) can be extruded through the needle without breaking. In some embodiments, the formulation can include a crosslinked protein matrix having a flexible matrix structure. For example, in some embodiments, the flexible matrix structure of the crosslinked protein matrix included in the formulation can facilitate the production of a stable biomaterial (e.g., a stable biomaterial that retains flexibility (e.g., substantial and / or sufficient flexibility)) and can enable the formulation and / or biomaterial to be ejected through a needle (e.g., a fine gauge needle).

[0125] (Preparation method) In some embodiments, the concentration of polysaccharide in the reaction can be about 0.1% to about 5% (eg, about 0.25% to about 3%, about 0.5% to about 3%, or about 0.25% to 3.5%).

[0126] In some embodiments, the reagent stoichiometry can vary depending on the chemistry and polysaccharide selected. For example, the ratio of HA:EDC:NHS can be 1:1:1, 1:1:2, 1:1:3, 1:0.5:2, or 1:0.5:3. A ratio of 1:1:1 has been shown to provide good results for the incorporation of NHS into soluble polysaccharide crosslinkers.

[0127] In some embodiments, the molar ratio of polysaccharide to activating agent can be at least 1:1 to at least 1:4. In some embodiments, the molar ratio of activating agent to carboxylic acid units of the polysaccharide can be from about 2% to about 200% (e.g., from about 5% to about 100%). In some embodiments, the molar ratio of modifying agent to activating agent can be from about 1:1 to about 3:1 (e.g., from about 1.5:1 to about 2.5:1 (e.g., about 2:1)).

[0128] In some embodiments, the pH at which the polysaccharide crosslinker reactant preparation is carried out can be at least 4, 5, 6, 7, 8.0, or 8.5. In some embodiments, the pH at which the polysaccharide crosslinker reactant preparation is carried out can be from about 5 to about 15, from about 6.5 to about 9, or from about 7 to about 8.6. Other pH values ​​can also be used.

[0129] In some embodiments, the temperature range for activation, coupling, and / or crosslinking is controlled at about 15° C. to about 30° C., 20° C. to about 25° C., or room temperature.

[0130] In some embodiments, the methods for purifying and / or isolating derivatized polysaccharides can be robust, simple, high-yielding, or a combination thereof. For example, the methods can result in at least 40% isolated derivatized polysaccharide relative to the starting polysaccharide. In other methods, the yield is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isolated derivative polysaccharide relative to the starting polysaccharide or modified polysaccharide.

[0131] Therapeutic Use The crosslinked protein matrix compositions, materials, formulations, methods of use, systems, and / or kits disclosed herein can be used in a variety of therapeutic contexts, including, but not limited to, human or veterinary medicine (e.g., surgical procedures). For example, the crosslinked protein matrix compositions, materials, formulations, methods of use, systems, and / or kits disclosed herein can be used therapeutically in reparative surgery, cosmetic surgery, aesthetics, tissue augmentation (e.g., incontinence or skin replacement products), dermatology (skin surgery), eye surgery, rheumatology, pharmacology, or cosmetics. Other therapeutic applications may include hemostasis in general surgery, nerve and vascular reconstruction in neurosurgery and cosmetic surgery reconstructive surgery, fixation of skin, vascular, or cartilage transplants or grafts in orthopedic surgery (treatment of osteoarthritis (knee inflammation)), vascular, and cosmetic surgery procedures. Some embodiments are useful as vehicles for the delivery of bioactive molecules (eg, growth factors that stimulate cells or localized repair). Growth factors, when combined with crosslinked protein matrix compositions, materials, and / or formulations, can be delivered locally to promote wound healing and tissue regeneration in many situations (e.g., promoting bone formation, promoting cartilage repair in cosmetic procedures, treating pathological wound conditions (chronic ulcers)), and / or serve as a scaffold for the formation of artificial tissues via the growth of autologous cells in culture. Crosslinked protein matrix compositions, materials, and / or formulations are injectable, and in some embodiments, these properties make them suitable for causing tissue expansion, for example, as inert biocompatible fillers (e.g., for filling skin wrinkles or lip restoration). In some embodiments, crosslinked protein matrix compositions, materials, and / or formulations are useful as prosthetic materials for filling body cavities or defects. In some embodiments, crosslinked protein matrix compositions, materials, and / or formulations are useful for cosmetic applications, plastic surgery, restoring volume lost during surgery (e.g., ophthalmic surgery), and / or for topical application to healthy or injured tissue (e.g., topical application in cosmetic and / or dermatological settings). In some embodiments, the crosslinked protein matrix compositions, materials, and / or formulations are useful for filling facial wrinkles and fine lines, treating depressions on the skin, such as aging skin, scar tissue, and / or lipodystrophy.

[0132] Some embodiments may be used to stabilize proteins (eg, bioactive proteins utilized to deliver one or more stabilized proteins (eg, bioactive proteins)).

[0133] Some embodiments may include pharmaceutical active substances dispersed throughout and may be useful as drug delivery systems, such as proteins, growth factors, enzymes, drugs, biopolymers, biocompatible synthetic polymers, and / or combinations, derivatives, or variations thereof.

[0134] (Characteristics; Stability) In some embodiments, the crosslinked protein matrix may include at least one or more of the following properties, but are not limited to: injectability, biocompatibility, substantially biocompatible, stability, substantially stability, maintaining bioactivity, substantially maintaining bioactivity, maintaining bioactive conformation, providing elasticity or substantial elasticity, modulus of elasticity, modulus of viscosity, providing structural rigidity or substantial rigidity, heat resistance or substantially heat resistance, resistance or substantially resistance to thermal degradation, resistance or substantially resistance to biodegradation, optionally biodegradable, not eliciting a foreign body reaction or significant foreign body reaction (i.e., self-recognition), having a purity level of at least about 25%, extrudability, extrudability through a needle, extrudability through a fine gauge needle.

