Modified gelatin, hydrogels and processes for producing them

Abstract

Provided herein are modified gelatins, cross-linked modified gelatins and hydrogels, and processes for making the same. Also provided herein is the use of such materials as matrices for cell growth.

Description

[Technical Field]

[0001] This patent / patent application claims priority from Australian Provisional Patent Application No. 2022903674, filed December 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] This disclosure relates to modified gelatin, cross-linked modified gelatin and hydrogels, and processes for making the same. This disclosure also relates to the use of such materials as matrices for cell growth. This disclosure also relates to the use of such materials in bioprinting. [Background technology]

[0003] Hydrogels are three-dimensional networks of hydrophilic polymers that have broad applications across many fields of endeavor. Because many hydrogels are biocompatible, one particular area of ​​use is biomedicine, including in applications such as medical bandages, tissue engineering scaffolds, superabsorbent materials, and drug delivery systems.

[0004] Gelatin is a partially hydrolyzed collagen derivative that can form hydrogels and has a wide range of potential applications in biomedicine, including proposed uses as a matrix for cell growth and in 3D bioprinting. Gelatin can be obtained from many different sources, including bovine and porcine sources, and many derivatives are known.

[0005] However, commonly used mammalian gelatin and its derivatives exhibit unfavorable rheological properties in solution, including thermal, non-covalent, and reversible crosslinking below their melting point of approximately 30°C, and high viscosity above 30°C (see, for example, Non-Patent Document 1). Furthermore, the mechanical and biochemical properties of some cross-linked gelatin derivatives can be significantly affected by the temperature at which the derivatives are cross-linked (Non-Patent Document 2). From a practical perspective, these properties create the need to heat and maintain gelatin and derivative solutions at a constant temperature above 30°C to enable liquid handling using manual and automated pipetting methods and to promote predictable, reproducible, and controllable physicochemical properties of cross-linked formulations. Furthermore, the high viscosity of solutions above 30°C adversely affects pipetting accuracy and reproducibility, as well as the ability to be 3D printed using various printing methods.

[0006] As a result, the handling and cross-linking of some gelatin and gelatin derivative solutions are difficult to control in a precise, predictable, and reproducible manner, and these difficulties increase with the concentration of the gelatin or gelatin derivative. Furthermore, these properties limit the ability of such gelatin and gelatin derivative solutions to be accurately handled using automated liquid handling techniques used in pharmaceutical drug discovery and development and other laboratory processes.

[0007] Approaches to modifying gelatin include chemical derivatization with photocrosslinkable moieties that allow radical polymerization in the presence of a photoinitiator and an appropriate light source. However, photoinitiated crosslinking requires specialized equipment and additional reagents to promote crosslinking, which increases complexity and cost. Furthermore, such photocrosslinking processes and equipment are difficult to integrate into applications, including, but not limited to, automated high-throughput drug discovery and inkjet, multijet, or polyjet 3D printing.

[0008] It would be beneficial to provide additional hydrogels that are useful as matrices for cell growth and / or 3D bioprinting applications, that can be conveniently used in a wide range of operating conditions, and that can be easily produced from starting materials without the need for specialized equipment. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Van Den Bulcke et al. [Non-patent document 2] Loessner et al. Summary of the Invention

[0010] The present inventors have identified modified gelatin polymers derived from natural sources of cold-water-adapted marine species, such as, but not limited to, salmon skin gelatin, and incorporating chemical functional groups such as thiol, amino, maleimide, or vinyl sulfone, as novel covalently crosslinkable biomaterials capable of producing hydrogels with advantageous properties for the biomedical and food industries. Solutions of the resulting gelatin derivatives exhibit low viscosity, low melting temperatures, and can be easily crosslinked via click chemistry reactions with crosslinker molecules containing appropriate reactive moieties. Exemplary materials according to the present disclosure have been found to possess low and substantially temperature-independent viscosity characteristics over a wide temperature range, as well as low melting points and the ability to adjust crosslinking times to relatively short or long lengths as required for the intended application, depending on the reactive moieties in the crosslinker molecules. This allows solutions of the material to be easily handled using manual and automated pipetting methods, as well as various 3D printing methods. For manual pipetting methods, a longer crosslinking time may be beneficial, for example, allowing for the preparation of larger master volumes that do not require immediate use. For 3D printing applications, shorter curing times may be beneficial in some embodiments, allowing desired three-dimensional structures to be printed quickly with high geometric fidelity.

[0011] Thus, in one aspect, there is provided a modified gelatin, wherein the gelatin is derived from a marine source and has been modified to incorporate crosslinkable groups that comprise reactive moieties, wherein the reactive moieties are reactive in click chemistry reactions.

[0012] In some embodiments, the number of proline residues in the gelatin is 20% or less of the total number of amino acid residues in the modified gelatin.

[0013] In some embodiments, the number of hydroxyproline residues in the gelatin is 20% or less of the total number of amino acid residues in the modified gelatin.

[0014] In some embodiments, the modified gelatin comprises: a) the content of reactive moieties in the modified gelatin is about 25 μmol / g to about 1000 μmol / g; b) the content of reactive moieties in the modified gelatin is about 300 μmol / g to about 700 μmol / g; c) an aqueous solution of 20% (wt / v) or less of the modified gelatin has a viscosity of less than 100,000 mPa·s over a shear rate range of 10 to 1,000 (1 / s); and d) Aqueous solutions of 20% (wt / v) or less modified gelatin are heated to 100°C over the temperature range of 0°C to 40°C. 8 have a complex viscosity of less than mPa·s, It can be characterized by one or more of the following:

[0015] In some embodiments, the gelatin is cold-water adapted fish gelatin, hi some embodiments, the cold-water adapted fish is from a genus selected from the group consisting of Salmo, Gadus, Oncorhynchus, and Merlucius, preferably Salmo or Oncorhynchus.

[0016] In some embodiments, the reactive moiety is selected from the group consisting of alkynes, amines, alkenes, conjugated dienes, thiols, isonitriles, and tetrazines. In some embodiments, the reactive moiety is selected from the group consisting of amines, thiols, alkenes, conjugated dienes, azides, and alkynes. In some embodiments, the reactive moiety is a thiol.

[0017] In some embodiments, the gelatin is i) reaction of gelatin amine groups with Traut's reagent; ii) reaction of gelatin carboxylic acid groups with diamines; iii) reaction of gelatin carboxylic acid groups with diamines and subsequent reaction with Traut's reagent; iv) amide coupling of gelatin carboxylic acids or carboxylates with additional cysteines; is modified by a reaction selected from the group consisting of:

[0018] In another aspect, there is provided a process for producing modified gelatin as defined herein, the process comprising reacting gelatin derived from a marine source with a crosslinkable group precursor.

[0019] In some embodiments, the gelatin derived from marine sources is i) reaction of gelatin amine groups with a crosslinkable group precursor, Traut's reagent; ii) reaction of gelatin carboxylic acid groups with a crosslinkable group precursor, which is a diamine; iii) reaction of gelatin carboxylic acid groups with diamines and subsequent reaction with Traut's reagent; iv) amide coupling of gelatin carboxylic acids or carboxylates with a crosslinkable group precursor which is cysteine; The crosslinkable group precursor is reacted with the compound by the following procedure.

[0020] In another aspect there is provided a modified gelatin produced or preparable by the process defined herein.

[0021] In another aspect there is provided a crosslinked modified gelatin produced by reacting a modified gelatin as defined herein with a crosslinker comprising two or more further reactive moieties in a click chemistry reaction, wherein the further reactive moieties are reactive with the reactive moieties of the crosslinkable groups incorporated into the modified gelatin in the click chemistry reaction.

[0022] In some embodiments, the further reactive moiety of the crosslinker is selected from the group consisting of alkenes, alkynes, and thiols. Examples of further reactive moieties of alkenes include maleimides, vinyl sulfones, acrylates, acrylamides, and methacrylates. In some embodiments, the further reactive moiety of the crosslinker is selected from the group consisting of maleimides, vinyl sulfones, acrylates, acrylamides, and methacrylates.

[0023] In some embodiments, the crosslinker has the formula: Core-(Spacer-R) n wherein Core is an atom or group that provides bonds to n Spacer-R groups, Spacer is a spacer group, R is a group that includes an additional reactive moiety, and n is an integer from 2 to 8. In some embodiments, Core is C(—CHO—), n is 4, and Spacer is (—CHCHO—). m where m is an integer from 2 to 150, and R is [ka] In some embodiments, the Core is C(—CH2O—), n is 4, and the Spacer is (—CH2CH2O—). m where m is an integer from 2 to 50, and R is [ka] In some embodiments, the Core is C(—CH2O—), n is 4, and the Spacer is (—CH2CH2O—). m where m is an integer from 2 to 150, and R is [ka] is.

[0024] In another aspect, there is provided a process for producing a crosslinked modified gelatin as defined herein, comprising reacting a modified gelatin as defined herein with a crosslinker comprising two or more further reactive moieties in a click chemistry reaction, wherein the reactive moieties are reactive with the reactive moieties of the crosslinkable groups incorporated into the modified gelatin in the click chemistry reaction.

[0025] In some embodiments, the process is carried out under ambient light conditions.

[0026] In another aspect there is provided a cross-linked modified gelatin produced or preparable by the process defined herein.

[0027] In another aspect, there is provided a hydrogel comprising cross-linked modified gelatin as defined herein and water.

[0028] In some embodiments, the hydrogel comprises: a) 2.5% (wt / v) or more of the hydrogel has a relaxed mass swelling ratio of 15 or less; b) 2.5% to 10% (wt / v) hydrogel has an equilibrium swelling ratio of 10 to 25; and c) 2.5%-10% (wt / v) hydrogels have an equilibrium water content of 80%-100%; It can be characterized by one or more of the following:

[0029] In some embodiments, the hydrogel comprises: a) 2.5% (wt / v) or more of the hydrogel has a relaxed mass swelling ratio of 15 or less; b) 2.5% to 10% (wt / v) hydrogels have an equilibrium swelling ratio of 10 to 25; c) 2.5%-10% (wt / v) hydrogels have an equilibrium water content of 80%-100%; d) During preparation, the 2.5% to 10% (wt / v) hydrogel has a crosslinking time of 1 to 10 seconds at room temperature; and e) During preparation, the 2.5% to 10% (wt / v) hydrogel has a crosslinking time of 10 to 30 minutes at room temperature; It can be characterized by one or more of the following:

[0030] In another aspect, there is provided a method of making a hydrogel comprising combining a cross-linked modified gelatin as defined herein with water.

[0031] In another aspect, there is provided a method of making a hydrogel, the method comprising carrying out the method of making cross-linked modified gelatin as defined herein in the presence of water.

[0032] In another aspect, there is provided a hydrogel produced or preparable by the process defined herein.

[0033] In another aspect, there is provided the use of a hydrogel as defined herein as a matrix for cell growth or for 3D bioprinting.

[0034] In another aspect, there is provided a method of growing cells, the method comprising providing a cell growth matrix comprising a hydrogel as defined herein and growing cells in and / or on the cell growth matrix.

