Method for functionalizing biopolymer and method for cross-linking biopolymer

Abstract

The invention relates to a method for functionalizing a biopolymer, in which a biopolymer having-OH or-NH2 groups is reacted with at least one compound containing at least one C = C or C = C bond and a UV-Vis radiation-absorbing chromophore directly adjacent to the bond. The invention also relates to a process for obtaining a solid biopolymer material, wherein the functionalized biopolymer obtained by the above process undergoes reversible crosslinking upon exposure to light in the UV-VIS range. The invention also relates to the use of the functionalized biopolymer as a support material for 3D bioprinting and to the use of the functionalized biopolymer for producing a structure selected from spheroids, organoids, artificial organs, envelopes and tissue models.

Description

Technical Field

[0001] This invention relates to a method for functionalizing biopolymers, wherein a biopolymer having -OH or -NH2 groups reacts with at least one compound containing at least one C=C or C≡C bond and a chromophore directly adjacent to that bond that absorbs UV-Vis radiation. The invention also relates to a method for obtaining solid biopolymer materials, wherein the functionalized biopolymer obtained by the above method undergoes reversible crosslinking upon exposure to light in the UV-VIS range. Furthermore, the invention relates to the use of functionalized biopolymers as 3D bioprinting support materials, and their use in producing structures selected from spheres, organoids, artificial organs, coatings, and tissue models. This invention is applicable to tissue engineering, transplantation, and medical and pharmacological research. Background Technology

[0002] Chemical compounds that can participate in cycloaddition reactions are used for crosslinking reactions of natural polymers (PelláMCG, Lima-Tenório MK, Tenório-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, Isabel 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. Gattás-Asfura, Eric Weisman, Fotios M. Andreopoulos, Miodrag Micic, Bill Muller, Sanjeev Sirpal, SiM. 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;KoshyST,Desai RM,Joly P,Li J,Bagrodia RK,Lewin SA,Joshi NS,Mooney DJ.Click-Crosslinked Injectable GelatinHydrogels.Adv Healthc Mater.2016,5(5),541-7,doi:10.1002 / adhm.201500757; Jasper Van Hoorick, Liesbeth Tytgat, Agnes Dobos, Heidi Ottevaere, Jürgen Van Erps, Hugo Thienpont, Aleksandr Ovsianikov, Peter Dubruel, Sandra Van Vlierberghe, (Photo-) crosslinkable gelatin derivatives for biofabrication applications, ActaBiomaterialia 2019,97,46-73,doi:10.1016 / j,actbio.2019.07.035). .

[0003] However, new methods are still needed to functionalize biopolymers that can crosslink under light alone (whether in 3D bioprinting or other techniques for forming solid biopolymer structures, such as molding), i.e., without using initiating additives for crosslinking, such as photoinitiators, metal salts, transition metal complexes, and high-valent iodine compounds, which constitute undesirable contaminants in the target structure. In particular, the elimination of photoinitiators is desirable, as they have the greatest cytotoxic effect on cells that may constitute components of the crosslinked biopolymer material.

[0004] Furthermore, there is a constant need for novel functionalized biopolymers capable of crosslinking under longer wavelengths (i.e., lower energy) of light, as this not only reduces the energy consumption of the crosslinking process but, more importantly, limits the negative impact of UV radiation on the viability of cells contained in the crosslinked biopolymer materials. Additionally, there is a constant need for novel functionalized biopolymers capable of reversible crosslinking under light irradiation, which can significantly facilitate the improvement of the resulting biopolymer structure or its more detailed analysis in certain applications. Summary of the Invention

[0005] The subject of this invention is a method for functionalizing biopolymers, wherein a biopolymer having a -OH or -NH2 group reacts with at least one compound containing at least one C=C or C≡C bond and a chromophore that absorbs UV-Vis radiation, with the chromophore immediately adjacent to the bond. In the context of this document, "immediately adjacent" means that the chromophore is attached to a carbon atom directly adjacent to the carbon atom of the multi-bonded carbon atom. Preferably, the compound containing at least one C=C or C≡C bond is a carboxylic acid derivative, preferably selected from reactive esters, acid anhydrides, and acyl 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-hydroxysuccinimide ester, which is obtained by reacting a carboxylic acid containing at least one C=C or C≡C bond with an N-hydroxysuccinimide in the presence of a coupling agent. The coupling agent is preferably selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexyl-carbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC).