[0135] In some embodiments, the crosslinked protein matrix, composition, material, and / or formulation may include a sugar-bearing molecule that has at least one of the following properties, including, but not limited to: substantially soluble, soluble in water, substantially soluble in aqueous and / or buffered aqueous solutions, physiologically soluble, substantially physiologically soluble, injectable, biocompatible, substantially biocompatible, stable, substantially stable, maintained biologically active, substantially maintained biologically active, maintained biologically active conformation, resistant or substantially resistant to biodegradation, may be biodegradable, does not elicit a foreign body reaction or significant foreign body reaction (i.e., autorecognition), or has a purity level of at least about 25%.

[0136] In some embodiments, the crosslinked protein matrix composition, material, and / or formulation may have or include at least one of the following properties, but is not limited to: injectability, biocompatibility, substantial biocompatibility, stability, substantial stability, maintaining bioactivity, substantially maintaining bioactivity, maintaining a bioactive conformation, providing elasticity or substantial elasticity, elastic modulus, viscous modulus, providing structural rigidity or substantial rigidity, heat resistance or substantially heat resistance, resistance or substantially resistance to thermal degradation, resistance or substantially resistance to biodegradation, may be biodegradable, does not elicit a foreign body reaction or significant foreign body reaction (i.e., self-recognition), has a purity level of at least about 25%, extrudability, extrudable through a needle, or extrudable through a fine gauge needle.

[0137] Some embodiments may have an elastic modulus of about 500 Pa to about 50 Pa, about 450 Pa to about 100 Pa, about 400 Pa to about 125 Pa, about 400 Pa to about 150 Pa, or about 385 Pa to about 150 Pa. The elastic modulus will vary depending on the concentration and factors used. For example, for a 4% tropoelastin matrix product crosslinked with 1% HA, the elastic / storage modulus is stable at about 80 Pa to 100 Pa over a range of frequencies and is dominated by a loss modulus that begins at about 5 Pa to 10 Pa and gradually increases with increasing angular frequency.

[0138] In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 25G needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 25G needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm.

[0139] In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 27G needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 27G needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm.

[0140] In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 30G needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 30G needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm.

[0141] In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 31G needle can be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length that remains substantially coherent and unsupported when extruded through a 31G needle can be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm.

[0142] In some embodiments, the extrudable length obtained when extruded through a fine gauge needle may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length may range from about 5 cm to about 30 cm, from about 10 cm to about 20 cm, from about 10 cm to about 15 cm, or from about 15 cm to about 30 cm. In some embodiments, the extrudable length obtained when extruded through a fine gauge needle that remains substantially coherent and unsupported may be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length that remains coherent and unsupported when extruded through a fine gauge needle can be at least about 5 cm, at least about 10 cm, at least about 12 cm, at least about 15 cm, at least about 18 cm, at least about 20 cm, or at least about 25 cm. In some embodiments, the extrudable length that remains substantially coherent and unsupported can range from about 5 cm to about 30 cm, from about 10 cm to about 20 cm, from about 10 cm to about 15 cm, or from about 15 cm to about 30 cm.

[0143] In some embodiments, the crosslinked protein matrix composition, material, and / or formulation may be stabilized with a product protein component. The stability of the crosslinked protein matrix composition, material, and / or formulation may be a result of combining a full-length protein component with a crosslinking component (a polysaccharide component). In some embodiments, the properties of the final material may be determined independently of the viscosity of the polysaccharide component (the starting polysaccharide molecule component). The crosslinked protein matrix composition, material, and / or formulation may be stabilized with a product crosslinking component (such as a sugar-containing crosslinking component; e.g., a polysaccharide crosslinking component).

[0144] In some embodiments, the composition, material, and / or formulation of the cross-linked protein matrix may include biocompatible cross-linked protein residues.

[0145] In some embodiments, the composition, material, and / or formulation of the cross-linked protein matrix may be suitable for incorporation into a syringe.

[0146] In some embodiments, fine gauge needles may be used. For example, 25G, 27G, 29G, 30G, or 31G needles may be used. However, some embodiments may be used with larger gauge needles, such as 20G-25G, 15G-25G, 10G-20G, or 10G-15G needles. In some embodiments, needle size may be determined based on the material being injected, e.g., the type and / or concentration of the material being injected, as desired to deliver a specific volumetric amount of material and / or mixture or modified material. In some embodiments, fine gauge needles may be used to allow the disclosed materials to retain sufficient coherence after extrusion through the needle, thereby allowing strings of material greater than 15 cm in length to be extruded without breaking. For example, in some embodiments, strings of material greater than 15 cm in length may be extruded without breaking through a 25G, 27G, 29G, or 30G needle.

[0147] For certain applications (e.g., applications targeting a distending effect such as on the bladder neck), an 18G, 19G, 20G, 21G, 22G, or 23G needle may be used. For certain applications, a larger needle may be used because the longer length of the needle (typically several inches) creates greater resistance to the material flowing through the needle. For certain applications, the injection volume may be increased by several milliliters, e.g., at least 1.5 ml, at least 2 ml, at least 2.5 ml, at least 3 ml, or at least 4 ml. For certain applications requiring smaller injection volumes (e.g., "threading" procedures, fine wrinkle filling, or thinning skin augmentation), smaller injection volumes may be used, e.g., less than 2 ml, less than 1.5 ml, less than 1.0 ml, less than 0.75 ml, less than 0.5 ml, or less than 0.1 ml. Typically, shorter, finer gauge needles are used in these applications, such as 1 / 2 inch long 29G, 30G, or 31G needles.