[0035] In another embodiment, there is provided a kit for producing cross-linked modified gelatin, comprising: a) a modified gelatin as defined herein; and b) a cross-linking agent as defined herein; and A kit is provided comprising: [Brief explanation of the drawings]

[0036] [Figure 1]

[0023] Figure 1 shows exemplary synthetic routes for gelatin functionalization with click-reactive thiol groups: A - thiolation of native cold-water-adapted fish gelatin with Traut's reagent to produce Traut-thiolated cold-water-adapted gelatin; B - amination of native cold-water-adapted fish gelatin with ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce aminated cold-water-adapted fish gelatin; C - amination of native cold-water-adapted fish gelatin with ethylenediamine and EDC to produce aminated cold-water-adapted gelatin, followed by thiolation of the aminated gelatin with Traut's reagent to produce two-step Traut-thiolated cold-water-adapted fish gelatin. [Figure 2]1 is a graph showing the chemical and spectroscopic properties of native and click-reactive thiolated cold-water fish skin gelatins synthesized using various routes. A - Amine content of native cold-water adapted fish gelatin ("Native FG"), aminated cold-water adapted fish gelatin ("Aminated"), Traut's thiolated cold-water adapted gelatin ("Traut's"), two-step Traut's thiolated cold-water adapted fish gelatin ("Aminated Traut's"), and L-cysteine ​​coupled cold-water adapted fish gelatin (formed using EDC / NHS chemistry) ("EDC / NHS L-cys"). B - Thiol content of Native FG, Traut's, Aminated Traut's, and EDC / NHS L-cys cold-water adapted fish gelatin. C - 1H-NMR spectra of Native FG, Traut's, Aminated Traut's, and EDC / NHS L-cys cold-water adapted fish gelatin. [Figure 3] Graphs showing the rheological properties of solutions of mammalian (pig skin type A) gelatin ("Gelatin") and thiolated cold-water-adapted fish skin gelatin ("Gel-SH") at various concentrations: A - viscosity versus shear rate; B - complex viscosity versus temperature for the gelatin from Figure 3A (mean ± SEM; n=3). [Figure 4] Graphs showing the mechanical properties of hydrogels formed from thiolated cold-water-adapted fish skin gelatin and PEG-4MAL. A - Stress-strain curves using three different concentrations of thiolated cold-water-adapted fish skin gelatin ("Gel-SH" in the figure), n=1; B - Young's modulus determined using the height, area, and slope of the stress-strain curve of the hydrogel at compressive strains of 10% to 15% (n=8; mean ± SEM; ***=P≤0.0001, ****=P≤0.0001). [Figure 5]FIG. 1 shows a click hydrogel reaction scheme, a graph depicting the precursor pH and crosslinking time using various concentrations of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, and the hydrogel. A - Schematic of hydrogel crosslinking to produce Gel-SH / PEG-4MAL hydrogels from thiolated cold-water-compatible fish skin gelatin (Gel-SH) and PEG-4MAL crosslinked by Michael-type addition with a 1:1 molar ratio of thiol (SH) to maleimide (MAL); B - pH of hydrogel precursors and Gel-SH / PEG-4MAL hydrogels prepared using 100 mM, 200 mM, and 300 mM HEPES buffer (mean ± SEM; n = 3); C - Crosslinking time of Gel-SH / PEG-4MAL hydrogels with Gel-SH concentrations reported as the final concentration in the hydrogel (mean ± SEM; n = 8; significance test = one-way ANOVA; *** = P ≤ 0.0005); D - Gel-SH / PEG-4MAL hydrogels imaged with a Nikon SMZ25 stereomicroscope. Scale = 1 cm. [Figure 6] 1 shows the swelling properties of Gel-SH / PEG-4MAL hydrogels. A - Relaxed mass swelling ratio; B - Equilibrium swelling ratio; C - Equilibrium water content (n≦4; mean±SEM; significance test=One-way ANOVA; **=P≦0.001, ***=P≦0.0001). [Figure 7] Figure 1 shows images and graphs depicting the viability of MCF-7 breast cancer cells in Gel-SH / PEG-4MAL click hydrogels compared to photocrosslinkable gelatin methacryloyl hydrogels ("GelMA" in the figure). A - Maximum intensity projection images of live (FDA) and dead (PI) MCF-7 breast cancer cells encapsulated in Gel-SH / PEG-4MAL hydrogels at an encapsulation density of 1 x 10 cells / mL. Scale = 500 µm. B - Viability of MCF-7 cells in Gel-SH / PEG-4MAL hydrogels (Gel-SH concentration reported as the final concentration of thiolated cold-water-adapted fish skin gelatin in Gel-SH / PEG-4MAL hydrogels (% (wt / v)). N = 3. Significance test = one-way ANOVA. * = P ≤ 0.05. Error bars = SEM). [Figure 8]Figure 1 shows images depicting spheroid formation of MCF-7 breast cancer cells encapsulated in Gel-SH / PEG-4MAL hydrogels. Nuclei (DAPI) and F-actin (phalloidin) of encapsulated MCF-7 cells were visualized by confocal microscopy and maximum intensity projection images. (Gel-SH concentration reported as final Gel-SH % (wt / v) in click hydrogels. Imaged with a Leica SP5 confocal microscope. N=1. Objective = 4x. Scale bar = 250 μm.) [Figure 9] Figure 1 shows that crosslinking time and gel formation can be controlled by the choice of click-reactive PEG moiety, where Gel-SH hydrogels crosslinked with 4-arm PEG-vinyl sulfone (PEG4-4VS) crosslinked more slowly than gels crosslinked with PEG-4MAL, resulting in extended liquid handling times. [Figure 10] Figure 1 shows the assessment of viability and metabolic activity of MCF-7 breast cancer cells in Gel-SH hydrogels crosslinked with PEG-4MAL or PEG-4VS. A - Bright-field microscopy images showing the growth and morphology of MCF-7 cells encapsulated in 10% (w / v) Gel-SH hydrogels with PEG-4MAL or PEG-4VS at days 1 and 7 of culture. B - Quantitative assessment of metabolic activity of MCF-7 cells encapsulated in 10% (w / v) Gel-SH hydrogels using PrestoBlue™ Cell Viability Reagent (ThermoFisher Scientific) at days 1 and 7 of culture. C - Metabolic activity of MCF-7 cells encapsulated in hydrogels containing either 5% or 10% (w / v) Gel-SH and equimolar concentrations of PEG-4VS assessed at days 1 and 7 of culture. DETAILED DESCRIPTION OF THE INVENTION

[0037] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described. For purposes of this disclosure, a number of terms are defined herein.

[0038] This disclosure refers to the entire contents of certain documents, which are incorporated herein by reference.

[0039] Where any prior art publication is referred to herein, it should be understood that such reference is not an admission that the publication forms part of the common general knowledge in the art.

[0040] As used herein, the term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be considered to explicitly endorse both meanings or either meaning.

[0041] As used herein, the term "about," when referring to a numerical value or range, allows for a certain degree of variation of the value or range, for example, within 10% of the stated range limits.

[0042] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps, or group of compositions shall be deemed to include one and a plurality (i.e., one or more) of that step, composition, group of steps, or group of compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects unless the context clearly indicates otherwise. For example, reference to "a" includes the single and more than one, reference to "an" includes the single and more than one, reference to "the" includes the single and more than one, etc.

[0043] Unless otherwise indicated, the terms "first," "second," etc. are used herein merely as labels and are not intended to impose any order, position, or hierarchy on the items to which they refer. Furthermore, a reference to a "second" item does not require or exclude the presence of lower-numbered items (e.g., the "first" item) and / or higher-numbered items (e.g., the "third" item).

[0044] As used herein, the phrase "at least one of," when used in conjunction with a list of items, means that different combinations of one or more of the listed items may be used, or that only one of the items in the list may be required. The items may be specific objects, things, or categories. In other words, "at least one of" means that any combination or number of items from the list may be used, but not all of the items in the list are required. For example, "at least one of item A, item B, and item C" may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, "at least one of item A, item B, and item C" may mean, for example, but not limited to, two of item A, one of item B, and ten of item C; four of item B, and seven of item C; or some other suitable combination.

[0045] As used herein, the word "comprise" and other forms such as "comprising" and "comprises" mean including but not limited to, and are not intended to exclude, for example, other additives, components, integers, or steps.

[0046] As used herein, a "protein" is a polymer of amino acid residues and includes peptides, including oligopeptides.

[0047] As used herein, "gelatin" is a protein obtainable by at least partial hydrolysis of collagen.

[0048] "Collagen" as used herein is the major structural protein of animal connective tissue and is most commonly found in animal cartilage, bone, tendons, ligaments and skin.

[0049] As used herein, "marine resources" refers to marine species of animals.

[0050] As used herein, a "cold-water adapted marine species" is a marine species of animal that lives in the wild in waters with an average temperature of 22°C or below, and is distinct from terrestrial animals.

[0051] As used herein, a "fish" is a marine species of animal having a backbone, gills and fins, and includes mammals that fit this description, such as whales, but are distinct from land mammals.

[0052] As used herein, "click chemistry" refers to a concept introduced by K. Barry Sharpless of the Scripps Research Institute to describe chemical reactions that proceed under high thermodynamic driving forces between reactive functional groups to form covalently bonded linking functional groups. These generally have high yields, are often stereospecific, can produce non-toxic by-products, and can proceed in the presence of water. See, e.g., Nair et al. For the avoidance of doubt, reactions involving photoinitiated crosslinking of methacryloyl groups are not click chemistry reactions. In some embodiments, click chemistry reactions are not photoinitiated. In some embodiments, click chemistry reactions are reactions that can be performed under ambient light conditions. In some embodiments, click chemistry reactions are reactions that can be performed in the dark.

[0053] As used herein, "classical click chemistry" refers to click chemistry reactions in the form of [3+2] cycloadditions, [4+1] cycloadditions, thiol-ene reactions, thiol-yne reactions, Diels-Alder cycloadditions (between conjugated dienes and alkenes), nucleophilic substitution or addition reactions between alkenes or alkynes, and the formation of thiol ether or disulfide bonds. Additionally, this category encompasses Michael addition reactions, in which a nucleophile such as a thiol adds to the double bond of an α,β-unsaturated carbonyl compound.

[0054] Modified Gelatin Processes for producing natural gelatin by at least partial hydrolysis of collagen are generally known in the art. These are generally chemical or enzymatic processes. Chemical processes often involve mild acid or alkaline treatment. Typical processes result in partially hydrolyzed collagen (gelatin) molecules with molecular weights between 5 kDa and 200 kDa.

[0055] In particular, the present disclosure relates to marine gelatins modified to incorporate crosslinkable groups containing reactive moieties that are reactive in click chemistry reactions with crosslinkers containing suitable additional reactive moieties, and hydrogels formed by crosslinking thereof.

[0056] The gelatin is modified to incorporate crosslinkable groups that contain reactive moieties, in other words, the gelatin is reacted with additional chemical species that are not naturally part of the gelatin.

[0057] The crosslinkable groups incorporated into gelatin to obtain modified gelatin can be incorporated by reaction of the gelatin molecule with a chemical species that consists of or contains a crosslinkable group. In many embodiments, the gelatin is modified so that pendent groups are covalently attached to the gelatin.

[0058] That is, when forming a covalent bond, the gelatin molecule and the crosslinkable group species are chemical species that comprise functional groups that consist of or contain reactive moieties, and react with each other to form a covalent bond upon linkage of the functional groups. Specifically, the reaction between the gelatin molecule and the crosslinkable group species can occur between the exposed reactive moiety of the gelatin molecule and the complementary reactive moiety of the crosslinkable group species to form a modified gelatin.

[0059] Reactive moieties involved in chemical reactions are known to or can be determined by those skilled in the art, many of which are described below. The reactions described herein may be assisted by facilitators such as additional energy sources (e.g., light, heat) or additional reagents (e.g., catalysts, bases, acids, initiators, coupling agents, etc.). Auxiliary chemical reactions are known to or can be determined by those skilled in the art. One example is the copper-catalyzed azide-alkyne click chemistry cycloaddition reaction between an azide moiety and an alkyne moiety. Another example is amide bond formation between a carboxylic acid and an amine using a coupling agent. A variety of coupling agents are known in the art. Reaction conditions, including temperature, pressure, and other physical parameters, such as the use of solvents, purification, and characterization methods, are known to or can be determined by those skilled in the art.

