Method for functionalizing biopolymers and crosslinking thereof

Abstract

The present invention relates to a method for functionalizing a biopolymer having an -OH group or an -NH2 group by reacting the biopolymer with at least one compound having at least one C=C bond or C≡C bond and containing a chromophore that absorbs UV-Vis radiation directly adjacent to this bond. The present invention also relates to a method for obtaining a solid biopolymer material by reversibly crosslinking the functionalized biopolymer obtained by the above method under UV-Vis light irradiation. Furthermore, the present invention relates to the use of functionalized biopolymers as support materials for 3D bioprinting, or as materials for manufacturing structures selected from spheroids, organoids, artificial organs, coatings, and tissue models.

Description

[Technical Field]

[0001] The present invention relates to a method for functionalizing a biopolymer, comprising reacting a biopolymer having an -OH group or an -NH2 group with at least one compound having at least one C=C bond or C≡C bond and a chromophore group that absorbs UV-visible light directly adjacent to the bond. The present invention also relates to a method for obtaining a solid biopolymer material, wherein the functionalized biopolymer obtained by the method is reversibly crosslinked under UV-visible light irradiation. The present invention also relates to using the functionalized biopolymer as a support material for 3D bioprinting, and to using the functionalized biopolymer for the manufacture of structures selected from spheroids, organoids, artificial organs, coatings, and tissue models. The present invention is used in tissue engineering, transplantation, medical, and pharmacological research. [Background technology]

[0002] The crosslinking reaction of natural polymers utilizes compounds that can participate in cycloaddition reactions (Pella MCG, Lima-Tenorio MK, Tenorio-Neto ET, Guilherme MR, Muniz EC, Rubira AF. Chitosan-based hydrogels: From preparation to biomedical applications. Carbohydr Polym. 2018, 196, 233-245, doi: 10.1016 / j.carbpol.2018.05.033; Eva Mueller, Isabelle Poulin, William James Bodnaryk, and Todd Hoare, Click Chemistry Hydrogels for Extrusion Bioprinting: Progress, Challenges, and Opportunities, Biomacromolecules 2022 23 (3), 619-640, doi: 10.1021 / acs.biomac.1c01105; Kerim M. Gattas-Asfura, Eric Weisman, Fotios M. Andreopoulos, Miodrag Micic, Bill Muller, Sanjeev Sirpal, Si M. Pham, and Roger M. Leblanc, Nitrocinnamate-Functionalized Gelatin: Synthesis and “Smart” Hydrogel Formation via Photo-Cross-Linking, Biomacromolecules 2005 6 (3), 1503-1509, doi: 10.1021 / bm049238w; Koshy ST, Desai RM, Joly P, Li J, Bagrodia RK, Lewin SA, Joshi NS, Mooney DJ. Click-Crosslinked Injectable Gelatin Hydrogels. Adv Healthc Mater. 2016, 5(5), 541-7, doi: 10.1002 / adhm.201500757; Jasper Van Hoorick, Liesbeth Tytgat, Agnes Dobos, Heidi Ottevaere, Jurgen Van Erps, Hugo Thienpont, Aleksandr Ovsianikov, Peter Dubruel, Sandra Van Vlierberghe, (Photo-) crosslinkable gelatin derivatives for biofabrication applications, Acta Biomaterialia 2019, 97, 46-73, doi: 10.1016 / j.actbio.2019.07.035). .

[0003] However, there remains a need to provide new methods for functionalizing biopolymers. Such methods would allow crosslinking solely by light irradiation (during 3D bioprinting or other techniques for forming solid biopolymer structures, such as casting), eliminating the need for crosslinking initiators such as photoinitiators, metal salts, transition metal complexes, and high-valent iodine compounds, which are undesirable contaminants in the final structure. Particularly desirable is the elimination of photoinitiators, which have the greatest cytotoxic effect on cells that may be components of the biopolymer material to be crosslinked.

[0004] Furthermore, there is always a need to provide novel functionalized biopolymers that can be crosslinked by irradiation with higher wavelengths (i.e., lower energy) of light. This is not only to reduce the energy consumption of the crosslinking process, but above all to suppress the adverse effects of ultraviolet light on the viability of cells contained in the biopolymer material to be crosslinked. Moreover, there is always a need to provide novel functionalized biopolymers that can be reversibly crosslinked by light irradiation, which can significantly facilitate modification of the formed biopolymer structure or its more detailed analysis in certain applications. [Overview of the project]

[0005] The subject of the present invention is a method for the functionalization of biopolymers, which comprises reacting a biopolymer having -OH groups or -NH2 groups with at least one compound having at least one C=C bond or C≡C bond and having a chromophore group that absorbs UV-visible light in the immediate vicinity of said bond. "In the immediate vicinity" in this context means that the chromophore group is bonded to a carbon atom directly adjacent to the carbon atom having the multiple bond. Preferably, the compound containing at least one C=C bond or C≡C bond is a carboxylic acid derivative, more preferably selected from active esters, acid anhydrides, and acid chlorides. Preferably, the carboxylic acid derivative is selected from coumarin-3-carboxylic acid derivatives, exo-5-norbornene-carboxylic acid derivatives, and trans-cinnamic acid derivatives. Preferably, the active ester is an N-hydroxysuccinimidyl ester obtained by reacting a carboxylic acid containing at least one C=C bond or C≡C bond with N-hydroxysuccinimide in the presence of a coupling agent, and the coupling agent is preferably selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC).

[0006] Preferably, the biopolymer is selected from proteins and polysaccharides. In a preferred embodiment of the present invention, the biopolymer is selected from gelatin, hyaluronic acid, alginic acid, chitosan, dextran, starch, cellulose, collagen, chitin, carrageenan, inulin, glycogen, and heparin, preferably selected from gelatin, chitosan, and hyaluronic acid, and most preferably gelatin.

[0007] The subject of the present invention is also a method for obtaining a solid biopolymer material, which is characterized by reversibly crosslinking the functionalized biopolymer obtained by the above method under irradiation with UV-visible light, preferably in the range of light having a wavelength of 280 to 800 nm, and most preferably by irradiation with a wavelength selected from 365 nm and 405 nm to reversibly crosslink.

[0008] The subject of the present invention is also the use as a support material for 3D bioprinting of the functionalized biopolymer obtained by the above method.

[0009] The subject of the present invention is also the use of the functionalized biopolymer obtained by the above method for the production of structures selected from spheroids, organoids, artificial organs, coatings, and tissue models.