[0148] The gauge and length of the needle used may vary depending on the specific application and / or formulation. For example, formulations with high derivatization levels (e.g., 20%-30% of available sites modified and with 1.5% and 5% polysaccharide and protein contents, respectively) used to provide more structurally durable tissue support are typically injected using shorter, larger needles (e.g., 27G or 25G needles, 1 / 2 or 1 inch long). Another example is formulations with low derivatization levels (approximately 5%) and low HA (less than 1%) and / or protein (less than 3%); these formulations are typically delivered through finer gauge needles, such as 31G.

[0149] In some embodiments, the extruded material may be extruded without a support, typically perpendicular to the initial plane or at a 45° angle relative to the perpendicular. The ability to form coherent threads of the extruded material makes it particularly attractive for applications where the graft is threaded in a matrix or lattice pattern within the skin to provide structural support.

[0150] Other delivery methods may be used, for example, using a cannula, catheter, flexible polymer catheter, and / or needleless syringe.

[0151] In some embodiments, at least one advantage of the methods and / or crosslinked protein matrix compositions, materials, and / or formulations disclosed herein is that the amount of protein contained in the resulting material formulation is not a limiting factor. For example, the crosslinked protein matrix may contain about 35 mg / ml or about 40 mg / ml more protein residue than would be present in a protein crosslinking method other than those disclosed herein (e.g., glutaraldehyde, or other crosslinking agents), which would result in excessively high crosslinked material concentrations and increased resistance to extrusion through a needle. The protein residues may be intramolecularly crosslinked, intermolecularly crosslinked, and / or a combination thereof. The protein residues may be substantially intermolecularly crosslinked, or may be exclusively intermolecularly crosslinked. The crosslinked protein matrix formulation may be one that has higher elasticity and / or is suitable for injection with high protein concentrations.

[0152] The crosslinked protein matrix compositions, materials, and / or formulations disclosed herein may be used in kits or packages. In some embodiments, the kits or packages include a syringe prefilled with the crosslinked protein matrix composition and an appropriately sized needle or needle delivery system (e.g., a needle rollerball system, an auto-injection pen system, or a mesotherapy injection gun system). The packages or kits may also include instructions for injecting the adjunct compositions. In other embodiments, the kit or package may include at least one syringe, at least one separate container (a vial or ampoule containing the composition to be used), multiple needles, and instructions for use of the kit. [Example]

[0153] The following examples and protocols are provided as some embodiments of the present disclosure to demonstrate these advantages: The above examples and protocols are provided for illustrative purposes and are not intended to limit the scope of the specification or the claims that follow.

[0154] (Procedure for derivatization of hyaluronic acid (HA) using EDC and NHS) Dissolve HA in water to a final concentration of 1.1% (probably less than 2%). 2. Add 1 g of N-hydroxysuccinimide (NHS) per g of HA to be derivatized. 3. Add 1 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) per g of HA. 4. Stir thoroughly (approximately 10-20 minutes) to completely dissolve the reactants. 5. Allow to react at 20-25°C for 60 minutes. Add NaCl to a final concentration of 6.1%. The derivatized HA is precipitated by the addition of 7.2 volumes of isopropanol (IPA). 8. Recover the precipitated derivatized HA by filtration, gentle centrifugation, or other suitable method and discard the supernatant. 9. Apply gentle pressure to the recovered derivatized HA to remove excess liquid. 10. Wash precipitated derivatized HA in 60% IPA. 11. Remove and discard the wash solution. 12. Apply gentle pressure to the collected derivatized HA to remove excess liquid. 13. Weigh the recovered derivatized HA. 14. Dissolve the recovered derivatized HA in sterile water to a final concentration of 2.5% based on the initial amount of HA dissolved in step 1. 15. Analyze the amount of NHS derivatization (based on chemical modification and UV analysis). 16. Analyze the concentration of dissolved derivatized HA (based on chemical modification and UV analysis or dry weight). 17. Adjust the concentration of derivatized HA to 2% (20 mg / ml). 18. Sterile filter the derivatized HA.

[0155] NOTE: Steps 7–14 should be performed as quickly as possible; less than 30 min total is acceptable for these steps (although the actual dissolution of the precipitated HA may take longer).

[0156] Procedure for the preparation of cross-linked protein matrices 1. Dissolve protein in sterile PBS to a final concentration of 100 mg / ml and filter sterilize. 2. Analyze protein concentration (e.g., based on UV analysis). 3. Mix an equal volume of 20 mg / ml derivatized HA with 100 mg / ml protein under thorough mixing / stirring without introducing any air bubbles. 4. Allow to stand for 30-60 minutes to gel (20-25°C). 5. Fill the syringe.

[0157] Below is a common procedure used in several of the examples that follow.

[0158] Example 1 Schematic for the generation of soluble hyaluronic acid crosslinkers using carbodiimide and N-hydroxysuccinimide (NHS). Hyaluronic acid (HA) is a polysaccharide composed of the disaccharide unit β-D-glucuronic acid-

[0013] -β-DN-acetyl-glucosamine. The idealized structure of HA is shown in Figure 1.