[0060] Gelatins derived from natural sources of cold-water-adapted marine species, such as, but not limited to, Salmo or Oncorhynchus, differ in amino acid composition from terrestrial mammalian gelatins, typically containing less than 20% proline and less than 20% hydroxyproline relative to the total amino acid content, whereas terrestrial mammalian gelatins contain approximately 30% of each of these amino acids. These natural gelatins also differ from their warm-water-adapted marine counterparts, which typically contain approximately 25% of each of these amino acids. Compared to terrestrial mammalian gelatins and warm-water marine gelatins and derivatives, the relatively low proline and hydroxyproline content of these natural gelatins results in significantly lower melting points (approximately 4°C) and significantly lower and more stable rheological properties over a broad temperature range, approximately 15°C to 40°C. This, as the inventors have discovered, results in a variety of properties that can be advantageously used in a variety of applications, including the preparation of hydrogels from modified cold-water-adapted marine gelatins containing click chemistry reactive moieties.

[0061] In a preferred embodiment, the number of proline and / or hydroxyproline residues is 20% or less of the total number of amino acid residues in the modified gelatin. Preferably, the content of proline and / or hydroxyproline is less than 20%, such as 19% or less, 18% or less, 17% or less, or 16% or less of the total number of amino acid residues in the modified gelatin.

[0062] In some embodiments, the gelatin comprises Ala, Gly, Pro, and 4-Hyp. In some embodiments, at least half of the amino acids present in the gelatin are selected from the group consisting of Ala, Gly, Pro, and 4-Hyp.

[0063] In some embodiments, the gelatin contains 8 mol% to 14 mol% Ala, 30 mol% to 40 mol% Gly, 4 mol% to 10 mol% 4-Hyp, and 10 mol% to 16 mol% Pro.

[0064] In some embodiments, the gelatin contains 8 mol% to 14 mol% Ala, 30 mol% to 40 mol% Gly, 4 mol% to 10 mol% 4-Hyp, 10 mol% to 16 mol% Pro, and the remaining amino acids selected from the group consisting of Arg, Asp, Glu, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, and Val.

[0065] In some embodiments, the gelatin comprises Ala, Gly, Pro, 4-Hyp, and Glu. In some embodiments, at least half of the amino acids present in the gelatin are selected from the group consisting of Ala, Gly, Pro, 4-Hyp, and Glu.

[0066] In some embodiments, the gelatin contains 8 mol% to 13 mol% Ala, 7 mol% to 11 mol% Glu, 21 mol% to 37 mol% Gly, 5 mol% to 10 mol% 4-Hyp, and 8 mol% to 12 mol% Pro.

[0067] In some embodiments, the gelatin contains 9 mol% to 12 mol% Pro, 7 mol% to 9 mol% 4-Hyp, 10 mol% to 12 mol% Glu, 21 mol% to 23 mol% Gly, and 9 mol% to 10 mol% Ala.

[0068] In some embodiments, the gelatin contains 9 mol% to 12 mol% Pro, 7 mol% to 9 mol% 4-Hyp, 10 mol% to 12 mol% Glu, 21 mol% to 23 mol% Gly, 8 mol% to 9 mol% Arg, 9 mol% to 10 mol% Ala, and up to 7 mol% of any other amino acid.

[0069] "Cold-water adapted marine species" from which natural gelatin may be derived include the genus Salmo, including Salmo salar; the genus Oncorhynchus, including Oncorhynchus gorbuscha, Oncorhynchus tshawytscha, Oncorhynchus keta, Oncorhynchus kisutch, Oncorhynchus masou, and Oncorhynchus nerka; the genus Gadus, including Gadus chalcogrammus, Gadus morhua, and Gadus microcephalus; the genus Melanogrammus, including Melanogrammus aeglephinus; In a preferred embodiment, the natural gelatin may be derived from a species of the genus Salmo, in particular Salmo salar.

[0070] The intended purpose of the reactive moiety that is reactive in a click chemistry reaction is to undergo a click chemistry reaction with a crosslinkable group precursor to form crosslinks between gelatin molecules via the linking functional groups to form a hydrogel. The reactive moiety may be selected based on the desired click chemistry reaction, or the click chemistry reaction may be determined by the selection of the particular reactive moiety.

[0071] Click chemistry reactions and their reactive functional groups are known in the art. Examples include [3+2] cycloadditions, such as the Huisgen 1,3-dipolar cycloaddition or azide-alkyne cycloaddition between an azide moiety and an alkyne moiety, amide bond coupling between an amine group and a carbonyl group (e.g., esters, carboxylic acids, aldehydes, anhydrides, ketones, acyl halides), including via reductive amination, Diels-Alder cycloadditions between alkenes and conjugated dienes, ester bond formation between alcohols or alkoxides and carbonyl groups, ester bond formation between alcohols and nitriles, thioester bond formation between thiols and alkyl halides or alkenes, and the like. These include ether bond formation, disulfide bond coupling between thiols, alkenyl sulfide bond formation between thiols and alkynes, other nucleophilic substitution reactions, particularly between epoxides and alcohols, amines, or organometallic nucleophiles such as organolithium compounds, Michael addition between thiols and alkenes, other nucleophilic addition reactions, particularly between carbonyl groups and alcohols, alkoxides, amines, and organometallic nucleophiles, oxime or nitrone formation between alkoxyamines and aldehydes or ketones, and [4+1] cycloaddition between isonitriles and tetrazines.

[0072] Thus, representative reactive moieties of the crosslinkable groups of modified gelatin include alkynes, amines, carbonyl groups, alkenes including conjugated dienes, alcohols, alkoxides, nitriles, thiols, alkyl halides, epoxides, organometallic species, alkoxyamines, isonitriles, and tetrazines.

[0073] In preferred embodiments, the crosslinkable group is intended to undergo classical click chemistry. In these embodiments, the reactive moiety is preferably selected from the group consisting of alkynes, amines, alkenes, conjugated dienes, thiols, isonitriles, and tetrazines. In more preferred embodiments, the crosslinkable group is intended to undergo a Michael addition reaction between a thiol and an alkene or between a thiol and an alkyne, a disulfide bond coupling between thiols, a Diels-Alder cycloaddition between an alkene and a conjugated diene, or an azide-alkyne cycloaddition between an azide and an alkyne, in which case the reactive moiety is preferably selected from the group consisting of alkynes, alkenes, conjugated dienes, and thiols. In the most preferred embodiment, the crosslinkable group is intended to undergo a Michael addition reaction between a thiol and an alkene, in which case the reactive moiety is preferably selected from the group consisting of alkenes and thiols.

[0074] As used herein, reference to a particular reactive moiety is considered to encompass a chemical functional group that includes that moiety, regardless of whether the functional group includes or consists solely of a reactive moiety. For example, an amine-reactive moiety can refer to a functional group that includes an amine moiety, or primary, secondary, or tertiary amines, amides, guanidines, hydrazines, hydrazines, and other functional groups.

[0075] Crosslinking groups that are incorporated into gelatin to obtain modified gelatin can be incorporated by reaction of crosslinking group species with exposed reactive moieties on the gelatin molecule to form covalent bonds.

[0076] Gelatin generally contains the amino acids proline and / or hydroxyproline, and other amino acids, which may be any of the 20 known common amino acids, particularly glycine, glutamic acid, arginine, alanine, aspartic acid, and cysteine. The amino acids of gelatin may contain functional groups having exposed (i.e., available) reactive moieties for reaction with complementary reactive moieties of functional groups of cross-linking species. The exposed reactive moieties may be, for example, hydroxyl groups (hydroxyproline, serine, threonine), amines or amides (arginine, lysine, asparagine, glutamine, and terminal amino acids resulting from collagen hydrolysis), thiols (cysteine), and carboxylic acids (glutamic acid, aspartic acid, and terminal amino acids resulting from collagen hydrolysis).

[0077] Thus, in a preferred embodiment, the crosslinkable group species, in addition to providing a reactive moiety in a click chemistry reaction, includes a second reactive moiety that is reactive with an exposed reactive moiety on a gelatin molecule. As the available exposed reactive moieties on gelatin molecules are generally hydroxyl groups, amines or amides, thiols, and carboxylic acids, preferably the complementary reactive moiety of the crosslinkable group species is reactive with one or more of these functional groups. In these embodiments, the complementary reactive moiety is preferably a carboxylic acid (for reaction with hydroxyls, amines), epoxide (for reaction with hydroxyls, amines, amides), anhydride (for reaction with hydroxyls, amines, amides), aldehyde (for reaction with hydroxyls, amines, amides), ketone (for reaction with hydroxyls, amines, amides), ester (for reaction with hydroxyls, amines, amides), isocyanate (for reaction with hydroxyls, amines), isothiocyanate (for reaction with hydroxyls, amines), thioimidate (for reaction with amines), or acyl halide (for reaction with hydroxyls, amines), alkyne (for reaction with amines, thiols), alkene (for reaction with thiols), or amine, amide, hydroxyl, epoxide, anhydride, aldehyde, ketone, ester, isocyanate, isothiocyanate, acyl halide, alkyne, or alkene for reaction with an exposed reactive moiety as set forth above.

[0078] The click chemistry groups described herein may be equally applicable to exposed reactive moieties on gelatin molecules and complementary reactive moieties on crosslinkable group species.

[0079] Where cross-linking of gelatin molecules by the cross-linkable group species itself may occur, this can be avoided in a number of ways, typically by selecting appropriate cross-linkable group species or reaction conditions or agents as needed. In a typical example, the cross-linkable group species may contain only one complementary reactive moiety or may contain two or more, all but one of which contain a protecting group that can be removed after reaction of the exposed reactive moiety of the gelatin molecule with the complementary reactive moiety of the cross-linkable group species.

[0080] In preferred embodiments, the exposed reactive moiety is an amine, a carbonyl group, or a thiol. The complementary reactive moiety is preferably a carbonyl group (for reaction with an amine), an epoxide, a thioimidate, an isocyanate, or an isothiocyanate, a hydroxyl or an amine (for reaction with a carboxylic acid), or a thiol, an alkene, or an alkyne (for reaction with a thiol). Preferably, the exposed reactive moiety is an amine (preferably the amine of a guanidine) and the complementary reactive moiety is a thioimidate, and / or the exposed reactive moiety is a carbonyl group (preferably a carboxylic acid) and the complementary reactive moiety is an amine (preferably a primary amine).

[0081] Thus, the crosslinkable group species may essentially be a bifunctional molecule, i.e., containing a reactive moiety for modifying gelatin and a reactive moiety for click chemistry crosslinking with a crosslinker. The crosslinkable group species is otherwise not particularly limited in structure. The structure of the bifunctional molecule may alternatively consist of or include, for example, an alkylene, alkenylene, or alkynylene, optionally further functionalized (e.g., containing additional functional groups such as those described herein), which is preferably a C1-C6 alkylene, alkenylene, or alkynylene. 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 In a preferred embodiment, the structure of the bifunctional crosslinkable group species may further include a non-functionalized C1-C 10 It consists of alkylene, preferably non-functionalized C1 to C6 alkylene.

[0082] Examples of preferred crosslinkable group species are Traut's reagent (2-iminothiolane), other cyclic thioimidates, ethylenediamine, propylenediamine, etc., ethylene glycol, propylene glycol, etc., malonic acid, succinic acid, etc., amino acids such as cysteine, lactones, and lactams.

[0083] An intermediate step that can be performed in forming modified gelatin involves treating gelatin with a first crosslinkable group species containing a reactive moiety that is reactive with a complementary reactive moiety of a second crosslinkable group species, the second crosslinkable group species containing a reactive moiety that is reactive in a click chemistry reaction. For example, a gelatin molecule can be reacted with a first crosslinkable group species, the reaction occurring between exposed reactive moieties of one species of gelatin, using a species that provides an exposed crosslinkable group reactive moiety that is the same as an exposed reactive moiety of another species of gelatin, followed by reacting the exposed reactive moiety with a second crosslinkable group species containing a reactive moiety for click chemistry reaction with a crosslinker. This has the advantage of increasing the content of reactive moieties for click chemistry crosslinking and controlling the degree of crosslinking formed when preparing a hydrogel.