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

[0007] The subject of this invention is also a method for obtaining solid biopolymer materials, wherein the functionalized biopolymer obtained by the above method undergoes reversible cross-linking under UV-VIS light irradiation, preferably with light in the wavelength range of 280-800 nm, and most preferably with wavelengths selected from 365 nm and 405 nm.

[0008] The subject of this invention is still the use of functionalized biopolymers obtained by the above method as support materials for 3D bioprinting.

[0009] The subject of this invention is still the use of the functionalized biopolymers obtained by the above method for producing structures selected from spheres, organoids, artificial organs, coatings, and tissue models.

[0010] The functionalized biopolymers obtained using the method according to the invention allow for the production of fully cross-linked solutions in the range of 0.5% to 50% without the addition of photoinitiators such as lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959). Functionalized biopolymers with molecular weights in the range of 1000 kDa to 500000 kDa, such as gelatin, hyaluronic acid, alginate, and other polymers of natural or semi-synthetic origin, having a degree of substitution of 10% to 100%, wherein the substituents are compounds containing at least one C=C or C≡C bond and a chromophore directly adjacent to that bond that absorbs UV-Vis radiation.

[0011] The functionalized biopolymers obtained by the method according to the invention remain completely soluble in aqueous environments (volume from 0.1 to 1000 ml), depending on the application, including physiological saline solutions or cell culture media with a pH of 4 to 8.

[0012] The functionalized biopolymers obtained by the method according to the invention are suitable for extrusion, volumetric printing, and other 3D printing technologies that require materials with appropriate viscosity and clear gel points to obtain uniform and coherent fibers, thereby maintaining print resolution. Depending on the concentration of the prepolymer solution used, the printing temperature can range from 5°C to 50°C, and in this case, the prepolymer should be understood as the functionalized biopolymer according to the invention before the crosslinking process begins. 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 / s. The needle diameter is typically in the range of 50 to 900 μm. The printed model can include any size expressed in millimeters, and an infill density of 5% to 100%, meaning that solid and cutout printing can be obtained.

[0013] When exposed to light with wavelengths from 280 nm to 800 nm and a power of 1 mW / cm² 2 Up to 1000mW / cm 2 When exposed to light for 10 to 720 seconds, the functionalized biopolymers obtained by the method according to the invention are fully crosslinked in the absence of a photoinitiator.

[0014] The functionalized biopolymers obtained by the method according to the invention are perfectly suitable for 3D culture, tissue engineering, and other live cell applications using UV-Vis light as a crosslinking factor. This technique eliminates the need for external initiators that may have potential cytotoxic effects.

[0015] The functionalized biopolymers obtained using the method according to the invention can be used as a single material in bio-inks or as an additive to mixtures of other printable materials. This means that during 3D bioprinting, the functionalized biopolymers undergo cross-linking, and the cross-linking reaction can occur not only between molecules of one type of functionalized biopolymer but also between molecules of two or more different functionalized biopolymers. The functionalized biopolymers, or mixtures of biopolymers functionalized with various compounds, can constitute 0.1% to 99.9% of the total mixture.