[0159] As shown in the figure, HA contains one carboxyl group per disaccharide unit, and it is this functional group that can be utilized in at least one of the cross-linking techniques disclosed herein. In this cross-linking technique, a covalent chemical bond is formed between the carboxyl group of HA and a free amino group on a protein. This is achieved by reacting HA with a carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)) to form an activated o-acylisourea ester. This compound is reactive with nucleophiles (e.g., primary amino groups) and is unstable, rapidly hydrolyzing in water in the absence of any suitable reactive groups. For this reason, it is often preferable to form a more stable intermediate activated ester from the activated o-acylisourea ester, which can be achieved by a condensation reaction with compounds such as N-hydroxysuccinimide (NHS) or sulfo-N-hydroxysuccinimide (sulfo-NHS). This results in the formation of NHS-activated HA, which can then react with primary amine groups from proteins to form highly stable amide bonds. A schematic of these reactions is shown in FIG.

[0160] One of the main advantages of this approach is that HA is derivatized before being mixed with the protein, limiting or avoiding the presence of residual derivatization reagent in the final formulation. The primary methods for removing excess reagent are either precipitation with a water-miscible organic solvent (e.g., isopropyl alcohol (IPA) or ethanol), followed by washing with a water / solvent mixture, or diafiltration using a semipermeable membrane with an appropriate molecular weight cutoff. Once purified, the derivatized HA can be dissolved in an appropriate buffer (e.g., water or phosphate-buffered saline (PBS)) and then mixed with the protein to form a cross-linked formulation via reaction between the NHS-activated carboxylic acid of HA and the amino groups of the protein. In this approach, HA essentially becomes the cross-linker for the protein. The properties of the resulting formulation are expected to differ from those in which the protein is cross-linked using a short cross-linker or when HA is cross-linked by a short cross-linker in the absence of the protein.

[0161] Example 2 Schematic for the generation of soluble hyaluronic acid crosslinkers using the heterobifunctional reagent allyl glycidyl ether (AGE). This approach is based on the derivatization of hyaluronan with heterobifunctional reagents, which allows for a two-step approach to crosslink proteins with HA. The method chosen in this example utilizes allyl glycidyl ether (AGE), which allows for the initial incorporation of AGE, forming a stable ether bond when the oxysilane group reacts with the hydroxyl groups of HA under strongly alkaline conditions. The allyl groups thus incorporated can then be converted to halohydrins by reaction with halogen compounds (e.g., bromine). These halohydrins can then react with proteins via the primary amine groups by forming stable secondary amine bonds. The reaction between halohydrins and primary amine groups occurs efficiently at high pH values, but some reaction begins to occur above pH 8.5-9.

[0162] [ka]

[0163] The reaction scheme for cross-linking proteins with HA is shown above: HA = hyaluronic acid, R = O-CH2-CH(OH)-CH2-O-CH2, AGE = allyl glycidyl ether, Pr = protein.

[0164] Example 3 (Preparation of Polysaccharide Crosslinker A) In this example, 5 ml of a 1% low-molecular-weight hyaluronic acid solution, 1 ml of HO, 100 mg of NHS (Sigma), and 100 mg of EDC (Sigma) were mixed and allowed to react for 1 hour at room temperature. The derivatized hyaluronic acid was then precipitated with 2 volumes of IPA, briefly pressed to reduce the water and solvent, washed with 66% ethanol, and redissolved in 4 ml of phosphate-buffered saline (Sigma) at room temperature. The derivatized HA completely dissolved within 1 hour. The concentration of the derivatized HA was measured using a moisture analyzer, and the derivatized HA was then diluted to a 2% solution before use in protein crosslinking. All preparations were sterile, and experiments were performed in a laminar flow hood when possible.

[0165] Example 4 (Preparation of Polysaccharide Crosslinker B) In this example, 5 ml of a 1% carboxymethylcellulose solution, 1 ml of HO, 100 mg of NHS (Sigma), and 100 mg of EDC (Sigma) were mixed and allowed to react for 1 hour at room temperature. The derivatized carboxymethylcellulose was then precipitated with 2 volumes of IPA, briefly pressed to reduce the water and solvent, washed with 66% ethanol, and redissolved in 4 ml of phosphate-buffered saline (Sigma) at room temperature. All preparations were sterile, and experiments were performed in a laminar flow hood when possible.

[0166] Example 5 (Preparation of Polysaccharide Crosslinker C) In this example, 4 ml of 2% HA was mixed with 0.5 ml of 10 M NaOH and 0.5 ml of AGE and allowed to react for 1 hour at room temperature. The solution was then neutralized with 1 ml of 9 M acetic acid, 40 mg of NaCl was added, and precipitated with two volumes of IPA. The precipitate was washed with 60% IPA, padded dry on filter paper, and then redissolved in 3 ml of water. The incorporated allyl groups were then converted with bromine water (250 μL was added, although 200 μL to 250 μL is required for complete conversion). To remove excess bromine, the solution was again precipitated with 30 mg of NaCl followed by two volumes of IPA. The precipitate was washed with 60% IPA, padded dry on filter paper, and then redissolved in 2 ml of water. 0.5 ml of the solution was used to determine the final concentration of derivatized HA in a moisture analyzer. The dry matter content was found to be 2.59%. The derivatized HA was diluted to a 2% solution before use in protein cross-linking.

[0167] Example 6 Preparation of Protein-Based Formulations Using rhTropoelastin 1 In this example, 250 μL of a 200 mg / mL rh-tropoelastin (Elastgen) solution in phosphate-buffered saline was mixed with 250 μL of phosphate-buffered saline, followed by the addition of 500 μL of the hyaluronic acid crosslinker from Example 3. The mixture was thoroughly mixed and briefly centrifuged to remove air bubbles. The material was allowed to stand at room temperature for 30 minutes before dispensing. The formulations were then loaded into sterile 1 mL syringes in a laminar flow hood. All of the formulations produced in this manner exhibited stable properties that allowed extrusion through a fine-gauge 31G needle as compact fibers 10-20 cm long.