[0010] The functionalized biopolymer obtained by using the method according to the present invention enables the obtaining of a completely crosslinkable solution in a concentration range of 0.5% to 50% without adding photoinitiators such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959). Functionalized biopolymers such as gelatin, hyaluronic acid, alginic acid, and other polymers of natural or semi-synthetic origin, after being functionalized according to the method of the present invention, have a molecular weight of 1,000 kDa to 500,000 kDa, a degree of substitution of 10% to 100%, and the substituent contains at least one C=C bond or C≡C bond and has a chromophore group that absorbs UV-visible light directly adjacent to the bond.

[0011] The functionalized biopolymer obtained by the method according to the present invention retains complete solubility in an aqueous environment (in a volume range of 0.1 to 1000 mL), and is the same in an environment containing physiological saline solution or cell culture medium with a pH of 4 to 8, or other environments according to the application.

[0012] The functionalized biopolymers obtained by the method according to the present invention are suitable for extrusion, volumetric printing, and other 3D printing technologies that require the use of materials with distinct gel points and appropriate viscosity in order to obtain uniform and consistent fibers and maintain printing resolution. The printing temperature ranges from 5°C to 50°C depending on the concentration of the prepolymer solution used, in which case the prepolymer is understood to mean the functionalized biopolymer according to the present invention before the crosslinking process is initiated. The printing pressure in extrusion printing is typically in the range of 5 to 150 kPa. The printing speed is typically in the range of 1 to 100 mm / second. The needle diameter is typically in the range of 50 to 900 μm. The printed model can include any dimensions expressed in millimeters, and the infill rate ranges from 5% to 100%, thereby making it possible to obtain both solid prints and watermarked prints.

[0013] The functionalized biopolymer obtained by the method according to the present invention can be exposed to light with wavelengths of 280 nm to 800 nm at a rate of 1 mW / cm² without the presence of a photoinitiator. 2 ~1000mW / cm 2 Complete cross-linking occurs when irradiated with light at a light intensity of 10 to 720 seconds.

[0014] The functionalized biopolymers obtained by the method according to the present invention are well-suited for 3D culture, tissue engineering, and other applications using living cells in which UV-visible light acts as a crosslinking factor. This technology does not require the use of external initiators that may potentially have cytotoxic effects. The functionalized biopolymers obtained by the method according to the present invention can be used as a single material in bio-inks or as an additive to mixtures with other printable materials. This means that in the 3D bioprinting process in which the functionalized biopolymers are crosslinked, crosslinking reactions can occur not only between molecules of one type of functionalized biopolymer, but also between molecules of two or more different types of functionalized biopolymers. Biopolymers or mixtures thereof functionalized with various compounds can constitute 0.1% to 99.9% of the overall blend.

[0015] The functionalized biopolymers or blends thereof according to the present invention can be used in early-stage biological studies using the reference cell line L929, or in studies using alternative cell lines compliant with ISO 10993-5, provided that identical or similar MTT test results are obtained, namely CCL 1 (NCTC clone 929), CCL 163 (Balb / 3T3 clone A31), CCL 171 (MRC-5), CCL 75 (WI-38), CCL 81 (Vero), and CCL 10 [BHK-21 (C-13) and V-79 379A], and can also be used in subsequent-stage studies using specific cell lines depending on the application. The functionalized biopolymers, blends thereof, and materials with added cell lines according to the present invention can be homogenized using syringe mixing or a dedicated cell mixer specialized for biological applications. [Brief explanation of the drawing]

[0016] One embodiment of the present invention is shown in the accompanying drawings as follows, without limiting the scope of the present invention. Figure 1 shows the crosslinking process of gelatin functionalized with coumarin-3-carboxylic acid, and the material obtained in Example 1. 1 The 1H NMR spectrum is shown. Figure 2a shows the exo-5-norbornenecarboxylic acid 1 The 1H NMR spectrum is shown. Figure 2b shows the crosslinking process of a mixture of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid and a 15% solution of gelatin functionalized with exo-5-norbornenecarboxylic acid, as obtained in Example 2. 1 The 1H NMR spectrum is shown. Figure 3a shows trans-cinnamic acid 1 The 1H NMR spectrum is shown. Figure 3b shows the crosslinking process of a mixture of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid and a 10% solution of gelatin functionalized with trans-cinnamic acid, as obtained in Example 3. 1 The 1H NMR spectrum is shown. Figure 3c shows the 1 1H NMR spectrum of the material obtained in Example 3 during the crosslinking process of a mixture of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid and a 15% solution of gelatin functionalized with trans-cinnamic acid. Figure 4 shows photographs of flaky scaffolds printed with different crosslinking times. Figure 5 shows the 1 1H NMR spectrum of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid (GelCM). Figure 6 shows the 1 1H NMR spectrum of a 12.5% GelCM solution. Figure 7 shows the 1 1H NMR spectrum of a 10% GelCM solution. Figure 8 shows a schematic diagram of the g.code file [template.gcode]. Figure 9 shows a schematic diagram of the fiber bending test platform. Figure 10 shows a microscopic image of the structure in the fiber bonding test. Figure 11 shows the ratio of the fiber diffusion rate Dfr in the fiber bonding test. Figure 12 shows the printability Pr in the fiber bonding test. Figure 13 shows the results of the alamar blue test for cells printed in structures of GelCM 10%, 12.5% and 15% and methacrylated gelatin (GelMA) 10%. Figure 14 shows a microscopic image of the printed structure containing L929 cells immediately after the printing process. Figure 15 shows a microscopic image of the printed structure containing L929 cells on the 3rd day of the experiment. Figure 16 shows a microscopic image of the printed structure containing L929 cells on the 7th day of the experiment. Figure 17 shows photographs of the biomaterial containing L-929 cells after transferring it to a 6-well plate containing supplemented medium, where A is GelMA 10%, GelCM 12.5%, GelCM 15%, and B is GelCM 10%. Figure 18 shows the percentage of lactate dehydrogenase (LDH) release from L-929 cells at days 1, 5, 7, and 14 as a result of interaction with the tested biomaterial. Figure 19 shows micrographs of cell cultures of the L-929 cell line exposed to the biomaterials GelCM 10%, GelCM 12.5%, GelCM 15%, and GelMA 10% after 14 days of incubation. [Modes for carrying out the invention]

[0017] The present invention relates to a method for functionalizing a biopolymer having an -OH group or an -NH2 group, by reacting it with at least one compound having at least one C=C bond or C≡C bond (which may be activated or deactivated), i.e., a bond capable of undergoing a 2+2 cycloaddition reaction, and a chromophore group that absorbs UV-visible light.