[0016] The functionalized biopolymers or blends thereof according to the invention have potential applications in biological research that uses a reference L929 cell line in the initial stages of the study, or, provided that the same or similar MTT test results are obtained, uses alternative cell lines according to ISO 10993-5 standards, namely CCL 1 (NCTC clone 929), CCL 163 (Balb / 3T3 clone A31), CCL171 (MRC-5), CCL 75 (WI-38), CCL81 (Vero), CCL10 (BHK-21[C-13]), and V-79 379A, and uses a specific cell line consistent with the application in subsequent stages of the study. The functionalized biopolymers according to the invention, blends thereof, and materials with added cell lines can be homogenized using syringe mixing techniques or specialized cell mixers for biological applications. Attached Figure Description

[0017] The subject matter of the invention in the embodiments is illustrated in the accompanying drawings in a manner that does not limit the scope of the invention, wherein:

[0018] Figure 1 The material obtained during the crosslinking process of coumarin-3-carboxylic acid-functionalized gelatin in Example 1 is shown. 1 HNMR spectrum:

[0019] Figure 2a This shows exo-5-norbornenic acid. 1 H NMR spectrum;

[0020] Figure 2b The material obtained during the crosslinking process of a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 15% solution of exo-5-norborneol-carboxylic acid-functionalized gelatin, as shown in Example 2, is illustrated. 1 H NMR spectrum;

[0021] Figure 3a Showing trans-cinnamic acid 1 H NMR spectrum:

[0022] Figure 3bThe material obtained during the crosslinking process of the mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 10% solution of trans-cinnamic acid-functionalized gelatin shown in Example 3 is an example of this. 1 H NMR spectrum;

[0023] Figure 3c The material obtained during the crosslinking process of a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 15% solution of trans-cinnamic acid-functionalized gelatin, as shown in Example 3, is illustrated. 1 H NMR spectrum;

[0024] Figure 4 Photographs showing printed flake-like scaffolds with different crosslinking times;

[0025] Figure 5 This shows a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin (GelCM). 1 H NMR spectrum;

[0026] Figure 6 The image shows a 12.5% ​​GelCM solution. 1 H NMR spectrum;

[0027] Figure 7 Showing a 10% GelCM solution 1 H NMR spectrum;

[0028] Figure 8 This shows the scheme for the g.code file [template.gcode].

[0029] Figure 9 A diagram showing the fiber bending test platform;

[0030] Figure 10 Microscopic images of the constructs during fiber bonding tests are shown;

[0031] Figure 11 The percentage of fiber diffusion rate Dfr is shown in the fiber bonding test.

[0032] Figure 12 The printability of Pr is shown in the fiber bonding test;

[0033] Figure 13 The results of the Almar Blue test for cells printed with GelCM 10%, 12.5%, and 15% and gelatin methacrylate (GelMA) 10% constructs are shown.

[0034] Figure 14 The image shows a microscopic image of a printed construct with L929 cells immediately following the printing process.

[0035] Figure 15 This shows a microscopic image of the printed construct containing L929 cells on day 3 of the experiment.

[0036] Figure 16 Microscopic images of the printed construct with L929 cells on day 7 of the experiment are shown.

[0037] Figure 17 Photographs show biological material containing L-929 cells transferred to 6-well plates containing supplemental culture medium, of which A-GELMA 10%, GelCM 12.5%, GelCM 15%, and B-GelCM 10%;

[0038] Figure 18 The percentage of lactate dehydrogenase (LDH) released in L-929 cells is shown as a result of interaction with the test biological material for 1, 5, 7, and 14 days.

[0039] Figure 19 The image shows a micrograph of L-929 cell line cultured after 14 days of incubation and exposed to biological materials of 10% GelCM, 12.5% ​​GelCM, 15% GelCM, and 10% GELMA. Detailed Implementation

[0040] This invention relates to a method for functionalizing biopolymers, wherein a biopolymer having a -OH or -NH2 group reacts with at least one compound containing at least one C=C or C≡C bond (activated or inactivated), i.e., a bond capable of undergoing a cycloaddition reaction 2+2, and a chromophore that absorbs UV-Vis radiation.