[0168] Example 7 Preparation of Protein-Based Formulations Using Bovine Serum Albumin (BSA) In this example, 250 μL of a 200 mg / mL BSA solution in phosphate-buffered saline (Sigma) was mixed with 250 μL of phosphate-buffered saline, and then 500 μL of the hyaluronic acid crosslinker from Example 3 was added. The mixture was thoroughly mixed and briefly centrifuged to remove air bubbles. The material was allowed to stand at room temperature for 30 minutes before dissolution. The resulting formulation exhibited stable properties that allowed it to be extruded through a fine-gauge 31G needle as compact fibers 10-20 cm long.

[0169] Example 8 Preparation of Protein-Based Formulations with rhHSA In this example, a 20 mg / ml solution of recombinant HSA (Sigma) in phosphate-buffered saline (Sigma) was mixed with an equal volume of the hyaluronic acid crosslinker from Example 3. The mixture was mixed thoroughly and then briefly centrifuged to remove air bubbles. The material was allowed to stand at room temperature for 30 minutes before dispensing. This resulted in a mildly crosslinked, clear, colorless HSA formulation that could be extruded through a fine-gauge 31G needle.

[0170] Example 9 Preparation of Protein-Based Formulations Using rhTropoelastin 2 In this example, 500 μL of a 100 mg / mL rhtropoelastin (Elastagen) solution in phosphate-buffered saline was mixed with 500 μL of 2% hyaluronic acid crosslinker from Example 5 at pH 8.5. The mixture was mixed thoroughly and briefly centrifuged to remove air bubbles. The material was allowed to stand at room temperature for 12 hours to dissolve. The resulting formulation was a clear, colorless, stable matrix formulation of HA-crosslinked tropoelastin.

[0171] Example 10 (Evaluation of Monomer Content in Formulations) In this example, an aliquot of the formulation made in Example 6 was soaked in PBS and the resulting supernatant was analyzed by SDS-PAGE (the resulting gel is shown in Figure 3).

[0172] Loading: Formulation made in Example 6, soaked in PBS Lane M: marker. Lanes A and B: supernatant. TE Lane: Pure TE.

[0173] As can be seen from the gel, no monomer was extracted from the HA-TE sample.

[0174] Example 11 (Evaluation of formulation hydrodynamics) In this example, the shear flow rheological behavior of the formulation prepared in Example 6 was investigated using a Haake RS150 rheometer utilizing a cone and plane geometry. A 35 mm / 1° titanium cone was used for the test, with the temperature maintained at 25°C.

[0175] The response of formulations to small amplitude oscillatory shear flow with varying angular frequency was determined by the storage modulus (G') over the angular frequency range of 0.1 to 100 rad / s. Over this range, the storage modulus was relatively insensitive to changes in angular frequency. In steady shear flow, strain increased linearly for stresses below 200 Pa. Above this value, small changes in applied stress led to significant increases in strain. When shear viscosity was plotted against shear rate, the fluid exhibited a constant viscosity of approximately 260 Pa at low shear rates. However, viscosity decreased rapidly with increasing shear rate above a shear rate of approximately 0.4 rad / s.

[0176] The rheological properties of the protein-based formulations made in Example 6 are significantly different from those of uncrosslinked polysaccharides that are subsequently micronized to allow extrusion through fine needles, and from polysaccharide products made with short crosslinkers (e.g., BDDE). These polysaccharide products behave more like viscous fluids, with a larger contribution to their rheological properties from the loss modulus (G'') across the same frequencies tested. The relatively constant value of the complex modulus of the formulations made in Example 6 makes them particularly different from polysaccharide-based products.

[0177] Further embodiments or examples are given below.

[0178] Embodiment 1. A method for producing a polymeric polymeric polymer comprising: (i) at least one protein residue, and (ii) a composition comprising at least one sugar-bearing residue.

[0179] 2. The composition of embodiment 1, which is an injectable composition.

[0180] 3. The composition of embodiment 1 or 2, delivered by a cannula, a catheter, a flexible polymer catheter, a syringe with a needle or a syringe without a needle.

[0181] 4. The composition of one or more of embodiments 1-3, which is extrudable to a length of at least 10 cm.

[0182] 5. The composition of one or more of embodiments 1-4, wherein the composition is extrudable through an 18G to 31G needle to a length of about 5 cm to about 30 cm, and wherein the extruded composition remains substantially coherent without surface support.

[0183] 6. The composition of one or more of embodiments 1-5, which is extrudable through a 25G needle to a length of at least 5 cm, 10 cm, 12 cm, 15 cm, 18 cm, 20 cm, or 25 cm.

[0184] 7. The composition of one or more of embodiments 1-6, which is extrudable through a fine gauge needle to a length of at least 10 cm, 15 cm, 20 cm, or 25 cm.

[0185] 8. The composition of one or more of embodiments 1-7, wherein the composition is extrudable without further surface support to a length of at least 5 cm to 30 cm, 10 cm to 20 cm, or 15 cm to 30 cm when extruded through a fine gauge needle, and the extruded composition is substantially coherent and substantially unitary.

[0186] 9. The composition of one or more of embodiments 1-8, wherein the composition is extrudable without additional physical support to a length of at least 10 cm, 15 cm, 20 cm, or 25 cm when extruded through a medium gauge, and the extruded composition is substantially coherent and substantially unitary.