[0018] The primary objective of the method according to the present invention is to obtain appropriately functionalized derivatives of natural polymers (hereinafter referred to and illustrated as Helix / P) that can be used in tissue engineering, particularly for 3D bioprinting, classical and 3D cell culture, formation of spheroids / organoids and artificial (bionic) organs, formation of material coatings, and printing of specific masses of artificial organs using specific tissue models (normal and cancerous tissues) and cell lines, including blood vessels, microorgans (including pancreatic islets), organoids, spheroids, and other three-dimensional cellular structures.

[0019] The proposed technology and the materials used in its implementation enable the crosslinking or curing of materials, primarily soft polymers, using UV-visible light without the use of crosslinking initiators such as photoinitiators, metal salts, transition metal complexes, and high-valent iodine compounds. Crosslinking of the said materials, i.e., the functionalized biopolymers obtained by the method according to the present invention, occurs via a [2+2] cycloaddition reaction of compounds containing carbon-carbon multiple bonds and activated double or triple bonds, which may be used as an independent source of copolymer or constitute an integral part of the polymer within the same molecule. A schematic diagram illustrating possible pathways for the functionalization and subsequent crosslinking processes of biopolymers is shown below. The presented pathways are examples, and crosslinking by the materials used may occur via any pathway that can arise based on the underlying chemical reaction mechanism of the proposed polymer crosslinking method.

[0020] A key feature of this method is the ability to use UV-visible light in the 280-800 nm wavelength range, depending on the structure and absorption characteristics of the chromophore substituent located on the multiple bond undergoing 2+2 cycloaddition. Shifting the wavelength range to longer wavelengths (lower energy) not only has the practical benefit of reducing energy consumption in the process, but also has a significant impact on the use of materials produced by this technology in the broad field of tissue engineering. Radiation close to the visible light range has a remarkably small impact on damaging cells that may constitute part of the cured material.

[0021] The functionalization method for biopolymers according to the present invention uses a compound containing at least one C=C bond or C≡C bond, i.e., a bond capable of undergoing a 2+2 cycloaddition reaction. Examples of such compounds include compounds having a coumarin skeleton in their structure, such as coumarin-3-carboxylic acid and coumarin-6-carboxylic acid. These compounds undergo a [2+2] cycloaddition reaction upon irradiation with light of an appropriate wavelength.

[0022] A schematic diagram of the process for obtaining the functionalized biopolymer according to the present invention and subsequent crosslinking is shown below. (i) Functionalization of biopolymers with compounds containing carbon-carbon multiple bonds [ka] (ii) Reversible crosslinking of functionalized biopolymers - in the case of variant (a) [ka] Here, Helix=P represents a biopolymer selected from oligopeptides, proteins, and polysaccharides, specifically including gelatin, hyaluronic acid, alginic acid, chitosan, dextran, starch, cellulose, collagen, chitin, carrageenan, inulin, glycogen, and heparin. X is either -OH or -NH2. R1, R2, R3, R4, R5, and R6 each independently contain hydrogen, C1-C 30 Alkyl, C2-C 30 Alkenyl, C2-C 30 Alkinyl, C6-C 10 C5-C containing aryl or 1-3 heteroatoms selected from N, S, and O 10 It is a heteroaryl compound.

[0023] Crosslinking in step (ii) is carried out using light with a wavelength of 280 to 800 nm, with preferred wavelengths being 365 nm or 405 nm. The irradiation time is 1 to 6000 seconds, preferably 10 to 360 seconds. The output of the light source used is 1 to 3000 mW / cm². 2 That is the case.

[0024] The key advantages of the biopolymer functionalization according to the present invention, as shown in (i) of the schematic diagram above, are that a hydrogel based on a properly functionalized polymer soluble in water or a buffer can be crosslinked without the use of an initiator, and that the cyclization reaction can be carried out reversibly at a appropriately selected wavelength, as shown in (ii) of the schematic diagram above.

[0025] Gelatin is one of the preferred biopolymers in the functionalization using the method according to the present invention. It is a natural polymer composed of protein chains. Gelatin's structure contains amino acids such as lysine, hydroxylysine, proline, and hydroxyproline, and the amino groups and hydroxyl residues of these amino acids play an important role in the functionalization reaction because they are involved in the reaction shown in the schematic diagram below. [ka]

[0026] dECM (Decellularized Extracellular Matrix): Based on the gelatin content in dECM, it is presumed that the cross-linking mechanism of dECM is similar to that of gelatin. R is synonymous with R1 as defined above. NHS: N-hydroxysuccinimide EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0027] Another preferred biopolymer in the functionalization according to the present invention is chitosan. This polysaccharide is a derivative of chitin produced in the deacetylation step. Chitosan is composed of β-glucosamine molecules linked by β-1,4-glycosidic bonds. The functionalization reaction of chitosan is carried out in which free hydroxyl groups present in the deacetylated β-glucosamine units are involved. The functionalization of chitosan is preferably carried out using an active ester of a suitable carboxylic acid, as is the case with the functionalization of gelatin and dECM, as shown in the schematic diagram below. [ka]

[0028] In the functionalization using the method according to the present invention, another preferred biopolymer is hyaluronic acid. This polysaccharide belonging to the glycosaminoglycan group is composed of D-glucuronic acid and N-acetyl-D-glucosamine units, which are linked by β-1,4-glycosidic bonds and β-1,3-glycosidic bonds. Functionalization of hyaluronic acid is carried out using the coupling reagent DMTMM [4-(4,6-dimethoxy[1,3,5]triazine-2-yl)-4-methylmorpholinium chloride]. DMTMM is obtained by reacting CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine) with morpholine. Functionalization of biopolymers can be carried out by two methods. The first method involves activating the carboxyl group of the D-glucuronic acid unit, and then reacting the activated carboxyl group with a compound containing an amino group or a hydroxyl group, as shown in the schematic diagram below. [ka] X: As defined above

[0029] A second method for functionalizing hyaluronic acid involves activating the carboxyl group of an arbitrary acid with DMTMM, and then reacting the activated acid with the hydroxyl group of an N-acetyl-D-glucosamine unit, as shown in the schematic diagram below. [ka]

[0030] As described above, one preferred example of the biopolymer functionalization method according to the present invention is a two-step reaction using a carboxylic acid derivative constituting an active ester. For this purpose, the carboxylic acid is reacted with NHS and a condensing agent such as EDC, DCC, or DIC. The active N-hydroxysuccinimidyl ester of the carboxylic acid obtained in this way is reacted with the biopolymer. The active ester reacts with free amino groups and hydroxyl groups present in the structure of the natural polymer, thereby obtaining a properly functionalized biopolymer. Instead of the active ester, an acid anhydride or acid chloride of a selected carboxylic acid can be used.