[0041] The primary objective of the method according to the invention is to obtain appropriately functionalized derivatives of natural polymers (described below and shown as Helix / P) for tissue engineering, including 3D bioprinting, classical and 3D cell culture, formation of spheroids / organoids and artificial (bionic) organs, formation of material coatings, and printing of specific tissue models (normal and cancerous), including using blood vessels of cell lines and specific quality artificial organs, microorganisms (including islets), organoids, spheroids and other three-dimensional cellular structures.

[0042] The proposed technology and the materials used in its implementation enable the crosslinking / hardening of materials (primarily soft polymers) using UV-VIS radiation without the need for crosslinking-initiating additives, namely photoinitiators, metal salts, transition metal complexes, and high-valent iodine compounds. The crosslinking of these materials, i.e., the functionalized biopolymers obtained by the method according to the invention, occurs due to the [2+2] cycloaddition reaction of compounds containing carbon-carbon multi-bonds and active double or triple bonds, which serve as a separate source of the copolymer or constitute components of the same intramolecular polymer. A schematic diagram of the functionalization process of the biopolymer and its possible subsequent crosslinking pathways is given below. The given pathways are examples, and crosslinking with the materials used can occur in any possible manner, resulting from the chemical reaction mechanisms that form the basis of the proposed polymer crosslinking method.

[0043] A key feature of this method is its ability to utilize light in the UV-Vis range of 280-800 nm, based on the structure and absorption characteristics of the chromophore substituents located on the multi-bonds undergoing 2+2 cycloaddition. Shifting the wavelength range to higher wavelengths (lower energies) not only offers practical advantages (reducing process energy consumption) but also significantly impacts the use of materials created in this technique for tissue engineering with broad understanding. Radiation near the visible light range has a much smaller damaging effect on cells that may be part of the hardening material.

[0044] The biopolymer functionalization method according to the invention uses compounds containing at least one C=C or C≡C bond (i.e., a bond capable of undergoing a 2+2 cycloaddition reaction). Examples of such compounds are those containing a coumarin moiety in their structure, such as coumarin-3-carboxylic acid and coumarin-6-carboxylic acid. These compounds undergo a [2+2] cycloaddition reaction when exposed to light of an appropriate wavelength.

[0045] The following is a scheme for obtaining functionalized biopolymers and then crosslinking them according to the present invention:

[0046] (i) Functionalization of biopolymers by compounds containing carbon-carbon multiple bonds

[0047]

[0048] (ii) Reversible crosslinking of functionalized biopolymers - for variant (a)

[0049]

[0050] in

[0051] Helix = P represents biopolymers selected from oligopeptides, proteins, and polysaccharides, such as gelatin, hyaluronic acid, alginate, chitosan, dextran, starch, cellulose, collagen, chitin, carrageenan, inulin, glycogen, and heparin.

[0052] X is -OH or -NH2;

[0053] R1, R2, R3, R4, R5, and R6 are each independently H, C1-C 30 -alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alynyl group, C6-C 10 -aryl or C5-C containing 1 to 3 heteroatoms selected from N, S and O 10 - Mixed aromatic compounds.

[0054] The crosslinking in step (ii) is performed using a light beam with a wavelength of 280-800 nm, preferably 365 nm or 405 nm. The exposure time is 1-6000 seconds, preferably 10-360 seconds. The power of the light source used is 1-3000 mW / cm². 2 .

[0055] As shown in point (i) of the above scheme, an important advantage of the functionalization of the biopolymer according to the present invention is that the water gelatin can be crosslinked based on the appropriately functionalized polymer that is soluble in water or buffer without the use of an initiator, and the cyclization reaction can be reversed at an appropriately selected wavelength, as shown in point (ii) of the above scheme.

[0056] One of the preferred biopolymers for functionalization using the method according to the invention is gelatin. It is a natural polymer composed of protein chains. Gelatin contains amino acids such as lysine, hydroxylysine, proline, and hydroxyproline, which play a key role in the functionalization reaction because the amino and hydroxyl residues of these amino acids participate in the reaction as shown in the following scheme:

[0057]

[0058] dECM - decellularized extracellular matrix [Due to the gelatin content in dECM, it can be assumed that the cross-linking mechanism of dECM is similar to that of gelatin].