[0187] 10. The composition of one or more of embodiments 1-9, wherein the composition is extrudable without further surface support to a length of at least 10 cm, 15 cm, 20 cm, or 25 cm when extruded through a large gauge needle, and the extruded composition is substantially compact and substantially coherent.

[0188] 11. The composition of one or more of embodiments 1-10, which is extrudable without further surface support at an angle of at least 45° from the vertical to a length of at least 10 cm, 20 cm, or 30 cm, forming tightly packed fibers of material.

[0189] 12. The composition of one or more of embodiments 1-11, wherein the extruded composition forms tightly packed fibers of material.

[0190] 13. The composition of one or more of embodiments 1-12, which can be extruded without substantial further processing.

[0191] 14. The composition of one or more of embodiments 1-13, which can be extruded through a fine gauge needle without substantial further processing, and when extruded, is substantially coherent and substantially holds together without further physical support.

[0192] 15. The composition of one or more of embodiments 1-14, wherein the strand of composition remains sufficiently tight after extrusion through a needle so that it does not break during extrusion.

[0193] 16. The composition of one or more of embodiments 1-15, wherein a string of the composition greater than 10 cm, 12 cm, 15 cm, 18 cm, or 20 cm retains sufficient compactness after extrusion through a needle such that the string of the composition can be extruded through the needle without breaking the string of the composition.

[0194] 17. The composition according to one or more of the preceding embodiments, wherein at least one of the cross-linked protein matrices comprises full-length protein residues.

[0195] 18. The composition according to one or more of the preceding embodiments, wherein at least one of the cross-linked protein matrices comprises substantially full-length protein residues.

[0196] 19. The composition of one or more of embodiments 1-18, which is substantially flexible so that it can be removed through a needle.

[0197] 20. The composition of one or more of embodiments 1-19, which has sufficient flexibility to allow removal through a fine gauge needle.

[0198] 21. The composition of one or more of embodiments 1 to 20, wherein at least one of the protein residues is allowed to remain full-length or substantially full-length, and at least one of the protein residues is protected from rapid absorption or degradation.

[0199] 22. The composition of one or more of embodiments 1 to 21, wherein at least one of the protein residues is allowed to remain full-length or substantially full-length, is injectable through an injection needle, retains a tight structure, and is sufficiently cross-linked to delay absorption of the composition in vivo.

[0200] 23. The composition according to one or more of embodiments 1 to 22, wherein at least one of said protein residues is substantially full-length and substantially devoid of intramolecular crosslinks.

[0201] 24. The composition of one or more of embodiments 1-23, which is tissue compatible, promotes tissue ingrowth, promotes tissue regeneration, or a combination thereof.

[0202] 25. A composition according to one or more of embodiments 1 to 24, which can be remodeled into a standard desired structure and incorporated into new tissue.

[0203] 26. The composition according to one or more of the preceding embodiments, wherein at least one of the sugar-bearing moieties remains soluble or substantially soluble in water or saline.

[0204] 27. The composition according to one or more of the preceding embodiments, wherein at least one of the sugar-bearing moieties is substantially soluble in an aqueous medium or a physiological medium.

[0205] 28. The composition according to one or more of embodiments 1 to 27, wherein at least one of the cross-linked protein residues is substantially intermolecularly cross-linked.

[0206] 29. The composition of one or more of embodiments 1 to 28, wherein at least one of the sugar-containing moieties has one or more of the following properties: substantial bioavailability, substantial biodegradability, substantial bioresorbability, or substantial bioresorbability.

[0207] 30. The composition according to one or more of the preceding embodiments, wherein the at least one sugar-bearing moiety comprises at least one sugar residue, at least one oligosaccharide residue, or a combination thereof.

[0208] 31. The composition of any one of embodiments 1 to 30, wherein at least one of the polysaccharide residues comprises a low molecular weight polysaccharide residue, a medium molecular weight polysaccharide residue, a high molecular weight polysaccharide residue, or a combination thereof.

[0209] 32. The composition of one or more of embodiments 1 to 31, wherein at least one of the polysaccharide residues has a molecular weight of about 50,000 to about 275,000 daltons.

[0210] 33. The composition according to one or more of the preceding embodiments, wherein at least one of the polysaccharide residues is derived from or comprises a residue of hyaluronic acid, a cellulose derivative, carboxycellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose carboxymethylamylose, xanthan gum, guar gum, α-glucan, β-glucan, β-1,4-glucan, β-1,3-glucan, alginate, carboxymethyldextran, a glycosaminoglycan derivative, chondroitin-6-sulfate, dermatin sulfate, heparin, heparin sulfate, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), tricalcium phosphate or 1-hydroxyapatite, a pharmaceutically acceptable salt thereof, a derivative thereof or a combination thereof.

[0211] 34. The composition according to one or more of the preceding embodiments, wherein at least one of the polysaccharide residues is derived from or comprises a residue of hyaluronic acid.

[0212] 35. The composition of one or more of embodiments 1-34, wherein at least one of the polysaccharide residues is derived from or comprises a residue of carboxymethylcellulose.

[0213] 36. The composition of any one of embodiments 1 to 35, wherein at least one of the cross-linked protein matrices comprises at least one sugar-bearing residue at a concentration of about 0.01% to about 30%.

[0214] 37. The composition of one or more of embodiments 1-36, wherein at least one of the protein residues comprises an amine-bearing side chain residue, and the side chain residue comprises at least one lysine residue, at least one arginine residue, or a combination thereof.