[0084] The crosslinkable group species may be referred to as a "crosslinkable group precursor."

[0085] In one example, exposed carboxylic acid groups of gelatin that are not amines are reacted with a first crosslinkable group species that provides exposed amine-reactive moieties, and the native gelatin and the amines of the first crosslinkable groups are reacted with a second crosslinkable group species that provides exposed reactive moieties for click chemistry reaction with a crosslinker. This represents a preferred embodiment that includes the following steps: i) reaction of exposed carboxylic acid groups of (native) gelatin with a diamine, and ii) subsequent reaction (of the native exposed amines and diamine-exposed amines) with Traut's reagent.

[0086] Applicable reactive moieties may be as described herein. In a specific example, gelatin may be treated with ethylenediamine (first crosslinkable group species), which reacts with carboxylic acids to increase the exposed amine content, followed by treatment with Traut's reagent, which reacts with amines, to increase the reactive moiety content compared to that of native gelatin, resulting in a high content of thiol moieties for click chemistry reaction with crosslinkers.

[0087] Third and subsequent treatments with crosslinkable species are applicable as well.

[0088] Another intermediate step that can be performed in forming modified gelatin involves chemically converting an exposed reactive moiety from one species to another. Another intermediate step that can be performed in forming modified gelatin involves chemically converting a reactive moiety of a crosslinkable group from one species to another. For example, the reactive moiety can be converted to another so that it can be applicable to a specific click chemistry reaction with a crosslinking agent. Another intermediate step that can be performed is the addition or removal of a protecting group, as needed. This intermediate step helps provide flexibility in the reagents selected for the crosslinkable group species and crosslinking agent. The intermediate step may include treating gelatin or modified gelatin with a reactive reagent. Examples of chemical conversion of functional groups with a reactive reagent include the conversion of an epoxide to an amine using an ammonia reagent and the conversion of an epoxide to a hydroxide using a sulfuric acid reagent. Many other examples are known to or can be determined by those skilled in the art.

[0089] In some embodiments, the modified gelatin is gelatin that has been modified by reacting carboxylic acid groups present in unmodified gelatin with a crosslinkable group precursor that contains an amine group (e.g., capable of reacting with carboxylic acid groups present in gelatin). For example, gelatin may be modified by reacting a C alkylene diamine (e.g., C alkylene diamine, such as ethylene diamine or propylene diamine) with a C alkylene diamine (e.g., C alkylene diamine, such as ethylene diamine or propylene diamine) to introduce crosslinkable groups via amide bond formation. 2~6 In such embodiments, the crosslinkable group may be modified by reaction with a monoprotected alkylenediamine, or a monoprotected alkylenediamine, which is subsequently deprotected. In such embodiments, the crosslinkable group may be of the formula -NH-C 2~6 As another example, gelatin may have a crosslinkable group of the formula -NH-C after deprotection. 2~6 alkylene -SH, such as 2~6 It may be modified by reaction with an alkylene-S-PG group, where PG represents a thiol protecting group.

[0090] In some embodiments, gelatin may be modified by reaction with a crosslinkable group precursor containing a carboxylic acid group (e.g., capable of reacting with an amino group present in gelatin). For example, gelatin may be a crosslinkable group precursor of the formula HO2C-C 2~6 It may be modified by reaction with a group of the formula alkylene-NH-PG, where PG represents an amine protecting group, followed by deprotection of the protecting group to introduce a crosslinkable group via amide bond formation. In another example, gelatin may be modified by reaction with a group of the formula HO2C-C 2~6 It may be modified by reaction with an alkylene-S-PG group (where PG represents a thiol protecting group), followed by deprotection of the protecting group to introduce a crosslinkable group, again via amide bond formation.

[0091] In some embodiments, the gelatin comprises a first functional group capable of reacting with a reactive group present in the gelatin, and a second functional group or a protected form of the second functional group, which second functional group may be modified by reaction with a crosslinking group that is an amino acid or a protected amino acid that constitutes a reactive moiety that is reactive with a reactive moiety in the crosslinker. One example of such an amino acid is cysteine, e.g., L-cysteine.

[0092] The modified gelatin may be characterizable by one or more properties, which may be reactive moiety content and viscosity, as described below.

[0093] The modified gelatin may be characterized by a reactive moiety content of about 25 μmol / g to about 1000 μmol / g, preferably about 50 μmol / g or 100 μmol / g to about 950 μmol / g, preferably about 150 μmol / g or 200 μmol / g to about 900 μmol / g, preferably about 250 μmol / g or 300 μmol / g to about 850 μmol / g, preferably about 350 μmol / g or 400 μmol / g to about 850 μmol / g, which is preferred when the reactive moiety is an amine or thiol.

[0094] The modified gelatin may be characterized by a reactive moiety content of about 300 μmol / g to about 700 μmol / g, preferably about 350 μmol / g to about 650 μmol / g, preferably about 400 μmol / g to about 600 μmol / g, preferably about 450 μmol / g to about 550 μmol / g, which is preferred when the reactive moiety is a thiol.

[0095] The following examples describe methods for determining reactive moiety content that represent preferred methods.

[0096] The modified gelatin may be characterized by a viscosity such that an aqueous solution of 20% (wt / v) or less, preferably about 5% (wt / v) to about 20% (wt / v) of the modified gelatin has a viscosity of less than 100,000 mPa·s over a shear rate of 10 to 1,000 1 / s, preferably less than 10,000 mPa·s over a shear rate of 10 to 1,000 1 / s, preferably less than 1,000 mPa·s over a shear rate of 10 to 1,000 1 / s, preferably less than 100 mPa·s over a shear rate of 10 to 1,000 1 / s, preferably less than 100 mPa·s over a shear rate of 10 to 1,000 1 / s, preferably less than 50 mPa·s over a shear rate of 10 to 1,000 1 / s. The modified gelatin may also be characterized by a viscosity where an aqueous solution of 10% (wt / v) or less, preferably about 5% (wt / v) to about 10% (wt / v), of the modified gelatin has a viscosity of less than 10 mPa·s over a shear rate (1 / s) of 10 to 1000. The modified gelatin may also be characterized by a viscosity where an aqueous solution of 5% (wt / v) or less, preferably about 5% (wt / v), of the modified gelatin has a viscosity of less than 5 mPa·s over a shear rate (1 / s) of 10 to 1000. These viscosities are preferred when crosslinkable groups are formed using Traut's reagent, including combinations, and the reactive moiety is a thiol, particularly when native gelatin is first functionalized with ethylenediamine (i.e., when ethylenediamine is used in an intermediate step as the first crosslinkable group species).

[0097] The following examples describe methods for measuring viscosity that represent preferred methods.

[0098] The modified gelatin is prepared by subjecting an aqueous solution of 20% (wt / v) or less, preferably about 5% (wt / v) to about 20% (wt / v) of modified gelatin, to a temperature range of 0°C to 40°C for 10 minutes. 8 Complex viscosity less than 10 mPa·s, preferably over the temperature range 0°C to 40°C 7 Complex viscosity less than 10 mPa·s, preferably over a temperature range of 20°C to 40°C 6 The modified gelatin may be characterized by a complex viscosity of less than mPa·s. The modified gelatin may be characterized by a complex viscosity of less than 10% (wt / v), preferably about 5% (wt / v) to about 10% (wt / v), of an aqueous solution of the modified gelatin having a complex viscosity of less than 10 mPa·s over a temperature range of 0°C to 40°C. 6 It may also be possible to characterize the gelatin by its complex viscosity, i.e., having a complex viscosity of less than mPa·s. These complex viscosities are preferred when the crosslinkable groups are formed using Traut's reagent and the reactive moiety is a thiol, including combinations, particularly when the native gelatin is first functionalized with ethylenediamine.

[0099] The following examples describe methods for measuring complex viscosity that represent preferred methods.

[0100] The gelatin and modified gelatin disclosed herein may exist in the form of a salt. In many cases, groups present in gelatin are capable of forming acidic and / or basic salts due to the presence of amino and / or carboxyl groups, or groups similar thereto. Salts can be formed with inorganic and organic acids and bases. Examples include salts with hydrochloric acid, sulfuric acid, acetic acid, propionic acid, sodium and potassium bases, and amines. Many others are known in the art.

[0101] Crosslinked Gelatin and Hydrogels Crosslinked modified gelatin can be produced by reacting the modified gelatin described herein with a crosslinking agent in a click chemistry reaction, where the crosslinking agent comprises two or more reactive moieties, which are reactive with the reactive chemical moieties of the crosslinkable groups of the modified gelatin in the click chemistry reaction.

[0102] Benefits of the crosslinked modified gelatin and hydrogels described herein are obtained through the ease and speed of formation via click chemistry reactions and the biocompatibility of many click chemistry reactions, which generally do not require any or significant external inputs for initiation. For example, compared to photoinitiated crosslinking of gelatin modified to contain methacryloyl groups, production of the crosslinked modified gelatin and hydrogels described herein can proceed in the absence of photoinitiation and in the absence of agents such as free radical scavengers that are often required for photoinitiated chemical reactions, without the need for specialized equipment.

[0103] Thus, in a preferred embodiment, the reaction is carried out under ambient light conditions.

[0104] An additional benefit is that the crosslinking time for hydrogel formation can be tailored by using various crosslinkers. For example, a crosslinker may contain maleimide groups as two or more reactive moieties, which are reactive with reactive chemical moieties on modified gelatin, such as thiols, in a click chemistry reaction. Such systems can have relatively short crosslinking times at room temperature (e.g., less than 1 minute, or less than 30 seconds, or less than about 10 seconds). In contrast, crosslinkers containing, for example, vinyl sulfone groups as two or more reactive moieties, can, in some embodiments, result in longer crosslinking times (e.g., 10 minutes or more, or 20 minutes or more, or about 30 minutes). The advantage of having a short crosslinking time is when rapid curing and hydrogel formation are desired, such as in 3D printing applications. On the other hand, the advantage associated with longer crosslinking times (e.g., greater than about 10 minutes) is that they may be suitable for applications where more time is required for handling and processing of components, such as during manual or automated liquid handling.

[0105] In some embodiments, the modified gelatin and crosslinking agent are selected to provide a crosslinking time ranging from 1 second to 1 minute, or from 1 second to 30 seconds, or from 1 second to 10 seconds. In some embodiments, the modified gelatin and crosslinking agent are selected to provide a crosslinking time ranging from 1 minute to 5 minutes. In some embodiments, the modified gelatin and crosslinking agent are selected to provide a crosslinking time ranging from 5 minutes to 1 hour, or from 10 minutes to 1 hour, or from 20 minutes to 1 hour, or from 30 minutes to 1 hour, or from 10 minutes to 30 minutes, or from 10 minutes to 20 minutes, or from 20 minutes to 20 minutes.

[0106] The click chemistry reaction between the crosslinkable group reactive moiety and the crosslinker reactive moiety forms a covalent bond. That is, the modified gelatin molecule containing the crosslinker is a chemical species that consists of reactive moieties or contains functional groups that contain reactive moieties, which react with each other in a click chemistry reaction to form a covalent bond to the linking functional group. Specifically, the click chemistry reaction between the crosslinkable group of the modified gelatin and the crosslinker can occur between the reactive moiety of the crosslinkable group and the reactive moiety of the crosslinker.