[0059] R = R1 as defined above

[0060] NHS stands for N-hydroxysuccinimide.

[0061] EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0062] Another preferred biopolymer for functionalization according to the invention is chitosan. This polysaccharide is a derivative of chitin produced during deacetylation. Chitosan is composed of β-glucosamine molecules linked by β-1,4-glycosidic bonds. The chitosan functionalization reaction involves the free hydroxyl groups present in the deacetylated β-glucosamine units. The functionalization of chitosan, as with gelatin and dECM, is preferably carried out using an active ester of a suitable carboxylic acid, as shown in the following scheme:

[0063]

[0064] Another preferred biopolymer for functionalization using the method according to the invention is hyaluronic acid. This polysaccharide derived from a glycosaminoglycan group consists of D-glucuronic acid and N-acetyl-D-glucosamine units linked by β-1,4-glycosidic and β-1,3-glycosidic bonds. The functionalization of hyaluronic acid is carried out using the coupling agent DMTMM (4-(4,6-dimethoxy[1,3,5]triazine-2-yl)-4-methylmorpholine chloride). DMTMM is obtained by reacting CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine) with morpholine. The functionalization of biopolymers can be carried out in two ways. The first way involves the activation of the carboxyl group of the D-glucuronic acid unit, followed by a reaction of the activated carboxyl group with a compound containing an amino or hydroxyl group, as shown in the following scheme:

[0065]

[0066] X - as defined above.

[0067] A second method for functionalizing hyaluronic acid involves activating the carboxyl group of any acid with DMTMM, and then reacting the activated acid with the hydroxyl group of an N-acetyl-D-glucosamine unit, as shown in the following scheme:

[0068]

[0069] As described above, one preferred embodiment of the biopolymer functionalization method according to the present invention is a two-step reaction using a carboxylic acid derivative constituting the active ester. For this purpose, the carboxylic acid reacts with NHS and a condensing agent such as EDC, DCC, or DIC. The active N-hydroxysuccinimide ester of the carboxylic acid used in this manner reacts with the biopolymer. The active ester reacts with the free amino and hydroxyl groups present in the natural polymer structure to produce a suitably functionalized biopolymer. An anhydride or acyl chloride of the selected carboxylic acid can be used instead of the active ester.

[0070] The degree of substitution of derivatives can be controlled within the range of 20%-100% by adjusting the pH, temperature, concentration, and amount of the substrate. The degree of substitution affects the mechanical properties of the obtained material.

[0071] Exemplary embodiments

[0072] Example 1 - Material Synthesis

[0073] Step 1. Synthesis of active esters

[0074] 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 substances were dissolved in 12.5 mL of DMF. Then, N,N'-dicyclohexylcarbodiimide (DCC, 4 equivalents) was added to the flask in portions while stirring (1000 rpm). The reaction mixture was allowed to stand at room temperature (25°C, 1000 rpm) for 4 hours. After this, the reaction mixture containing the active ester was filtered through a Schott funnel (G5) and used for the next stage of synthesis.

[0075] Step 2. Functionalization of Gelatin

[0076] Place a three-necked round-bottom flask equipped with a mixing element in a heating block on a magnetic stirrer. Pour 50 mL of carbonate buffer (CB, pH 9.55) into the flask using a funnel and heat to 50 °C. Then, add 5 g of gelatin (1 equivalent) to the buffer and stir continuously (1000 rpm). Dissolve the mixture completely (50 °C, 1000 rpm). Then, add the DMF solution of the active ester dropwise to the solution. Incubate the mixture for 24 hours (50 °C, 1000 rpm). After this, dilute the reaction mixture with 200 mL of phosphate-buffered saline (PBS xl, pH 7.40) and pour into a 50 mL Falcon tube. Centrifuge the mixture (10000 rpm, 30 min), and filter the supernatant through a 0.22 μm filter. Dialyze the clear solution at 40 °C (12-14 kDa tube) for 3 days, changing the water 3 times a day. Freeze and lyophilize the purified solution (10 °C, 48 h, 0.01 mbar). To the finished product 1 1H NMR analysis was used to determine the degree of substitution.