[0031] By controlling pH, temperature, concentration, and substrate mass, the degree of substitution of the derivative can be controlled within a range of 20-100%. The degree of substitution affects the mechanical properties of the resulting material. [Examples]

[0032] Example 1: Material Synthesis Step 1: Synthesis of activated esters 1.3324 g of coumarin-3-carboxylic acid (CAS: 531-81-7) (4.8 equivalents) and 0.9674 g of N-hydroxysuccinimide (4.8 equivalents) were placed in a round-bottom flask equipped with a stirrer. All of these were dissolved in 12.5 mL of DMF. Next, N,N'-dicyclohexylcarbodiimide (DCC) (4 equivalents) was added to the flask in several portions while stirring (1000 rpm). The reaction was left at room temperature (25°C, 1000 rpm) for 4 hours. After this, the post-reaction mixture containing the active ester was filtered through a shot funnel (G5) and used in the next step of the synthesis.

[0033] Step 2: Functionalization of gelatin A three-necked round-bottom flask equipped with a stirring bar was placed on a heating block on a magnetic stirrer. 50 mL of carbonate buffer (CB, pH 9.55) was poured into the flask using a funnel and heated to 50°C. Next, 5 g (1 equivalent) of gelatin was added to the buffer and stirring (1000 rpm) was continued. The mixture was maintained at 50°C and 1000 rpm until completely dissolved. Next, the active ester solution in DMF was added dropwise. The mixture was maintained at 50°C and 1000 rpm for 24 hours. After this, the post-reaction mixture was diluted with 200 mL of phosphate-buffered saline (PBS × 1, pH 7.40) and dispensed into 50 mL Falcon tubes. The mixture was centrifuged at 10,000 rpm for 30 minutes, and the upper layer was filtered through a 0.22 μm filter. The resulting clear solution was dialyzed at 40°C for 3 days (using 12-14 kDa tubing), with the water changed three times a day. The purified solution was frozen and freeze-dried at 10°C for 48 hours at 0.01 mbar. The resulting product was then processed. 1 The degree of substitution was measured by 1H NMR analysis.

[0034] Synthesis optimization: To optimize the synthesis, a series of experiments were conducted by changing parameters such as the pH of the buffer, the type of solvent in the first step (DMF, DMSO, CHCl3), the type of condensing agent (EDC and DCC), the reaction time (2, 4, 8, 16, 24, and 48 hours), and the ratio of reactants in the reaction.

[0035] 1 H NMR analysis: The analytical solution was prepared by weighing an appropriate amount (usually about 5 mg) of sample, dissolving it in 600 μL of heavy water to which 0.0916 mmol of tetramethylsilylpropionic acid (TMSP, quantitative and chemical shift criteria) had been added, and placing it in a 5 mm NMR tube. The sample was then placed in an NMR spectrometer (Agilent DirectDrive2 700 MHz). The temperature was set to 60°C. After the temperature stabilized, the sample was mixed, the probe was adjusted, pulses were measured, and magnetic field inhomogeneity was corrected. Next, 1The H spectrum was measured (measurement conditions: 8 scans, 15 seconds repetition time, 2.5 μs 45° pulse time). The material obtained in this example in the crosslinking process of gelatin functionalized with coumarin-3-carboxylic acid. 1 The 1H NMR spectrum is shown in Figure 1.

[0036] Degree of substitution: Materials 1 The 1H NMR spectra were analyzed using the NMRGlue package in a Python environment. After importing the data, exponential weighting (line broadening: 2Hz), Fourier transform, phase adjustment, and baseline correction were performed on the regions 8.85–8.6 ppm, 1.05–0.8 ppm, and 0.1–-0.1 ppm. Subsequently, the integral value of the peak in the 8.85–8.6 ppm region (corresponding to protons on double bonds) was calculated, and the peak in the 1.05–0.8 ppm region (peaks of protons originating from the parent molecule) was integrated. Based on these parameters, DS was performed. NMR The (degree of substitution) value was calculated using the following formula.

number

[0037] A simplified crosslinking scheme using gelatin functionalized with coumarin-3-carboxylic acid (the final step in the above process, PX- = gelatin): [ka]

[0038] Example 2: Material Synthesis The procedure was the same as in Example 1, but in the first step, in addition to the synthesis of the active ester using coumarin-3-carboxylic acid, an active ester using exo-5-norbornenecarboxylic acid (CAS: 934-30-5) was synthesized in parallel, with the same amount used in molar equivalents. As a result, two different active esters were obtained, and by using these in parallel in the second step (functionalization of gelatin), appropriately functionalized gelatin derivatives were obtained.

[0039] Next, the crosslinking process for both functionalized gelatin derivatives was carried out simultaneously. A simplified schematic diagram of cross-linking of a mixture of gelatin solutions functionalized with coumarin-3-carboxylic acid and gelatin solutions functionalized with exo-5-norbornenecarboxylic acid is shown below (PX- = gelatin). [ka]

[0040] Figure 2a shows the exo-5-norbornenecarboxylic acid 1 Figure 2b shows the 1H NMR spectrum and the crosslinking step of a mixture of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid and a 15% solution of gelatin functionalized with exo-5-norbornenecarboxylic acid, which yielded the material obtained in this example. 1 The 1H NMR spectrum is shown.

[0041] In this example and in Examples 3 and 4 below, the functionalized gelatin solution was prepared in PBS. Example 3: Material Synthesis The procedure was the same as in Example 1, but in the first step, in addition to the synthesis of the active ester using coumarin-3-carboxylic acid, an active ester using trans-cinnamic acid (CAS: 140-10-3) was synthesized in parallel, with the same amount used in molar equivalents. As a result, two different active esters were obtained, and by using these in parallel in the second step (functionalization of gelatin), appropriately functionalized gelatin derivatives were obtained.

[0042] Next, the crosslinking process for both functionalized gelatin derivatives was carried out simultaneously. A simplified schematic diagram of cross-linking of a mixture of gelatin solution functionalized with coumarin-3-carboxylic acid and gelatin solution functionalized with trans-cinnamic acid is shown below (PX- = gelatin). [ka]

[0043] Figure 3a shows trans-cinnamic acid 1 The 1H NMR spectrum is shown, and Figure 3b shows the crosslinking process of a mixture of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid and a 10% solution of gelatin functionalized with trans-cinnamic acid, resulting in the material obtained in this example. 1 The 1H NMR spectrum is shown.