[0107] As noted above, the reactive moieties involved in the click chemistry reaction may be selected based on the desired click chemistry reaction, or the click chemistry reaction may be determined by the selection of a particular reactive moiety. Click chemistry and its reactive functional groups are known in the art. Many examples, including representative examples that may be applicable, have been described above.

[0108] In preferred embodiments, the crosslinkable group and crosslinker are intended to undergo classical click chemistry reactions. In these embodiments, the reactive moiety of the crosslinkable group is selected from the group consisting of alkynes, amines, alkenes, conjugated dienes, thiols, isonitriles, and tetrazines. In this case, preferred reactive moieties of the crosslinker are selected from the group consisting of azides or thiols (to react with alkynes), carbonyl groups or epoxides (to react with amines), conjugated alkenes or thiols (to react with alkenes, including, for example, α,β-unsaturated carbonyl systems, including maleimides, and vinyl sulfones), alkenes (to react with conjugated dienes or thiols), thiols (to react with thiols, alkyl halides, or alkenes, including, for example, α,β-unsaturated carbonyl systems, including maleimides, and vinyl sulfones), isonitriles (to react with tetrazines), and tetrazines (to react with isonitriles).

[0109] In more preferred embodiments, the crosslinkable group and crosslinker are intended to undergo a Michael addition reaction between a thiol and an alkene or a thiol and an alkyne, a disulfide bond coupling between thiols, a Diels-Alder cycloaddition between an alkene and a conjugated diene, or an azide-alkyne cycloaddition between an azide and an alkyne. In preferred embodiments, when the reactive moiety of the crosslinkable group is an alkyne, an alkene, a conjugated diene, or a thiol, the reactive moiety of the crosslinker is an azide, a thiol, an alkene, or an alkene, respectively. In a most preferred embodiment, when the reactive moiety of the crosslinkable group is intended to undergo a Michael addition reaction between a thiol and an alkene, the reactive moiety of the crosslinkable group is a thiol, and the reactive moiety of the crosslinker is an alkene. In a preferred embodiment, the functional group comprising an alkene-reactive moiety is selected from the group consisting of maleimide, vinyl sulfone, acrylate, acrylamide, and methacrylate.

[0110] Since the crosslinker is intended to form at least one bond with at least two modified gelatin molecules, the crosslinker comprises two or more reactive moieties that are reactive with the reactive chemical moieties of the crosslinkable groups of the modified gelatin in a click chemistry reaction.

[0111] In some embodiments, the click chemistry reaction is a reaction between a thiol-reactive group present on the crosslinkable group and an alkene-reactive moiety present on the crosslinker.

[0112] In some embodiments, the click chemistry reaction is a reaction between a thiol reactive group present on the crosslinkable group and either a vinyl sulfone or maleimide reactive group present on the crosslinker.

[0113] Thus, the crosslinker species is essentially at least a difunctional molecule, and may be a trifunctional molecule, a tetrafunctional molecule, etc., meaning that it contains at least two reactive moieties for click chemistry crosslinking with the modified gelatin. The crosslinker species is otherwise not particularly limited structurally. The crosslinker species may alternatively consist of or include, for example, an alkylene, alkenylene, alkynylene, alkyloxirene, alkenyloxirene, alkynyloxirene, or combinations thereof, optionally further functionalized (e.g., containing additional functional groups such as those described herein). The structure between the reactive moieties or functional groups containing them may be referred to as a "spacer."

[0114] In a preferred embodiment, the crosslinker has the following formula: Core-(Spacer-R) n wherein Core is an atom or group that provides bonding to n Spacer-R groups, Spacer is a spacer group, R is a functional group containing a reactive moiety, and n is an integer.

[0115] In a preferred embodiment, n is an integer from 2 to 8, for example, n can be 2, 3, 4, 5, 6, 7, or 8. Preferably, the crosslinker is tetrafunctionalized in that it contains four reactive moieties for click chemistry crosslinking with the modified gelatin, in which case, in a preferred embodiment, n is 4.

[0116] The crosslinker may alternatively consist of or include, for example, an alkylene, alkenylene, alkynylene, alkyloxylene, alkenyloxylene, alkynyloxylene, or combinations thereof, optionally further functionalized (e.g., containing additional functional groups such as those described herein).

[0117] In a preferred embodiment, Core consists of a central tetrasubstituted carbon atom having the formula C(Link)4, where Link is alkylene, alkenylene, alkynylene, alkyloxirene, alkenyloxirene, or alkynyloxirene, and is preferably C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene, C1-C6 alkyloxirene, C2-C6 alkenyloxirene, or C2-C6 alkynyloxirene, and is preferably C1-C6 alkyloxirene. In a preferred embodiment, Link is -CHO-, in which case Core is C(-CHO-)4 and n is 4.

[0118] In a preferred embodiment, the spacer is an alkylene, alkenylene, alkynylene, alkyloxirene, alkenyloxirene, or alkynyloxirene, preferably an alkyloxirene. In a preferred embodiment, the spacer is (-CH2CH2O-) m where m is an integer. In these embodiments, the spacer may be referred to as polyethylene glycol (PEG). In some preferred embodiments, m is an integer from 2 to 150, preferably from about 25 to about 140, preferably from about 50 to about 130, preferably from about 75 to about 120, or preferably from about 100 to about 115. In some other preferred embodiments, m is an integer from 2 to 50.

[0119] R is a group comprising a further reactive moiety. For example, R may comprise not only the further reactive moiety itself, but also a linking moiety that provides connectivity between the further reactive moiety and the spacer. R may be, for example, an alkylene, alkenylene, alkynylene, alkyloxirene, alkenyloxirene, or alkynyloxirene, optionally further functionalized (e.g., comprising a further functional group such as a functional group described herein, preferably an amide bond) further comprising a reactive moiety, and preferably a C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C1-C 10 Alkyloxylylene, C2-C 10 Alkenyl oxirane or C2-C 10 Alkynyloxirane, preferably C1-C further comprising a reactive moiety 10 It may be alkylene.

[0120] In a preferred embodiment, R is [ka] In another preferred embodiment, R is [ka] is.

[0121] A representative preferred embodiment of the cross-linker is the species known as PEG-4 maleimide (PEG4-MAL). PEG4-MAL has the structure Core-(Spacer-R) n where Core is C(-CH2O-)4 and Spacer is (-CH2CH2O-) m where m is an integer of 2 to 150, or 25 to 150, or 75 to 125, and R is [ka] and is available, for example, from JenKem™.

[0122] Another exemplary preferred embodiment of a cross-linking agent is the species known as PEG-4 vinyl sulfone (PEG-4VS). PEG-4VS has the structure Core-(Spacer-R) n where Core is C(-CH2O-)4 and Spacer is (-CH2CH2O-) m where m is an integer of 2 to 150, or 25 to 150, or 75 to 125, and R is [ka] and is available, for example, from JenKem™.

[0123] The alkene reactive moiety of the maleimide or vinyl sulfone functional group can undergo a click chemistry reaction with the thiol group of the modified gelatin to form a thioether linkage between the crosslinker and the modified gelatin.

[0124] In certain preferred combinations, the exposed reactive moieties of the gelatin are amines, carbonyl groups or thiols, preferably amines or carbonyl groups, and the complementary reactive moieties of the crosslinkable groups are carbonyl groups, epoxides, thioimidates, isocyanates, isothiocyanates, hydroxyls, amines, thiols, alkenes or alkynes, preferably thioimidates or amines, the crosslinkable groups are intended to undergo a classical click chemistry reaction, the reactive moieties in the click chemistry reaction being selected from the group consisting of alkynes, amines, alkenes, conjugated dienes, thiols, isonitriles and tetrazines, preferably alkenes or thiols, and the crosslinker has the formula Core-(Spacer-R) n wherein n is an integer of 2 to 8, preferably 4, the core is C(—CHO—) and the spacer is (—CHCHO—). m where m is an integer of 2 to 150, for example, 2 to 50, and the crosslinking agent is PEG-4MAL or PEG-4VS.

[0125] Thus, cross-linked modified gelatin can be produced by cross-linking the cross-linking agent through the reaction of the modified gelatin, which allows the production of a hydrogel by applying water during or after the cross-linking step.

[0126] The hydrogel may be characterized by one or more properties, which may be the content of cross-linked modified gelatin, swelling ratio, equilibrium water content, cross-linking time, and sol-gel transition temperature, as described below.

[0127] The hydrogels may be characterized by a cross-linked modified gelatin content ranging from 1% to 25% (w / v).

[0128] The hydrogels may be characterized by a relaxed mass swelling ratio such that 2.5% (wt / v) or greater hydrogels have a relaxed mass swelling ratio of 15 or less, preferably 14 or 13 or less, and preferably 12 or less. The hydrogels may also be characterized by a relaxed mass swelling ratio such that 5% (wt / v) or greater hydrogels have a relaxed mass swelling ratio of 9 or less, preferably 8 or 7 or less, and preferably 6 or less. The hydrogels may also be characterized by a relaxed mass swelling ratio such that 10% (wt / v) or greater hydrogels have a relaxed mass swelling ratio of 8 or less, preferably 7 or 6 or less, and preferably 5 or less. These relaxed mass swelling ratios, including combinations, are preferred when the crosslinkable groups are formed using Traut's reagent and the reactive moiety is a thiol, particularly when native gelatin is first functionalized with ethylenediamine and the crosslinker is PEG-4MAL.

[0129] The following examples describe methods for measuring relaxation mass swelling ratios that represent preferred methods.

[0130] The hydrogels may be characterized by an equilibrium swelling ratio such that a 2.5% to 10% (wt / v) hydrogel has an equilibrium swelling ratio of 10 to 25, preferably 11 to 24 or 12 to 23, preferably 13 to 22 or 14 to 21, and most preferably 15 to 20. This equilibrium swelling ratio is preferred when the crosslinkable groups are formed using Traut's reagent and the reactive moiety is a thiol, particularly when native gelatin is first functionalized with ethylenediamine and the crosslinker is PEG-4MAL.

[0131] The following examples describe methods for measuring equilibrium swelling ratios that represent preferred methods.

[0132] The hydrogels may be characterized by an equilibrium water content such that a 2.5%-10% (wt / v) hydrogel has an equilibrium water content of 80%-100%, preferably 85%-100%, preferably 90%, or in other words, 88%-92%. This equilibrium water content is preferred when the crosslinkable groups are formed using Traut's reagent and the reactive moiety is a thiol, particularly when native gelatin is first functionalized with ethylenediamine and the crosslinker is PEG-4MAL.

[0133] The following examples describe methods for measuring equilibrium swelling ratios that represent preferred methods.

[0134] The hydrogels may be characterized by a crosslinking time such that a 2.5% to 10% (wt / v) hydrogel has a crosslinking time at room temperature of about 1 second to about 5 seconds, preferably about 1 second to about 4 seconds, or about 1 second to about 3 seconds, preferably about 1.2 seconds or about 1.8 seconds, or in other words, 1 second to 2 seconds. This crosslinking time is preferred when the crosslinkable reactive moiety is a thiol, particularly when native gelatin is first functionalized with L-cysteine ​​and the crosslinker is PEG-4MAL.

[0135] The following examples describe methods for measuring crosslinking time that represent preferred methods.

[0136] Hydrogels can be characterized by their crosslinking time. For example, in some embodiments, 2.5% to 10% (wt / v) hydrogels can be prepared from systems (i.e., modified gelatin and crosslinker) that have crosslinking times at room temperature (e.g., 25°C) of about 1 second to 1 minute, or about 1 second to 30 seconds, or about 1 second to 10 seconds, or about 10 seconds to 30 seconds, or about 20 seconds to 30 seconds. Such crosslinking times can be achieved, for example, in some embodiments, when the crosslinkable reactive moiety is a thiol (e.g., when native gelatin is first functionalized with L-cysteine) and the crosslinker is PEG4-MAL.