[0077] Synthesis Optimization:

[0078] To optimize the synthesis, a series of experiments were conducted, changing parameters such as: buffer pH, first-stage solvent type (DMF, DMSO, CHCl3), condensation reagent type (EDC and DCC), reaction time (2 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 ​​hours), and the proportion of reactants in the reaction.

[0079] 1 H NMR analysis:

[0080] Weigh an appropriate amount of sample (typically about 5 mg), dissolve it in 600 μL of deuterated water, add 0.0916 mmol of tetramethylsilylpropionic acid (TMSP, quantitative and chemical transfer standard), and place it in a 5 mm NMR tube. Then, place the sample in an NMR spectrometer (Agilent DirectDrive2 700 MHz). The temperature is set to 60 °C. After the temperature stabilizes, mix the sample, adjust the probe, measure the pulse, and correct for magnetic field inhomogeneities. Then, measure... 1 H spectrum (measurement parameters: 8 scans, 15-second repetition time, 45° pulse time 2.5μs). Figure 1 The diagram shows the material obtained during the crosslinking process of coumarin-3-carboxylic acid-functionalized gelatin in this embodiment. 1 H NMR spectrum.

[0081] Substitutability:

[0082] Analyzing materials using the NMRGlue package in a Python environment 1 1H NMR spectra. After importing the data, exponential weighting (linewidth: 2Hz), Fourier transform, phase, and baseline correction were performed on the regions of 8.85ppm÷8.6ppm, 1.05ppm÷0.8ppm, and 0.1ppm÷0.1ppm. Then, the integrals of the peaks in the 8.85ppm÷8.6ppm region (corresponding to protons in double bonds) and the peaks in the 1.05ppm÷0.8ppm region (peaks from protons in mere bonds) were calculated. Based on these parameters, DS was calculated using the following formula. NMR Value (degree of substitution):

[0083]

[0084] Figure 1 The middle shows 1 H NMR spectrum.

[0085] The functionalization schemes for gelatin are as follows:

[0086]

[0087] A simplified cross-linking scheme using gelatin functionalized with coumarin-3-carboxylic acid (the last step in the above process, PX- = gelatin):

[0088]

[0089] Example 2 - Material Synthesis

[0090] The process is similar to that of Example 1, but in step 1, regardless of whether coumarin-3-carboxylic acid is used to synthesize the active ester, exo-5-norbornene carboxylic acid (CAS: 934-30-5) (in the same molar equivalent) is used for the parallel synthesis of the active ester. As a result, two different active esters were obtained, which were then used in the parallel process of stage 2 (functionalization of gelatin) to obtain appropriately functionalized gelatin derivatives.

[0091] Then, the two functionalized gelatin derivatives were cross-linked together.

[0092] A simplified crosslinking scheme for a mixture of coumarin-3-carboxylic acid-functionalized gelatin and exo-5-norborneol-enylcarboxylic acid-functionalized gelatin solution is shown below, where PX is gelatin:

[0093]

[0094] Figure 2a This shows exo-5-norbornenic acid. 1 H NMR spectrum, Figure 2b This embodiment illustrates the material obtained during the crosslinking process of a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 15% solution of exo-5-norbornene carboxylic acid-functionalized gelatin. 1 H NMR spectrum.

[0095] In this example and in Examples 3 and 4 below, solutions of functionalized gelatin were prepared in PBS.