[0044] Example 4: Material Synthesis The procedure was the same as in Example 3, but instead of a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 15% solution of trans-cinnamic acid-functionalized gelatin, a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 10% solution of trans-cinnamic acid-functionalized gelatin was crosslinked.

[0045] Figure 3c shows the crosslinking process of a mixture of a 15% solution of gelatin functionalized with coumarin-3-carboxylic acid and a 15% solution of gelatin functionalized with trans-cinnamic acid, and the material obtained in this example. 1 The 1H NMR spectrum is shown.

[0046] Example 5: Determination of the crosslinking profile This experiment involved the following variants: material preparation, digestion of the material with a 0.5% type II collagenase solution, freeze-drying process, and 1 This includes 1H NMR analysis.

[0047] Variant 1: Four layers of 10x10 mm flake-like scaffolds were printed using a 15% solution with different crosslinking times (60 seconds, 120 seconds, 240 seconds, 360 seconds) and a lamp output of 13.0 mW / cm². 2 The irradiation was performed at a wavelength of 365 nm.

[0048] Variant 2: For 10% and 12.5% ​​solutions, 10x10 mm single-layer flake scaffolds were printed at different crosslinking times (120 seconds, 240 seconds, 360 seconds) with a lamp output of 13.0 mW / cm². 2 The irradiation was performed at a wavelength of 365 nm.

[0049] Solution preparation To prepare a 15% GelCM solution, 1.0515 g of GelCM (coumarin-3-carboxylic acid-functionalized gelatin obtained in Example 1 above) (AZ-041-9) was weighed into a 50 mL bottle using an analytical balance. Next, 5.959 mL of PBS × 1 was added using an automatic pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a thermoblock (40°C, 400 rpm) and held for approximately 30 minutes to allow the substance to dissolve.

[0050] To prepare a 12.5% ​​GelCM solution, 250.0 mg of GelCM (AZ-049-9) was weighed into a 5 mL bottle using an analytical balance. Next, 1.750 mL of PBS × 1 was added using an automated pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a thermoblock (40°C, 400 rpm) and held for approximately 30 minutes to allow the substance to dissolve. The pH of the resulting solution was then adjusted to pH 7.37 using 5 μL of 5 M NaOH solution.

[0051] To prepare a 10% GelCM solution, 200.0 mg of GelCM (AZ-049-9) was weighed into a 5 mL bottle using an analytical balance. Next, 1,800 mL of PBS × 1 was added using an automated pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a thermoblock (40°C, 400 rpm) and held for approximately 30 minutes to allow the substance to dissolve. The pH of the resulting solution was then adjusted to pH 7.31 using 5 μL of 5 M NaOH solution.

[0052] Printing of flake-shaped scaffolding The tests were conducted using a BioX CELLINK bioprinter and a Polbionica UV-Vis lamp.

[0053] (a) 15% GelCM solution Using the 10×10×4_05_G4S10 file, four 10×10mm four-layer flake-like scaffolds were printed. Appropriate printing conditions (13.5~14℃, 10mm / sec, 175kPa) and material crosslinking conditions were also selected. P1: 360 seconds; 365 nm; 13.0 mW / cm 2 P2: 240 seconds; 365 nm; 13.0 mW / cm 2 P3: 120 seconds; 365 nm; 13.0 mW / cm 2 P4:60 seconds;365nm;13.0mW / cm 2 A 580μm needle (a pink plastic needle) was used for printing.

[0054] Figure 4 shows photographs of flake scaffolding printed at different bridging times. (b) 12.5% ​​GelCM solution Three flake-shaped scaffolds measuring 10 x 10 x 5 mm were printed. Appropriate printing conditions (14°C, 81-87 kPa, 20 mm / sec and 15°C, 63-68 kPa, 20 mm / sec) and material crosslinking conditions were also selected. P5: 360 seconds; 365 nm; 13.0 mW / cm 2 P6:240sec;365nm;13.0mW / cm 2 P7:120sec;365nm;13.0mW / cm 2

[0055] (c) 10% GelCM solution Three flake-shaped scaffolds measuring 10 x 10 x 5 mm were printed. Appropriate printing conditions (14°C, 33-40 kPa, 20 mm / sec and 15°C, 21-27 kPa, 20 mm / sec) and material crosslinking conditions were also selected. P8:360sec;365nm;13.0mW / cm 2 P9:240sec;365nm;13.0mW / cm 2 P10:120sec;365nm;13.0mW / cm 2

[0056] Disposal of flake-shaped scaffolding Printed flake scaffolds were placed in a 24-well plate. 1 mL of 0.5% Ty II collagenase solution was added to each flake scaffold, and the plate was incubated at 37°C for 48 hours.

[0057] Freeze drying The digested flake scaffold was frozen at -80°C. The 15% variant was then freeze-dried for 48 hours, while the 12.5% ​​and 10% variants were freeze-dried for 24 hours. Freeze-drying conditions: 10°C, 0.01 mbar.

[0058] 1 H NMR analysis 1 To perform 1H NMR analysis, the lyophilized product was weighed into 10 2 mL Eppendorf tubes, and 590 μL of heavy water and 10 μL of TMSP standard solution at a concentration of 0.95 mg / mL were added. The sample was placed in a thermoblock (40°C, 400 rpm) and heated for 10 minutes to dissolve the substance. After that, the sample was... 1 The sample was subjected to 1H NMR analysis (outsourced to Spektrino). Weight of materials: P1: 6.0 mg P2: 5.7mg P3: 5.9mg P4: 5.4 mg Q5: 10.7mg P6: 11.5mg P7: 11.6 mg P8: 12.0 mg P9: 15.5mg P10: 17.4 mg

[0059] result: The analytical signals of the flake-like scaffold after digestion were: 1 The 1H NMR spectrum shows signals in the ranges of 8.85–8.6 ppm and 3.95–4.0 ppm. The peak at 8.85–8.6 ppm corresponds to a proton on the double bond of the coumarin moiety. When the cyclization reaction at this moiety proceeds, this signal disappears, and a new signal appears around 3.95–4.0 ppm. This new signal corresponds to a proton on the cyclobutane ring formed by the cyclization reaction of the coumarin moiety. When the cyclization reaction at this moiety proceeds, this signal disappears, and a new signal appears around 3.95–4.0 ppm. This signal corresponds to a proton on the cyclobutane ring formed by the cyclization reaction of the coumarin moiety.