[0137] In some embodiments, 2.5% to 10% (wt / v) hydrogels can be prepared from systems having crosslinking times at room temperature (e.g., 25°C) of about 1 minute to about 5 minutes, or about 5 minutes to 10 minutes, or about 10 minutes to about 30 minutes, or about 20 minutes to about 30 minutes, or about 22 minutes to about 28 minutes, or about 25 minutes to about 27 minutes. Such crosslinking times may be achievable, for example, in some embodiments, when the crosslinkable reactive moiety is a thiol, particularly when native gelatin is first functionalized with L-cysteine ​​and the crosslinker is PEG-4VS.

[0138] The following examples describe methods for measuring crosslinking time that represent preferred methods.

[0139] kit Also provided is a kit for producing cross-linked modified gelatin. The kit may include natural gelatin derived from a marine resource and one or more cross-linkable group species for producing the modified gelatin described herein. Alternatively or additionally, the kit may include the modified gelatin and, optionally, a cross-linking agent for producing the cross-linked modified gelatin described herein.

[0140] In a preferred embodiment, the kit comprises a modified gelatin as described herein and a cross-linking agent as described herein.

[0141] In another preferred embodiment, the kit comprises natural gelatin derived from a marine source and a crosslinkable group species for producing the modified gelatin described herein, optionally together with a crosslinking agent for producing the crosslinked modified gelatin described herein.

[0142] The kit may include water for producing the hydrogels described herein. The kit may include a buffer, such as HEPES. For example, the kit may include an aqueous solution of a buffer, such as HEPES. The kit may include instructions for producing one or more of the modified gelatin, cross-linked gelatin, and hydrogels described herein.

[0143] Uses of Cross-Linked Gelatin and Hydrogels The cross-linked modified gelatins and hydrogels described herein are useful as biomaterials, particularly cell growth / storage matrices, and for 3D bioprinting.

[0144] In particular, the inventors have found that cell growth matrices comprising hydrogels of the present disclosure are essentially as useful in growing, preserving, and maintaining encapsulated cells in a viable state under growth conditions (growth medium, 37°C, 5% CO2) in the short term (up to 2 weeks), and actually more effective in the long term (beyond 2 weeks) than hydrogels made from photoinitiator-crosslinked methacryloyl-functionalized gelatin.

[0145] In a preferred embodiment, the cell growth matrix (regardless of the growth medium) consists of a hydrogel as described herein.

[0146] Cells can be encapsulated by forming a hydrogel around the cell population, which can include providing the cell population, suspending the cell population in an aqueous solution of a modified gelatin as described herein, and mixing with a cross-linking agent as described herein under conditions suitable to effect a cross-linking reaction, or subsequently subjecting the suspension to conditions suitable to effect a cross-linking reaction.

[0147] The following examples describe methods for encapsulating cells, which represent preferred methods.

[0148] In a preferred embodiment, a population of cells encapsulated under growth conditions in a hydrogel formed from an aqueous solution of 2.5% (wt / v) to 10% (wt / v) modified gelatin is at least 82% viable, preferably 83% viable, and preferably 84% viable after 1 day, preferably 7 days, preferably 14 days, and preferably 21 days. Preferably, a population of cells encapsulated in a hydrogel formed from an aqueous solution of 2.5% (wt / v) to 5% (wt / v) modified gelatin is at least 84% viable, preferably 85% viable, after 1 day, preferably 7 days, and preferably 14 days, and preferably at least 86% viable after 1 day, preferably 7 days. Preferably, a population of cells encapsulated in a hydrogel formed from an aqueous solution of about 2.5% (wt / v) modified gelatin is at least 86% viable after 1 day, preferably 7 days. More preferably, after 21 days, the population of cells encapsulated in a hydrogel formed from an aqueous solution of 5% (wt / v) to 10% (wt / v) modified gelatin is at least about 84% viable, and the population of cells encapsulated in a hydrogel formed from an aqueous solution of about 2.5% (wt / v) modified gelatin is at least about 85%, preferably 86% viable.

[0149] The following examples describe methods for measuring cell viability that represent preferred methods.

[0150] In a preferred embodiment, a population of cells encapsulated under growth conditions in a hydrogel formed from an aqueous solution of 2.5% (wt / v) to 10% (wt / v) modified gelatin has a metabolic activity (AU) of about 400 AU to about 1000 AU, preferably about 450 AU to about 800 AU, and preferably about 500 AU to about 600 AU after 7 days. In some embodiments, the hydrogel is formed from modified gelatin and PEG-4MAL. In other embodiments, the hydrogel is formed from modified gelatin and PEG-4VS.

[0151] The following examples describe methods for measuring metabolic activity that represent preferred methods.

[0152] In a preferred embodiment, the cells are about 2 x 10 6 cells / mL, or in other words, the cell population was approximately 2 x 10 6 In another preferred embodiment, the cells comprise about 1 x 10 cells / mL. 6 cells / mL, or in other words, the cell population was approximately 1 x 10 6 Contains cells in number of cells / mL.

[0153] In a preferred embodiment, the cell is a mammalian cell, preferably a human cell, preferably a human cancer cell, with breast cancer cells being an exemplary embodiment. [Example]

[0154] Example 1 Synthesis of an exemplary modified gelatin: Thiolated cold-water fish skin gelatin by EDC / NHS coupling of L-cysteine 10 g of gelatin from cold-water fish skin (molecular weight approximately 60 kDa, Sigma™, lot number SLCG7135) was added to 500 mL of 0.1 mM HCl and stirred at room temperature until dissolved. 7.5 g of 1-ethyl-3(3-dimethylamino)propylcarbodiimide (EDC) (Sigma™) and 3.75 g of N-hydroxysuccinimide (NHS) (Sigma™) were then added to the solution. The EDC / NHS reaction was allowed to proceed for 30 minutes, after which 20 g of L-cysteine ​​(Sigma™) was added to the solution. The conjugation reaction was allowed to proceed for 24 hours at room temperature, protected from light. The pH of the solution was maintained at 5.0 throughout the reaction. The solution was then dialyzed against 0.1 mM HCl for 5 days using snakeskin dialysis tubing (Sigma™) with a 1 kDa molecular weight cutoff. After dialysis was completed, the samples were frozen at −80°C overnight and lyophilized for 5 days.

[0155] Example 2 Synthesis of an exemplary modified gelatin: Amination of cold-water fish skin gelatin Cold-water fish skin gelatin was aminated by the following method: 6 g of cold-water fish skin gelatin was dissolved in 150 mL of 0.1 M phosphate-buffered saline (PBS) (Sigma-Aldrich™, St. Louis). Then, 60 g of ethylenediamine (Sigma-Aldrich™, St. Louis) was added to the solution. The pH of the solution was adjusted to 5.0 using HCl and NaOH, and 2.3 g of EDC was added. The reaction mixture was stirred with a magnetic stir bar at room temperature for 24 hours, protected from light. After the reaction time, the sample was dialyzed, frozen, and lyophilized. The reaction scheme is shown in Figure 1B. The aminated gelatin was thiolated using Traut's reagent as follows:

[0156] Synthesis of an exemplary modified gelatin: Thiolated gelatin using Traut's reagent Thiolation of gelatin using Traut's reagent was adapted from the protocol originally described by Duggan et al. 1 g of cold-water fish skin gelatin was added to 100 mL of ultrapure water and dissolved with stirring at room temperature. After dissolution, the pH of the solution was adjusted to 7.0, and a 2-fold molar excess of 2-iminothiolane (Traut's reagent) (Sigma™) was subsequently added to the solution. The reaction mixture was stirred at room temperature for 24 hours, protected from light. The solution was then dialyzed against 0.1 mM HCl for 5 days using snakeskin dialysis tubing (Sigma™) with a 1 kDa molecular weight cutoff. After dialysis was complete, the sample was frozen at -80°C overnight and lyophilized for 5 days. The reaction scheme is shown in Figures 1A and 1C.

[0157] Example 3 Determination of the amine content in gelatin and gelatin derivatives The amine content of gelatin and gelatin derivatives was quantified by the 2,4,6-trinitrobenzenesulfonate (TNBS) assay as described by Meinert et al. Briefly, 0.1 M NaHCO3 buffer was prepared, and the pH of the solution was adjusted to 8.5 using HCl and NaOH. A 0.01% (wt / v) TNBS solution was prepared by diluting the TNBS stock 500-fold. Gel-SH and cold-water fish skin gelatin were then dissolved at 10 mg / mL in 0.1 M NaHCO3 buffer. 250 μL of each solution was diluted to 500 μg / mL using 0.1 M NaHCO3 buffer. Two-fold dilution series of Gel-SH and cold-water fish skin gelatin were prepared at concentrations ranging from 0 μg / mL to 500 μg / mL. A standard dilution series of L-cysteine ​​was prepared from 0.5 mM to 0.156 mM. 200 μL of each sample and standard dilution was added in triplicate to a clear 96-well plate (Costar™ from Corning™), and 100 μL of 0.01% (wt / v) TNBS solution was added. The samples were then mixed on a plate shaker for 5 minutes in the dark. The samples were then transferred to a 37°C laboratory oven and incubated for 2 hours in the dark. The absorbance of the well plates was read at 335 nm using a CLARIOstar™ spectrophotometer. The amine content of the samples was determined by comparing the absorbance of the samples to that of an L-cysteine ​​standard curve.

[0158] Figure 2A shows that the amine content of cold-water-adapted fish gelatin aminated with ethylenediamine (830.17 μmol / g) increased nearly fourfold compared to native (non-aminated) fish gelatin (215.91 μmol / g). The figure further demonstrates that the amine content of this aminated gelatin decreased (401.21 μmol / g) upon treatment with Traut's reagent. The amine content of native gelatin also decreased (97.40 μmol / g) upon treatment with Traut's reagent, and the amine content of native gelatin treated with EDC / NHS coupling of L-cysteine ​​was comparable to that of native gelatin (205.03 μmol / g).

[0159] Quantification of thiol content in gelatin and gelatin derivatives. The thiol content of native and thiolated cold-water-adapted fish gelatin (Gel-SH) was quantified by the 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) assay, as previously described by Deng et al. A two-fold L-cysteine ​​standard dilution series was prepared from 0 mM to 2 mM. Gel-SH and cold-water fish skin gelatin were dissolved in PBE at 5 mg / mL. Two-fold dilution series of samples were prepared at concentrations ranging from 500 μg / mL to 125 μg / mL. Then, 25 μL of each sample and standard was added in triplicate to a clear 96-well plate (Costar® from Corning®). Then, 125 μL of DTNB solution was added to each sample. The well plate was then shaken on a plate shaker and incubated at room temperature, protected from light, for 15 minutes. After incubation, the absorbance of the well plates was measured at 412 nm using a CLARIOstar™ well plate reader. The thiol content of Gel-SH was determined by comparing the absorbance of the samples with that of an L-cysteine ​​standard curve.

[0160] Figure 2B shows that natural cold-water-adapted fish gelatin showed increased thiol content (30.31 μmol / g) when reacted with Traut's reagent compared to natural gelatin (near baseline). The thiol content was further increased (497.41 μmol / g) in aminated (using ethylenediamine) natural cold-water-adapted fish gelatin treated with Traut's reagent. Natural gelatin treated with EDC / NHS coupling of L-cysteine ​​also showed increased thiol content (198.44 μmol / g) compared to natural gelatin.

[0161] 1 H-NMR Proton nuclear magnetic resonance ( 1H-NMR was performed to characterize the molecular profile of Gel-SH. Gel-SH and cold-water fish skin gelatin were dissolved in 90% HO / 10% DO to a final concentration of 1% (wt / v). 1 mL of each sample was added to its respective NMR tube, and sample spectra were collected with water suppression using a Bruker Avance 600 MHz NMR instrument. Sample spectra were analyzed using Bruker TopSpin 3.6.4. A comparison spectrum is shown in Figure 2C.