[0096] Example 3 - Material Synthesis

[0097] The process is similar to that of Example 1, but in step 1, regardless of whether coumarin-3-carboxylic acid is used to synthesize the active ester, trans-cinnamic acid (CAS: 140-10-3) is used for the parallel synthesis of the active ester (in the same molar equivalent amount). As a result, two different active esters were obtained, which were then used in the parallel process of stage 2 (functionalization of gelatin) to obtain appropriately functionalized gelatin derivatives.

[0098] Then, the two functionalized gelatin derivatives were cross-linked together.

[0099] A simplified crosslinking scheme for a solution mixture of coumarin-3-carboxylic acid-functionalized gelatin and trans-cinnamic acid-functionalized gelatin is shown below, PX-gelatin:

[0100]

[0101] Figure 3a Showing trans-cinnamic acid 1 H NMR spectrum, Figure 3bThis embodiment illustrates the material obtained during the crosslinking process of a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 10% solution of trans-cinnamic acid-functionalized gelatin. 1 H NMR spectrum.

[0102] Example 4 - Material Synthesis

[0103] The process is similar to 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, the crosslinked gelatin is a mixture of a 10% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 10% solution of trans-cinnamic acid-functionalized gelatin.

[0104] Figure 3c This embodiment illustrates the material obtained during the crosslinking process of a mixture of a 15% solution of coumarin-3-carboxylic acid-functionalized gelatin and a 15% solution of trans-cinnamic acid-functionalized gelatin. 1 H NMR spectrum.

[0105] Example 5 - Determination of Crosslinking Curve

[0106] The experiments included material preparation, digestion of the material using a 0.5% solution of type II collagenase, freeze-drying of the following variants, and... 1 H NMR analysis:

[0107] Variant 1: For a 15% solution, with different crosslinking times (time: 60 seconds, 120 seconds, 240 seconds, 360 seconds, lamp power: 13.0 mW / cm²). 2 (Wavelength: 365nm) Printed four-layer 10×10mm sheet-like scaffolds

[0108] Variant 2: For solutions with concentrations of 10% and 12.5%, with different crosslinking times (time: 120 seconds, 240 seconds, 360 seconds, lamp power: 13.0 mW / cm²). 2 (Wavelength: 365nm) Printing a single-layer 10×10mm sheet-like scaffold

[0109] Solution preparation

[0110] 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. Then, 5.959 ml of PBS was added using an automated pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a hot block (40°C, 400 rpm) for approximately 30 minutes to dissolve the substance.

[0111] 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. Then, 1.750 ml of PBS was added using an automated pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a hot block (40°C, 400 rpm) for approximately 30 minutes to dissolve the substance. The resulting solution was then adjusted to pH 7.37 with 5 μL of 5M NaOH solution.

[0112] 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. Then, 1800 ml of PBS was added using an automated pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a hot block (40°C, 400 rpm) for approximately 30 minutes to dissolve the substance. The resulting solution was then adjusted to pH 7.31 with 5 μL of 5M NaOH solution.

[0113] Printing of sheet-like scaffolds

[0114] The tests were conducted using a BioX CELLINK bioprinter and a Polbionica UV-Vis lamp.

[0115] (a) 15% GelCM solution

[0116] Four four-layer 10×10mm sheet-like scaffolds were printed from the 10×10×4_05_G4S10 file. Suitable printing parameters (13.5-14℃, 10mm / s, 175kPa) and material crosslinking were also selected.

[0117] P1.360 sec; 365 nm; 13.0 mW / cm 2 P2.240 sec; 365 nm; 13.0 mW / cm 2 P3.120 sec; 365 nm; 13.0 mW / cm² 2 P4.60 seconds; 365nm; 13.0mW / cm 2

[0118] Printing was performed using 580μm needles (pink plastic needles).

[0119] Figure 4 Photographs of sheet-like scaffolds printed at different crosslinking times are shown.