[0060] 1 The results of the 1H NMR measurement were analyzed, and the proportion of unreacted coumarin-3-carboxylic acid groups (DS of the crosslinking material) was calculated based on the following formula, taking into account the content of repeating units (lysine residues) in the sample.

number

[0061] The proportion of unreacted species was converted to the degree of crosslinking of the material, taking into account the degree of substitution of the non-crosslinked material (DS of the starting material). The results are summarized in the table below. [Table 1]

[0062] Figure 5 shows the 15% GelCM solution (a) 1 Figure 6 shows the 1H NMR spectrum of a 12.5% ​​GelCM solution (b). 1 Figure 7 shows the H NMR spectrum of a 10% GelCM solution (c). 1 The 1H NMR spectra are shown below.

[0063] Conclusion: 1 1H NMR analysis revealed that, for all concentration variants, longer crosslinking times resulted in higher crosslinking degrees. The conditions that yielded the highest degree of crosslinking were the following crosslinking parameters: 360 seconds, 13.0 mW / cm². 2 365 nm. These conditions were used in subsequent material tests.

[0064] Example 6: Printability Solution preparation (i) 15% (w / w) GELCM 2.5 mL of 15% (w / w) GelCM solution (AZ-057-9, DS=50%) was prepared.

[0065] Using an analytical balance, 378.6 mg of lyophilized GelCM was weighed onto a weighing dish. Next, the weighed lyophilized material was transferred to a 5 mL Falcon tube and supplemented with 2.145 mL of PBS × 1. The GelCM solution was maintained in a thermoblock (50°C, 400 rpm) for 30 minutes. After the GelCM had completely dissolved, the pH of the solution (pH 0 = 6.55) was measured, and 5 μL of 5 M NaOH solution and 1.5 μL of 5 M HCl solution were added to adjust the pH to 7.39. The prepared solution was filtered through a 0.22 μm syringe filter and transferred to a 5 mL sterile aluminum foil-wrapped Falcon tube.

[0066] (ii) 12.5% ​​(w / w) GELCM 2.5 mL of 12.5% ​​(w / w) GelCM solution (AZ-057-9, DS=50%) was prepared.

[0067] Using an analytical balance, 318.8 mg of lyophilized GelCM was weighed onto a weighing dish. Next, the weighed lyophilized material was transferred to a 5 mL Falcon tube, and 2.232 mL of PBS × 1 was added. The GelCM solution was kept in a thermoblock (50°C, 400 rpm) for 30 minutes. After the GelCM had completely dissolved, the pH of the solution (pH 0 = 6.58) was measured, and 4 μL of 5 M NaOH solution and 0.5 μL of 5 M HCl solution were added to adjust the pH to 7.39. The prepared solution was filtered through a 0.22 μm syringe filter and transferred to a 5 mL sterile aluminum foil-wrapped Falcon tube.

[0068] (iii) 10% (w / w) GELCM 2.5 mL of 10% (w / w) GelCM solution (AZ-057-9, DS=50%) was prepared.

[0069] Using an analytical balance, 254.3 mg of lyophilized GelCM was weighed onto a weighing dish. Next, the weighed lyophilized material was transferred to a 5 mL Falcon tube, and 2.289 mL of PBS × 1 was added. The GelCM solution was kept in a thermoblock (50°C, 400 rpm) for 30 minutes. After the GelCM had completely dissolved, the pH of the solution (pH 0 = 6.66) was measured, and 3 μL of 5 M NaOH solution and 0.5 μL of 5 M HCl solution were added to adjust the pH to 7.43. The prepared solution was filtered through a 0.22 μm syringe filter and transferred to a 5 mL sterile aluminum foil-wrapped Falcon tube.

[0070] (iv)10%(w / w)GELMA+0.25%(w / w)LAP 2.5 mL of 10% (w / w) GelMA solution (P10-01, DS=81 / 86%) was prepared.

[0071] Using an analytical balance, 318.8 mg of lyophilized GELMA was weighed onto a weighing dish. Next, the weighed lyophilized material was transferred to a 5 mL Falcon tube, and 2.291 mL of PBS × 1 was added. The GelMA solution was maintained in a thermoblock (50°C, 400 rpm) for 30 minutes. After the GelMA had dissolved, 6.5 mg of LAP, weighed in a weighing bottle, was added to the Falcon tube containing the solution. After the GELMA and LAP had completely dissolved, the pH of the solution (pH 0 = 7.33) was measured. The prepared solution was filtered through a 0.22 μm syringe filter and transferred to a 5 mL sterile aluminum foil-wrapped Falcon tube.

[0072] Printability Test Printability tests were conducted using a BioX CELLINK bioprinter. The printing conditions for each material are shown in Table 2 below. Uniform and dense fibers were obtained under the conditions used. The procedure for conducting the printability tests was based on the literature (Ahasan Habib, Venkatachalem Sathish, Sanku Mallik, Bashir Khoda, 3D Printability of Alginate-Carboxymethyl Cellulose Hydrogel, Materials (Basel), 2018 Mar 20; 11(3): 454. doi: 10.3390 / ma11030454). [Table 2]

[0073] This technology includes extrusion printing, volume printing, and other printing techniques, and requires a material viscosity suitable for obtaining uniform and integrated fibers to maintain print resolution. The printing temperature can vary in the range of 5 to 50°C depending on the material used. The printing pressure in extrusion printing is in the range of 5 to 150 kPa. The printing speed is in the range of 1 to 100 mm / second. The needle diameter is in the range of 50 to 900 μm. The printed model can have any dimensions expressed in millimeters, and the infill rate can be set in the range of 5% to 100%, so open structures can be obtained.

[0074] Fiber bonding test A suitable g-code, "template.gcode," was created for fiber bonding testing. This code assumes the continuous printing of two layers using the test material without crosslinking with an external lamp. Printing was performed using a BioX CELLINK printer. Printing was performed in a 0° to 90° pattern, which provides a two-dimensional effect and widens the spacing between fibers. The fiber spacing ranged from 1 to 5 mm, set in 1 mm increments. The printing speed, needle diameter, and extrusion width used in the test were 10 mm / sec, 25 G (0.250 mm), and 0.3 mm, respectively. During the test, the material was supplied within the appropriate pressure and temperature range shown in Table 2 above. The printed material was crosslinked using an external UV-visible light lamp (Polbionica), with a wavelength of 365 nm, a time of 360 seconds, and an output of 13 mW / cm² for GelCM. 2 In the case of GelMA, the wavelength is 405 nm, the crosslinking time is 30 seconds, and the output is 28.5 mW / cm². 2 The experiment was conducted under the following conditions. After printing, microscopic images were taken. The images were analyzed using ImageJ software. Based on the results obtained, two parameters, namely the diffusion rate (material spreading rate) ratio Dfr and printability Pr, were calculated using the following formulas. The pore diffusion rate when there is no material spreading is 0 (i.e., At=Aa), and the printability when the model is perfectly reproduced is 1.0.