[0162] Rheology The rheological properties of thiolated cold-water fish skin gelatin (Gel-SH) and pig skin gelatin (Type A, 300 Bloom, Sigma-Aldrich) were determined using an Anton-Paar modular compact rheometer (MCR) 302. Shear rate sweeps were performed at 25 °C using a 25 mm cone-plate (CP25) with a constant frequency of 1 Hz over the shear rate range of 0.1 / s to 1000 / s. Temperature sweeps were performed using a 25 mm parallel plate (PP25) with a constant frequency of 1 Hz and a constant strain of 1%, linearly varying the temperature from 40 °C to 0 °C at a rate of 2 °C / min.

[0163] Figure 3 demonstrates that the thiolated cold water fish skin gelatin solution has a lower viscosity and less temperature dependence compared to solutions of mammalian (porcine) gelatin, allowing for easier liquid handling and improved volume reproducibility.

[0164] Example 4 Crosslinking of modified gelatin by click chemistry: Preparation of Gel-SH / PEG-4MAL hydrogels Gel-SH and PEG-4MAL (molecular weight 20 kDa, JenKem™) were dissolved in 300 mM, 200 mM, or 100 mM (4-2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Gibco™, lot number 2185833) at 20%, 10%, or 5% (wt / v), respectively. To determine the effect of HEPES buffer concentration on the pH of the hydrogel precursor and the hydrogel crosslinking time, Gel-SH, PEG-4MAL, and Gel-SH / PEG-4MAL hydrogels were prepared using various concentrations of HEPES buffer. Figure 5B shows the pH of hydrogel precursor solutions (PEG-4MAL and Gel-SH) prepared at concentrations ranging from 5% to 20% (wt / v) in 100 mM, 200 mM, or 300 mM HEPES, respectively. The precursor solutions were mixed in equimolar amounts (based on the number of thiol groups in Gel-SH and the number of maleimide groups in the crosslinker PEG-4MAL) under ambient light conditions to produce crosslinked modified gelatin. The pH of each hydrogel precursor and hydrogel was determined using test strips. To prepare Gel-SH / PEG-4MAL hydrogels with a final Gel-SH concentration of 10% (wt / v), a 20% (wt / v) Gel-SH precursor solution and a 20% (wt / v) PEG-4MAL precursor solution were mixed in equimolar amounts (based on the number of thiol groups in Gel-SH and the number of maleimide groups in PEG-4MAL) in a 96-well plate (Corning™). The reaction scheme is shown in Figure 5A. The crosslinking time was determined by pipette mixing and was defined as the time when the hydrogel solution could no longer be pipetted. Figure 5C shows the crosslinking time after mixing of hydrogel precursor solutions (PEG-4MAL and Gel-SH) prepared with final Gel-SH concentrations ranging from 2.5% to 10% (wt / v) in either 100 mM, 200 mM, or 300 mM HEPES, respectively. The 300 mM HEPES allowed the pH of the combined hydrogel precursor solution (Gel-SH / PEG-4MAL) to be maintained at 7–7.5, a pH range suitable for highly stereoselective click chemistry reactions and compatible with cells.

[0165] Mechanical testing Gel-SH / PEG-4MAL hydrogels containing equimolar amounts of PEG-4MAL with final Gel-SH concentrations of 10%, 5%, and 2.5% (wt / v), respectively, were prepared in 300 mM HEPES buffer and swollen overnight in phosphate-buffered saline (PBS) at 37°C. Prior to compression testing, the hydrogels were imaged and their surface areas determined using a Nikon™ SMZ25 stereomicroscope. The hydrogels were then immersed in a 37°C water bath filled with PBS and compressed in an unconstrained configuration at a strain rate of 0.01 mm / s using an Instron 5567 (Instron) equipped with a 5 N load cell and a nonporous aluminum indenter. The compressive Young's modulus, E, of the hydrogels was determined as the slope of the stress-strain curve at strains between 0.1 mm / mm and 0.15 mm / mm, as described by Kahl et al.

[0166] Figure 4A shows representative stress-strain curves, and Figure 4B shows the Young's modulus obtained for hydrogels with final Gel-SH concentrations ranging from 2.5% to 10% (w / v), demonstrating that the mechanical properties of the crosslinked hydrogels increase in a concentration-dependent manner.

[0167] Equilibrium swelling ratio and mass swelling ratio Hydrogels containing 10% (wt / v), 5% (wt / v), and 2.5% (wt / v) Gel-SH with equimolar PEG-4MAL concentrations were prepared using 300 mM HEPES buffer and weighed immediately after crosslinking. The hydrogels were then allowed to swell overnight in PBS at 37°C. After swelling, the hydrogels were reweighed and subsequently lyophilized. The recorded weights of the hydrogels were used to calculate the following equation:

number

[0168] Equilibrium mass swelling ratio (Q m ) is expressed as:

number

[0169] Relaxation mass swelling ratio (Q mr ) is expressed as:

number

[0170] Relaxation mass swelling ratio (Q mr ) represents the relationship between the weight of the dry hydrogel and the weight of the hydrogel immediately after crosslinking. m ) represents the relationship between the weight of a dry hydrogel and the weight of the hydrogel after swelling. Equilibrium water content (EWC) represents the water-holding capacity of a hydrogel when the osmotic and ionic pressures of the solution outside the hydrogel matrix are in equilibrium with the pressures in the hydrogel matrix.

[0171] Figure 6A shows a negative correlation between hydrogel concentration and the relaxed mass swelling ratio, while Figures 6B and 6C demonstrate that the equilibrium swelling ratio and equilibrium water content, respectively, are not affected by hydrogel concentration within the range tested.

[0172] Example 5 Cell encapsulation in Gel-SH / PEG-4MAL hydrogels Stock solutions of 20% (wt / v), 10% (wt / v), and 5% (wt / v) Gel-SH and PEG-4MAL were prepared in 300 mM HEPES buffer. Cells were harvested using 0.25% trypsin / ethylenediaminetetraacetic acid (EDTA) and counted. MCF-7 breast cancer cells were cultured at 2 × 10 6The cells were resuspended in Gel-SH at a concentration of 1000 cells / mL. A 10 μL volume of PEG-4MAL solution was added to a 48-well plate (Corning™), and then an amount of the Gel-SH cell suspension resulting in an equimolar amount of Gel-SH (based on the number of thiol groups in Gel-SH and the number of maleimide groups in the crosslinker PEG-4MAL) was mixed with the PEG-4MAL solution by pipetting until crosslinking occurred. After crosslinking, the cell-laden hydrogels were incubated at 37°C in a 5% CO2 humidified cell incubator in RPMI 1640 medium (Gibco™) supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (v / v) P / S, 1% (v / v) non-essential amino acids, 1% (v / v) sodium pyruvate, and 0.1% (v / v) insulin-transferrin-selenium (all ThermoFisher™).

[0173] Cell viability The viability of cells encapsulated in Gel-SH / PEG-4MAL hydrogels was determined using a fluorescein diacetate (FDA) (ThermoFisher™) / propidium iodide (PI) (ThermoFisher™) assay. The cell culture medium was aspirated, and the samples were washed with PBS at room temperature for 5 minutes, followed by incubation with staining solution (10 μg / mL FDA and 5 μg / mL PI in PBS) for 2 minutes. The staining solution was aspirated, and the samples were washed with PBS for 2 minutes. The samples were then transferred to glass slides and imaged using either a Leica SP5 confocal microscope or a Nikon SMZ25 epifluorescence microscope. Z-stacks of the hydrogels were acquired with a 10 μm slice interval, and maximum intensity projection images of the hydrogel Z-stacks were obtained using ImageJ. Cell viability was determined by quantification of particles in the live and dead channels of the maximum intensity projection images.

[0174] Figure 7A shows representative live / dead images for cell-encapsulated Gel-SH / PEG-4MAL hydrogels with final Gel-SH concentrations ranging from 2.5% to 10% (wt / v) and a photocrosslinkable gelatin methacryloyl (GelMA; 5% (w / v)) control hydrogel. Figure 7B shows that high cell viability was maintained over a 21-day culture period, demonstrating the cytocompatibility of Gel-SH-based hydrogels.

[0175] Nuclear and F-actin staining Nuclei and f-actin filaments of MCF-7 cells encapsulated in Gel-SH / PEG-4MAL hydrogels were stained using diamidino-2-phenylindole (DAPI) (ThermoFisher™ / Alexa-Fluor™ 488-conjugated phalloidin stain). On days 1, 7, 14, and 21, the medium was removed from the MCF-7 hydrogel wells. The hydrogels were then washed with 1 mL of PBS for 10 minutes at room temperature. The PBS was then aspirated, and the samples were fixed with 1 mL of 4% (wt / v) paraformaldehyde (PFA) for 1 hour. After fixation, the PFA was aspirated, and the hydrogels were washed with 1 mL of PBS. The PBS for the washes was aspirated, and an additional 1 mL aliquot of PBS was added to the hydrogels. The hydrogels were then stored at 4°C. The PBS was aspirated from the wells, and the hydrogels were blocked with 300 μL of blocking buffer (5% (v / v) goat serum (Gibco™), 0.1% (v / v) Triton X-100 (Sigma-Aldrich™)) per hydrogel on a plate shaker overnight at 4°C. The blocking buffer was then aspirated, and the hydrogels were washed twice with PBS at RT for 5 minutes per wash. The PBS was aspirated, and 150 μL of 1:1000 DAPI, 1:200 phalloidin in PBS was added to each hydrogel. The hydrogels were incubated overnight at 4°C on a plate shaker. After incubation, the staining solution was aspirated, and the hydrogels were washed three times with 300 μL of wash buffer (20% (v / v) blocking buffer, 1% (v / v) goat serum) per hydrogel on a plate shaker for 8 hours at 4°C. After washing, the wash buffer was aspirated, and the hydrogels were washed three times with PBS and stored at 4°C until imaging. Hydrogels were imaged using a Leica SP5 confocal microscope with a 4x objective. The DAPI channel was acquired at an excitation wavelength of 405 nm, and the phalloidin channel was acquired at an excitation wavelength of 488 nm.

[0176] Figure 8 shows that Gel-SH / PEG-4MAL-based hydrogels with final Gel-SH concentrations ranging from 2.5% to 10% (wt / v) support the formation of multicellular spheroids similar to the GelMA control, which is considered the gold standard gelatin derivative for 3D cell culture applications.

[0177] Example 6 Crosslinking time of Gel-SH / PEG-4MAL hydrogel vs. Gel-SH / PEG-4VS hydrogel Hydrogels were formed by reacting Gel-SH with 4-arm PEG-maleimide (PEG-4MAL, MW 20 kDa, JenKem™) and Gel-SH with 4-arm PEG-vinylsulfone (PEG-4VS, MW 20 kDa, JenKem™), respectively, to assess the difference in crosslinking time between the two PEG-based activators, as outlined in Example 4.

[0178] For PEG-4MAL-based reactions, both Gel-SH and PEG-4MAL were dissolved in 300 mM HEPES buffer (Gibco™, lot number 2185833) at 20% (wt / v), respectively. For PEG-4VS-based reactions, both Gel-SH and PEG-4VS were dissolved in 300 mM HEPES containing 300 mM triethanolamine (TEA, Sigma-Aldrich). Solutions of Gel-SH and PEG-based activators were mixed in an equimolar ratio (based on the number of thiol groups in Gel-SH and the number of maleimide groups in the crosslinker PEG-4MAL or the number of vinyl sulfone groups in the crosslinker PEG-4VS) to produce hydrogels with final Gel-SH concentrations of 2.5%, 5%, and 10% (w / v), respectively. The crosslinking time at room temperature was determined as the time when the hydrogel precursor solution could no longer be drawn into a pipette due to gel formation. The results for Gel-SH cross-linked with PEG-4MAL are shown in FIG. 5C, and the results for Gel-SH cross-linked with PEG-4VS are shown in FIG.