[0215] 38. The composition of one or more of embodiments 1-37, wherein at least one of the protein residues is derived from or comprises a residue of tropoelastin, elastin, albumin, collagen, collagen monomer, immunoglobulin, insulin, derivatives or combinations thereof.

[0216] 39. The composition according to one or more of the preceding embodiments, wherein the amount of at least one said protein residue is from about 1 mg / ml to about 200 mg / ml.

[0217] 40. The composition according to one or more of the preceding embodiments, wherein at least one of the cross-linked protein matrices comprises a biologically active protein residue.

[0218] 41. The composition of one or more of embodiments 1 to 40, wherein at least one of the cross-linked protein matrices is derived from or comprises residues of a synthetic protein substantially identical to a naturally occurring human protein.

[0219] 42. The composition according to one or more of the preceding embodiments, wherein at least one of the cross-linked protein matrices is derived from or comprises residues of a stabilized protein.

[0220] 43. The composition according to one or more of the preceding embodiments, wherein at least one of the crosslinked protein matrices is derived from or comprises an extracellular protein.

[0221] 44. The composition of one or more of embodiments 1 to 43, wherein the extracellular protein is tropoelastin, elastin, collagen or a derivative thereof.

[0222] 45. The composition of one or more of embodiments 1 to 44, wherein at least one of the cross-linked protein matrices comprises at least one of the sugar-bearing residues at a concentration of about 0.1% to about 5%.

[0223] 46. ​​The composition of one or more of embodiments 1 to 45, wherein at least one of the cross-linked protein matrices comprises a ratio of at least one of the protein residues to at least one of the residues having a sugar of from about 0.1% to about 6%.

[0224] 47. The composition of one or more of embodiments 1 to 46, wherein at least one of the crosslinked protein matrices comprises from about 0.1% to about 1.5% of at least one of the protein residues to from about 0.1% to about 6% of at least one of the sugar-bearing residues, and the sugar-bearing residues comprise polysaccharide residues.

[0225] 48. The composition of one or more of embodiments 1 to 47, wherein at least one of the cross-linked protein matrices is prepared from at least one cross-linking molecule bearing a sugar, the cross-linking molecule comprising at least 5%, 10%, 20%, or 25% activated carboxyl and / or activated hydroxyl groups, modified carboxyl and / or modified hydroxyl groups, or a combination thereof.

[0226] 49. The composition of one or more of embodiments 1 to 48, wherein at least one of the cross-linked protein matrices is prepared from at least one cross-linking molecule bearing a sugar, the cross-linking molecule comprising at least 5% activated carboxyl and / or activated hydroxyl groups, modified carboxyl and / or modified hydroxyl groups, or a combination thereof.

[0227] 50. The composition of one or more of embodiments 1-49, wherein at least one of the crosslinked protein matrices comprises less than 5% monomeric protein residues.

[0228] 51. The composition according to one or more of the preceding embodiments, wherein at least one of the crosslinked protein matrices comprises less than 1% monomeric protein residues.

[0229] 52. The composition of one or more of embodiments 1-51, which is employed therapeutically in at least one of surgery, aesthetics, tissue augmentation, treatment of incontinence, skin replacement products, dermatology, dermatological surgery, cosmetics, or a combination thereof.

[0230] 53. A composition according to one or more of embodiments 1 to 52, for therapeutic use in dermatology.

[0231] 54. A composition according to one or more of embodiments 1 to 53, which is employed therapeutically in dermatological surgery.

[0232] 55. A composition according to one or more of embodiments 1 to 54, which is employed therapeutically for topical application in cosmetology, dermatology or a combination thereof.

[0233] 56. A composition according to one or more of embodiments 1 to 55, which is applied therapeutically in dermatology for topical use.

[0234] 57. The composition of one or more of embodiments 1 to 56, for use in the treatment of facial wrinkles, filling of facial wrinkles, treatment of fine lines, treatment of aging skin, treatment of scar tissue or treatment of skin depressions, or a combination thereof.

[0235] 58. A composition according to one or more of embodiments 1 to 57, which is used for implanting local deposits of protein residues that are substantially biologically active.

[0236] 59. A composition according to one or more of embodiments 1 to 58, which is used for implanting localized deposits of protein residues that are substantially biologically active.

[0237] 60. A composition according to one or more of embodiments 1 to 59, which is used for implanting a delayed release deposit of a protein residue having substantial biological activity.

[0238] 61. The composition of one or more of embodiments 1 to 60, wherein at least one of the cross-linked protein matrices comprises at least 90%, 95%, 98% or 99% of at least one of the protein residues cross-linked with a biomolecule and / or biopolymer, and the biomolecule and / or biopolymer comprises at least one of the residues bearing a sugar.

[0239] 62. The composition of one or more of embodiments 1 to 61, wherein at least one of the cross-linked protein matrices comprises at least one of the protein residues that is substantially cross-linked using a cross-linked biomolecule or a cross-linked biopolymer or a combination thereof.

[0240] 63. The composition of one or more of embodiments 1 to 62, wherein the number of crosslinks for at least one of the polysaccharide residues may be at least 5%, 10%, 15%, 20%, or 25% of the number of possible crosslinking sites for at least one of the polysaccharide residues.

[0241] 64. The composition according to one or more of the preceding embodiments, wherein the number of protein units remaining unbound and not incorporated into at least one of the cross-linked protein matrices may be at least 1%, 3%, 5%, or 7%.

[0242] 65. The composition of one or more of embodiments 1-64, wherein less than 20%, 15%, 10%, or 7% of the protein units are not incorporated into and remain unbound in at least one of the crosslinked protein matrices.