number

[0075] Figure 8 shows the structure of the gcode file [template.gcode]. Fiber bending test The central deflection of suspended fibers was analyzed to evaluate the material's collapse behavior. For the experiment, a special platform consisting of seven supports was designed, spaced at known intervals (1, 2, 3, 4, 5, 6 mm). The five central supports within the structure measured 2 × 10 × 6 mm. 3 The dimensions of the two support posts at both ends are 5 x 10 x 6 mm. 3 The following procedure was performed: A single fiber of the test material was deposited on the platform according to the g-code "MR_test1.gcode," and a photograph of the printed material was taken immediately afterward. The photographs were analyzed using ImageJ software. During the process, temperature and pressure conditions were adjusted according to the test material, and printing was performed using a 25G (0.250mm) needle at a speed of 10mm / second. Collapse area coefficient C f This represents the actual area after the suspension fiber has deflected, expressed as a percentage of the theoretical area.

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[0076] result Three fiber bonding tests were performed on each material variant. Microscopic images of the resulting structures were then taken and are shown in Figure 10. Based on the obtained measurement results, the percentages of fiber diffusivity (Dfr) and printability (Pr) were calculated. These results are shown in the graphs in Figures 11 and 12, respectively.

[0077] Conclusion: In all tested materials, continuous and dense fibers were formed, resulting in high-resolution printing. None of the tested materials could be printed with 1x1 mm holes (with the exception of 15%(w / w)GELCM, where small holes were formed in the second print). For each material variant, the printability was above 0.8, and the diffusion percentage was less than 50%. Diffusion decreased with increasing pore size. For all pore sizes except 1x1, the printability of all materials was in the range of 0.8 to 0.9. The highest printability was observed with 15%(w / w)GELCM and 10%(w / w)GELMA + 0.25%(w / w)LAP. The lowest diffusivity observed in 4x4 and 5x5 pores was 12.5% ​​GELCM, while the lowest diffusivity observed in 2x2 and 3x3 pores was the reference material, 10% (w / w) GELMA + 0.25% (w / w) LAP.

[0078] Example 4: Study on cell proliferation rate and toxic effects Alamar Silver Exam This method is based on a reaction in which a compound called Alamar Blue (resazurin) is converted to a resorphine compound in the presence of living cells. Resazurin is known as a redox blue dye that freely passes through the cell membrane and enters the cell, where it is reduced to resorphine, a fluorescent pink compound. Dead cells have lost their metabolic activity and therefore cannot reduce resazurin, and thus cannot generate a fluorescent signal. The resulting signal is detected by a fluorometer, and its intensity increases with increasing numbers of living cells. Figure 13 shows the results of the Alamar Blue test on cells printed within structures containing 10%, 12.5%, and 15% GELCM, and 10% GELMA. The control group consisted of L929 cells directly seeded on plates without the use of biomaterial. Measurements were performed at four time points: the day of printing, and 3, 7, and 14 days after printing.

[0079] Conclusion: Cells in 10% and 12% GELCM biomaterials proliferated at similar levels. The degree of cell proliferation (decrease in population growth rate) in 15% GELCM was significantly lower compared to cells in other biomaterials. The results indicated that the degree of cell proliferation in 10% GELCM was highest at the beginning of the experiment, and that cell division became less likely over time compared to K or 10% and 12.5% ​​GELCM.

[0080] Microscopic observation I. Imaging of biomaterials, including cells, in bright-field imaging. After printing, the structures were transferred to multiwell plates and imaged under a bright-field Olympus microscope using a 10x objective lens. II. Imaging of cell-containing biomaterials after FDA / PI staining Cell viability was assessed based on FDA / PI staining. This staining was performed using two fluorescent dyes (propidium iodide (PI) and fluorescein diacetate (FDA)) to distinguish between dead and living cells, following a pre-established procedure. Fluorescein diacetate can permeate the cell membrane. When FDA is taken up into cells, it is hydrolyzed by intracellular esterases to fluorescein, which has fluorescent properties. Living cells can accumulate this compound, thereby emitting strong green fluorescence. Propidium iodide is electrically charged and does not permeate undamaged cell membranes. It stains cells in a necrotic or late apoptotic state red. Samples were suspended in 1×PBS, stained with FDA / PI, and immediately observed under a fluorescence microscope. Two solutions were prepared for staining: FDA (5 mg / mL in acetone) and PI (2 mg / mL in PBS).

[0081] Figure 14 shows a microscopic image of the printed structure containing L929 cells immediately after printing, Figure 15 shows a microscopic image of the printed structure containing L929 cells on day 3 of the experiment, and Figure 16 shows a microscopic image of the printed structure containing L929 cells on day 7 of the experiment.

[0082] summary In the test groups of 10% GELCM, 12.5% ​​GELCM, and 10% GELMA, L929 cells showed high survival rates. High survival rates exceeding 90% were observed over subsequent days, confirming the proper development of cells printed in 10% GELCM, 12.5% ​​GELCM, and 10% GELMA (reference material).

[0083] Cytotoxicity evaluation of GELCM biomaterials using LDH release tests The purpose of this experiment was to evaluate the cytotoxicity of biomaterials containing 10% GELCM, 12.5% ​​GELCM, and 15% GELCM (batch GELCM AZ-050-9). The cytotoxicity of the biostructure was evaluated using mouse fibroblast cell line L-929 (ATCC®, catalog number: CCL-1). TM The lactate dehydrogenase (LDH) activity of the substance was evaluated based on tests analyzing this activity.

[0084] Lactate dehydrogenase (LDH) activity test: Principle of the method Cytotoxicity (cytotoxic activity) refers to the ability of a specific factor (chemical, physical, or biological factor) to inhibit cell function. This can include, for example, the inhibition of cell growth or proliferation, or the induction of cell death [Abe K., Matsuki N., Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT, Neurosci Res., 2000; 38(4): 325-329.]. Currently, numerous test methods are used in the market to evaluate cell viability after exposure to test substances.This includes the MTT test (MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), the neutral red test (NR), or the activity measurement of the cytoplasmic enzyme lactate dehydrogenase (LDH) [Abe K., Matsuki N., Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT, Neurosci Res., 2000; 38(4): 325-329; Jost LM, Kirkwood JM, Whiteside TL, Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells, J Immunol Methods., 1992; 147(2): 153-165; Wang S., Yu H., Wickliffe JK, Limitation of the MTT and XTTs for measuring assay cell viability due to superoxide formation induced by nano-scale TiO2, Toxicol Vitr., 2011; 25(8): 2147-2151.].