[0179] Example 7 Cell encapsulation in Gel-SH / PEG-4MAL hydrogel versus Gel-SH / PEG-4VS hydrogel MCF-7 breast cancer cells were encapsulated in hydrogels of 10% (w / v) Gel-SH containing PEG-4MAL or PEG-4VS, respectively, prepared as outlined in Example 5, at a density of 1 million cells per mL of hydrogel precursor solution. On days 1 and 7 of culture, MCF-7 cell viability and proliferation were assessed using bright-field microscopy with a Nikon Eclipse Ts2 inverted microscope (FIG. 10A) and PrestoBlue™ Cell Viability Reagent (ThermoFisher Scientific) (FIG. 10B) according to the manufacturer's instructions. Briefly, individual cell-laden hydrogels were incubated with 900 μL of cell culture medium and 100 μL of PrestoBlue™ Cell Viability Reagent for 1 hour at 37°C and 5% CO in a humidified cell culture incubator (Binder CB 170). After the incubation period, samples (100 μL) of cell culture medium containing PrestoBlue™ Cell Viability Reagent were transferred to a Nunc™ MicroWell™ 96-well plate (ThermoFisher Scientific), and cellular metabolic activity was assessed using fluorescence measurements (excitation wavelength: 560 nm; emission wavelength: 590 nm) on a CLARIOstar™ Plus (BMG LabTech) plate reader and expressed in arbitrary units (AU).

[0180] In a second experiment, the effect of Gel-SH concentration was assessed: MCF-7 cells were encapsulated at 1 million cells per mL in hydrogels containing 5% or 10% (w / v) Gel-SH and an equimolar amount of PEG-4VS, as outlined in Example 5, and metabolic activity was assessed at days 1 and 7 of culture according to the methods outlined above (Figure 10C).

[0181] This data demonstrates that the Gel-SH materials of the present invention can be crosslinked using a variety of Michael addition / click-reactive moieties. Here, PEG-vinyl sulfone was shown to crosslink Gel-SH much more slowly than PEG-maleimide, allowing more time for liquid handling. The slower kinetics of PEG-vinyl sulfone are particularly useful for high-throughput liquid handling, where the cell-containing Gel-SH suspension can be premixed with the PEG-vinyl sulfone solution in a larger master volume and subsequently dispensed robotically. This further enhances the benefits of low-viscosity precursor solutions, which themselves are ideal for automated liquid handling. This contrasts with gels formed with PEG-MAL, which crosslink very rapidly and therefore must be used immediately and mixed / formed separately.

[0182] The above embodiments are merely preferred examples of the present disclosure. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present disclosure, and they shall fall within the protection scope of the present disclosure.

[0183] In the claims and the above detailed description of the invention, unless the context otherwise requires, either by express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in their inclusive sense, i.e., they are used to specify the presence of specified features but not to exclude the presence or addition of further features in various embodiments of the disclosure.

[0184] References 1. Deng et al., Development of Disulfide Bond Crosslinked Gelatin / ε-Polylysine Active Edible Film with Antibacterial and Antioxidant Activities, Food Bioprocess Technol., 13, 577-588. 2. Duggan et al., Synthesis of Mucoadhesive Thiolated Gelatin Ysing a Two-Step Reaction Process, European Journal of Pharmaceutics and Biopharmaceutics, 2015, 91, 75-81. 3. Kahl et al. MechAnalyze: An Algorithm for Standardization and Automation of Compression Test Analysis, Tissue Engineering Part C: Methods, 2021, 27. 4. Loessner et al, Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms, Nature Protocols, 2016, 11, 727-746. 5. Meinert et al., A Method for Prostate and Breast Cancer Cell Spheroid Cultures Using Gelatin Methacryloyl-Based Hydrogels, Methods in Molecular Biology, 2018, 1786, 175-194. 6. Nair et al., The Thiol-Michael Addition Click Reaction: A Powerful and Widely Used Tool in Materials Chemistry, Chem. Mater., 2014, 26, 1, 724-744. 7. Van Den Bulcke et al, Structural and rheological properties of methacrylamide modified gelatin hydrogels,Biomacromolecules, 2000, 1, 1, 31-38.

Claims

1. 1. A modified gelatin, wherein the gelatin is derived from a marine source and has been modified to incorporate crosslinkable groups that include reactive moieties, the reactive moieties being reactive in click chemistry reactions.

2. 2. The modified gelatin according to claim 1, wherein the number of proline residues is 20% or less of the total number of amino acid residues in said modified gelatin.

3. 3. The modified gelatin according to claim 1, wherein the number of hydroxyproline residues is 20% or less of the total number of amino acid residues in the modified gelatin.

4. a) the content of the reactive moiety in the modified gelatin is about 25 μmol / g to about 1000 μmol / g; b) the content of the reactive moiety in the modified gelatin is about 300 μmol / g to about 700 μmol / g; c) an aqueous solution of the modified gelatin at 20% (wt / v) or less has a viscosity of less than 100,000 mPa·s over a shear rate range of 10 to 1,000 (1 / s); and d) An aqueous solution of the modified gelatin at 20% (wt / v) or less is heated for 10 minutes over a temperature range of 0°C to 40°C. ８ having a complex viscosity of less than mPa s; 4. The modified gelatin according to claim 1, which can be characterized by one or more of the following:

5. The modified gelatin according to any one of claims 1 to 4, wherein the gelatin is a gelatin from cold-water adapted fish.

6. 6. The modified gelatin of claim 5, wherein the cold-water adapted fish is of a genus selected from the group consisting of Salmo, Gadus, Oncorhynchus and Merlucius, preferably Salmo or Oncorhynchus.

7. The modified gelatin according to any one of claims 1 to 6, wherein the reactive moieties are selected from the group consisting of alkynes, amines, alkenes, conjugated dienes, thiols, isonitriles and tetrazines.

8. 8. The modified gelatin of claim 7, wherein the reactive moieties are selected from the group consisting of amines, thiols, alkenes, conjugated dienes, azides and alkynes.

9. The modified gelatin of claim 8, wherein the reactive moiety is a thiol.

10. The gelatin is i) reaction of gelatin amine groups with Traut's reagent; ii) reaction of gelatin carboxylic acid groups with diamines; iii) reaction of gelatin carboxylic acid groups with a diamine and subsequent reaction with Traut's reagent; and iv) amide coupling of gelatin carboxylic acids or carboxylates with additional cysteines; 10. The modified gelatin of claim 9, which is modified by a reaction selected from the group consisting of:

11. A process for producing the modified gelatin according to any one of claims 1 to 10, comprising reacting gelatin derived from a marine resource with a crosslinkable group precursor.

12. The gelatin derived from the marine resource, i) reaction of gelatin amine groups with a crosslinkable group precursor, which is Traut's reagent; ii) reaction of gelatin carboxylic acid groups with a crosslinkable group precursor which is a diamine; iii) reaction of gelatin carboxylic acid groups with a diamine and subsequent reaction with Traut's reagent; and iv) amide coupling of gelatin carboxylic acids or carboxylates with a crosslinkable group precursor that is cysteine; The process of claim 11 , wherein the crosslinkable group precursor is reacted with the crosslinkable group precursor by

13. A modified gelatin produced or producible by the process according to any one of claims 10 to 12.

14. 14. A crosslinked modified gelatin produced by reacting the modified gelatin according to any one of claims 1 to 10 or 13 with a crosslinker comprising two or more further reactive moieties in a click chemistry reaction, wherein said reactive moieties are reactive with the reactive moieties of the crosslinkable groups incorporated into said modified gelatin in a click chemistry reaction.

15. 15. The cross-linked modified gelatin of claim 14, wherein the further reactive moiety of the cross-linker is selected from the group consisting of alkenes, alkynes and thiols.

16. 16. The crosslinked modified gelatin of claim 15, wherein the further reactive moiety of the crosslinker is selected from the group consisting of maleimides, vinyl sulfones, acrylates, acrylamides and methacrylates.

17. The crosslinker has the formula: Core-(Spacer-R) ｎ 17. The crosslinked modified gelatin according to any one of claims 14 to 16, having the formula: (wherein Core is an atom or group that provides bonds to n Spacer-R groups, Spacer is a spacer group, R is a group that contains a further reactive moiety, and n is an integer from 2 to 8).

18. Core is C(-CH ２ O-) ４ n is 4, and Spacer is (-CH ２ CH ２ O-) ｍ where m is an integer from 2 to 150, and R is 【Chemistry 1】 18. The cross-linked modified gelatin according to claim 17, wherein

19. Core is C(-CH ２ O-) ４ n is 4, and Spacer is (-CH ２ CH ２ O-) ｍ where m is an integer from 2 to 50, and R is 【Chemistry 2】 19. The cross-linked modified gelatin according to claim 17 or 18,

20. Core is C(-CH ２ O-) ４ n is 4, and Spacer is (-CH ２ CH ２ O-) ｍ where m is an integer from 2 to 150, and R is 【Transformation 3】 18. The cross-linked modified gelatin according to claim 17, wherein

21. 21. A process for producing a crosslinked modified gelatin according to any one of claims 14 to 20, comprising reacting a modified gelatin according to any one of claims 1 to 10 or 13 with a crosslinker comprising two or more further reactive moieties in a click chemistry reaction, said reactive moieties being reactive with the reactive moieties of the crosslinkable groups incorporated into said modified gelatin in a click chemistry reaction.

22. 22. The process of claim 21 carried out under ambient light conditions.

23. 23. A cross-linked modified gelatin produced or preparable by the process of claim 21 or 22.

24. A hydrogel comprising the crosslinked modified gelatin according to any one of claims 14 to 20 and water.

25. a) 2.5% (wt / v) or greater of the hydrogel has a relaxed mass swelling ratio of 15 or less; b) 2.5% to 10% (wt / v) hydrogels have an equilibrium swelling ratio of 10 to 25; and c) 2.5% to 10% (wt / v) hydrogels have an equilibrium water content of 80% to 100%; 25. The hydrogel of claim 24, which can be characterized by one or more of:

26. a) 2.5% (wt / v) or greater of the hydrogel has a relaxed mass swelling ratio of 15 or less; b) 2.5% to 10% (wt / v) hydrogels have an equilibrium swelling ratio of 10 to 25; c) 2.5% to 10% (wt / v) hydrogels have an equilibrium water content of 80% to 100%; d) during preparation, the 2.5% to 10% (wt / v) hydrogel has a crosslinking time of 1 to 10 seconds at room temperature; and e) During preparation, the 2.5% to 10% (wt / v) hydrogel has a crosslinking time of 10 to 30 minutes at room temperature; 25. The hydrogel of claim 24, which can be characterized by one or more of:

27. A method for producing a hydrogel, comprising mixing the cross-linked modified gelatin according to any one of claims 14 to 20 with water.

28. 23. A method for producing a hydrogel, comprising carrying out the process for producing cross-linked modified gelatin according to claim 21 or 22 in the presence of water.

29. 29. A hydrogel produced or producible by the method or process of claim 27 or 28.

30. 30. Use of the hydrogel according to any one of claims 25 to 27 and 29 as a matrix for cell growth or for 3D bioprinting.

31. 1. A method for growing cells, comprising: Providing a cell growth matrix comprising the hydrogel of any one of claims 25 to 27 and 29; Growing cells in and / or on said cell growth matrix; A method comprising:

32. A kit for producing cross-linked modified gelatin, comprising: a) a modified gelatin according to any one of claims 1 to 10 and 13; b) a crosslinking agent according to any one of claims 14 to 20; Includes a kit.

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