[0243] 66. The composition according to one or more of the preceding embodiments, wherein the protein monomers can be crosslinked such that about 90% to about 100% of the protein monomers can be incorporated into the composition.

[0244] 67. The composition according to one or more of the preceding embodiments, wherein at least one of the protein residues is derived from a full-length protein or a substantially full-length protein, and the structure of the protein residue is not substantially covered by at least one of the residues bearing a sugar.

[0245] 68. The composition according to one or more of the preceding embodiments, comprising at least one protein residue having a structure that is substantially not covered by at least one of the residues having a sugar, and which may be more biocompatible, may promote tissue ingrowth, or may promote tissue regeneration, or a combination thereof.

[0246] 69. A composition according to one or more of embodiments 1 to 68, comprising at least one of the protein residues having a structure that is substantially not covered by at least one of the residues having sugars, and which can be remodeled into a more standard desired structure and / or incorporated into new tissue.

[0247] 70. A method for preparing a composition according to one or more of embodiments 1 to 69, comprising cross-linking at least one protein with at least one soluble molecule comprising a sugar.

[0248] Embodiment 71. A method for preparing a composition comprising at least one crosslinked protein matrix, comprising: At least one of the cross-linked protein matrices comprises: (i) at least one protein residue, and (ii) contains at least one residue bearing a sugar; The crosslinking is (i) modifying at least one of the sugar-bearing molecules to include at least one reactive chemical group; (ii) combining at least one of the sugar-containing modified molecules with at least one of the proteins that includes a reactive chemical group complementary to the chemical group on the sugar-containing modified molecule; and (iii) forming at least one bond between at least one of the sugar-containing modified molecules and at least one of the proteins.

[0249] 72. The method according to embodiment 70 or 71, wherein at least one reactive chemical group is a chemical group capable of forming a covalent bond when combined with at least one of the proteins.

[0250] 73. The method according to one or more of embodiments 70-72, wherein at least one of the bonds is a covalent bond.

[0251] 74. The method according to one or more of embodiments 70 to 73, wherein at least one of the modified sugar-bearing molecules is soluble or substantially soluble in water and / or saline.

[0252] 75. The method according to one or more of embodiments 70 to 74, wherein at least one of the modified sugar-bearing molecules remains soluble or substantially soluble in water or saline.

[0253] 76. The method according to one or more of embodiments 70 to 75, wherein at least one of the sugar-bearing molecules contains a carboxyl group and / or a hydroxyl group.

[0254] 77. The method according to one or more of embodiments 70 to 76, wherein at least one of the sugar-bearing molecules is modified by activating a carboxyl group and / or a hydroxyl group.

[0255] 78. The method of one or more of embodiments 70 to 77, further comprising purifying the at least one sugar-bearing modified molecule by precipitation and / or filtration of the at least one sugar-bearing modified molecule to remove or substantially remove unreacted modification reactants.

[0256] 79. The method according to one or more of embodiments 70 to 78, wherein the at least one sugar-containing modified molecule is used as a cross-linking agent when the at least one sugar-containing modified molecule is combined with the at least one protein.

[0257] 80. The method according to one or more of embodiments 70 to 79, wherein a solution of at least one of the molecules modified with sugars is mixed with at least one of the proteins to form at least one of the cross-linked protein matrices.

[0258] 81. A method of use comprising injecting a composition according to one or more of embodiments 1-69.

[0259] 82. Human or veterinary medicine; surgical procedures; restorative surgery; cosmetic surgery; aesthetics; tissue augmentation; dermatological surgery; eye surgery; rheumatology; pharmacology; in the field of cosmetic surgery, hemostasis in general surgical procedures; nerve and vascular reconstruction in reconstructive surgery, neurosurgery; plastic surgery; fixation of skin, vascular or cartilage transplants or grafts in orthopedic surgery; treatment of osteoarthritis of the knee; vascular surgery; vehicles for delivery of cells or bioactive molecules (e.g., growth factors to stimulate local repair); local delivery of growth factors in combination with the above crosslinked protein matrix compositions to promote wound healing and tissue regeneration or enhance bone formation; stimulation of cartilage tissue repair in orthopedic procedures; treatment of pathological trauma conditions (e.g., chronic ulcers); acting as a scaffold for generating artificial tissue through the proliferation of autologous cells in culture; tissue augmentation in plastic surgery (e.g., filling skin folds or lip reconstruction); completion of body cavities or defects; aesthetic medicine 82. The method of embodiment 81, wherein the injection is used to augment, augment, or a combination thereof, tissue in at least one of: (a) augmenting or enhancing tissue; (b) restoring volume lost during surgery (e.g., eye surgery); or (c) restoring volume lost during surgery (e.g., eye surgery).

[0260] 83. A method of use comprising topically applying a composition according to one or more of embodiments 1-69.

[0261] 84. The method of embodiment 83, wherein the topical application is performed on healthy or damaged tissue in at least one of the following: cosmetic procedures; dermatology; filling of facial wrinkles; fine lines; treatment of aging skin; scar tissue; or skin depressions.

[0262] 85. A kit for administering a composition according to one or more of embodiments 1 to 69.

[0263] While the present disclosure has been described in connection with particular embodiments, it is understood that the disclosure is not limited to the disclosed embodiments and is intended to cover various modifications and equivalent arrangements. Also, various embodiments described herein may be implemented in conjunction with other embodiments, e.g., aspects of one embodiment may be combined with aspects of other embodiments to achieve yet further embodiments. Furthermore, each independent feature or component of an embodiment may constitute a further embodiment.

Claims

[Claim 1] The composition described herein.

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