[0085] Lactate dehydrogenase (LDH) is a soluble and stable cytoplasmic enzyme that is released into the culture medium when the integrity of the cell membrane is compromised. The released enzyme can be detected by various methods, including colorimetric, fluorescence, or the most sensitive luminescence method. In this study, we used the Promega LDH-Glo, based on the luminescence method, to quantitatively measure the released LDH. TM A cytotoxicity assay kit was used.

[0086] LDH released from damaged cells catalyzes the oxidation of lactate to pyruvate, and simultaneously NAD + It reduces to NADH. The reductase uses NADH and the reductase substrate to produce luciferin, and this luciferin is converted into rLuciferase Ultra-Glo TM It is converted into a bioluminescent signal. The generated luminescence signal is proportional to the amount of LDH present [Holmes RS, Goldberg E. Computational analysis of mammalian lactate dehydrogenase: LDH of human, mouse, opossum and platypus. Comput Biol Chem. Oct. 2009; 33(5): 379-385; Khan AA, Allemailem KS, Alhumaydhi FA, Gowder SJT, Rahmani AH, The Biochemical and Clinical Perspectives of Lactate Dehydrogenase: An Enzyme of Active Metabolism., Endocr Metab Immune Disord Drug Targets., 2020; 20(6): 855-868.] Measuring LDH activity in the supernatant is an indicator for evaluating the toxicity of a test substance to cells in culture. This method allows for clear determination of whether a particular substance damages the cell membrane of cells and consequently causes cell death.

[0087] Preparation of biomaterials for LDH testing The Bioprint team provided four types of biomaterials containing printed mouse fibroblasts: GELMA 10% (Gelma batch P10-01), GELCM 10%, GELCM 12.5%, and GELCM 15% (GELCM batch AZ-050-9). Each biomaterial was printed three times. 5 × 10⁶ cells per 1 mL of each bioink. 6Printing was performed using individual cells. The biostructures were placed in 6-well plates using DMEM medium (total volume of medium was 4 mL). Throughout the experiment, the structures were stored under standard culture conditions, i.e., 37°C and 5% CO2. The controls in this experiment were a negative control using only L-929 cells, a positive control using L-929 cells treated with 0.1% Triton X-100, and a print without cells. Figure 17 shows photographs of the biomaterial containing L-929 cells after being transferred to a 6-well plate replenished with medium. A shows GELMA 10%, GELCM 12.5%, and GELCM 15%, and B shows GELCM 10%.

[0088] LDH Test Procedure The toxicity of each biomaterial (GELMA 10%, GELCM 10%, GELCM 12.5%, and GELCM 15%) to L-929 cells was evaluated using an LDH assay. The tests were conducted at four time points: days 1, 5, 7, and 14. For the tests, culture medium samples were collected (on days 1, 5, 7, and 14) and diluted to a 1:100 ratio with LDH Storage Buffer. Each test consisted of three repetitions (three prints per biomaterial), forming one test set. Samples were stored at -20°C until testing. Before starting the tests, reaction mixtures were prepared according to the manufacturer's instructions. 50 μL of LD detection enzyme mixture and 0.25 μL of reductase substrate were added to each sample. A dilution series of LDH standard solutions was also prepared. The reaction mixture was dispensed into a white 96-well plate in a 1:1 ratio with the test sample. After incubation at room temperature for 60 minutes, the luminescence intensity was measured using a microplate reader.

[0089] LDH release analysis Figure 18 shows the percentage of lactate dehydrogenase (LDH) released from L-929 cells on days 1, 5, 7, and 14 in response to interaction with the tested biomaterial. The results for LDH enzyme activity were compared to a positive control (K + It is expressed as a relative value (percentage) to ).

[0090] Figure 19 shows micrographs of L-929 cell cultures exposed to biomaterials containing 10% GELCM, 12.5% ​​GELCM, 15% GELCM, and 10% GELMA after 14 days of incubation. The images were taken using an Olympus IX83 microscope in bright-field (BF) mode at magnifications of 4x, 10x, and 20x.

[0091] Observations and conclusions: In all biomaterials, the cytotoxic effect increased over time. Specifically, as the incubation time of cells in certain biomaterials increased, the LDH level in the culture medium rose. When cells were exposed to GELCM 10%, GELCM 12.5%, and GELCM 15% for 7 days, the amount of LDH released was less than 30%. Microscopic observations showed that cell morphology was normal up to day 7 (spindle-shaped, no granules, normal confluence), and no abnormalities were observed.

Claims

1. A method for functionalizing a biopolymer, comprising -OH group or -NH ２ A method characterized by reacting a biopolymer having a group with at least one compound having at least one C=C bond or C≡C bond and a chromophore group that absorbs UV-visible light directly adjacent to this bond.

2. The method according to claim 1, wherein the compound comprising at least one C=C bond or C≡C bond is a carboxylic acid derivative, preferably selected from active esters, acid anhydrides, and acid chlorides.

3. The method according to claim 2, wherein the carboxylic acid derivative is selected from coumarin-3-carboxylic acid derivatives, exo-5-norbornene-carboxylic acid derivatives, and trans-cinnamic acid derivatives.

4. The method according to claim 2 or 3, wherein the active ester is an N-hydroxysuccinimidinyl ester obtained by reacting a carboxylic acid containing at least one C=C bond or C≡C bond with N-hydroxysuccinimidide in the presence of a coupling agent, and the coupling agent is preferably selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC).

5. The method according to any one of claims 1 to 4, wherein the biopolymer is selected from proteins and polysaccharides.

6. The method according to claim 5, wherein the biopolymer is selected from gelatin, hyaluronic acid, alginic acid, chitosan, dextran, starch, cellulose, collagen, chitin, carrageenan, inulin, glycogen, and heparin, preferably selected from gelatin, chitosan, and hyaluronic acid, and most preferably gelatin.

7. A method for obtaining a solid biopolymer material, characterized in that a functionalized biopolymer obtained by the method described in any one of claims 1 to 6 is reversibly crosslinked under UV-visible light irradiation, preferably by irradiation with light in the range of wavelengths from 280 to 800 nm, most preferably at wavelengths selected from 365 nm and 405 nm.

8. Use of a functionalized biopolymer obtained by the method described in any one of claims 1 to 6 as a support material for 3D bioprinting.

9. Use of a functionalized biopolymer obtained by the method of any one of claims 1 to 6 for the production of structures selected from spheroids, organoids, artificial organs, coatings, and tissue models.

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