[0120] (b) 12.5% ​​GelCM solution

[0121] Three sheet-like supports measuring 10×10×5mm were printed. Suitable printing parameters (14℃, 81-87kPa, 20mm / s and 15℃, 63-68kPa, 20mm / s) and material crosslinking were also selected.

[0122] Q5.360 seconds; 365nm; 13.0mW / cm 2 P6.240 sec; 365 nm; 13.0 mW / cm² 2 P7.120 seconds; 365nm; 13.0mW / cm 2

[0123] (c) 10% GelCM solution

[0124] Three sheet-like supports measuring 10×10×5mm were printed. Suitable printing parameters (14℃, 33-40kPa, 20mm / s and 15℃, 21-27kPa, 20mm / s) and material crosslinking were also selected.

[0125] P8.360 seconds; 365 nm; 13.0 mW / cm 2

[0126] P9.240 sec; 365 nm; 13.0 mW / cm² 2

[0127] P10.120 sec; 365 nm; 13.0 mW / cm 2

[0128] digestion of sheet-like scaffolds

[0129] Place the printed sheet-like scaffolds in 24-well plates. Pour 1 mL of 0.5% type II collagenase solution into each sheet-like scaffold and incubate at 37°C for 48 hours.

[0130] freeze-drying

[0131] The digested sheet-like scaffolds were frozen at -80°C. They were then freeze-dried for 48 hours (15% variant) and 24 hours (12.5%, 10% variant). Freeze-drying conditions: 10°C, 0.01 mbar.

[0132] 1 H NMR analysis

[0133] In order to conduct 1For 1H NMR analysis, the lyophilized product was weighed into ten 2mL Eppendorf-Falcon tubes, and 590μL of deuterated water and 10μL of 0.95mg / mL TMSP standard solution were added. The sample was placed in a hot block (40℃, 400rpm) and heated for 10 minutes to dissolve the substance. The sample was then injected for further analysis. 1 1H NMR analysis (subcontractor Spektrino), material weights: P1. 6.0 mg; P2. 5.7 mg; P3. 5.9 mg; P4. 5.4 mg; Q5. 10.7 mg; P6. 11.5 mg; P7. 11.6 mg; P8. 12.0 mg; P9. 15.5 mg; P10. 17.4 mg.

[0134] result

[0135] exist 1 The analytical signal of the digested sheet-like scaffold was detected in the ¹H NMR spectrum 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 the proton at the double bond in the coumarin moiety. This signal disappears as the cyclization reaction of this moiety proceeds, while a signal reappears at 3.95 ÷ 4.0 ppm, which corresponds to the proton at the cyclobutane ring formed during the cyclization reaction of the coumarin moiety. The presence of the signal in the 3.95 ÷ 4.0 ppm range confirms the crosslinking of the GelCM material under the influence of UV-Vis radiation.

[0136] analyze 1 Based on the 1H NMR measurements and taking into account the monomer (lysine residue) content in the sample, the percentage of unreacted coumarin-3-carboxylic acid groups (DS of the crosslinked material) is determined according to the following formula:

[0137]

[0138] Taking into account the degree of substitution (DS) of the non-crosslinked material, the percentage of unreacted species was converted to the degree of crosslinking of the material. The results are summarized in the table below:

[0139] Table 1: 1 Summary of H NMR Analysis Results - Degree of Substitution (DS) Values ​​of Crosslinked Materials

[0140]

[0141]

[0142] Figure 5 Showing a 15% GelCM solution 1 H NMR spectrum (a), Figure 6 The 12.5% ​​GelCM solution is shown.1 H NMR spectrum (b), Figure 7 Showing a 10% GelCM solution 1 H NMR spectrum (c).

[0143] in conclusion

[0144] 1 1H NMR analysis showed that the longer the crosslinking time, the higher the degree of crosslinking for each concentration variant. The highest degree of crosslinking was obtained with the following crosslinking parameters: 360 seconds, 13.0 mW / cm². 2 365nm. These parameters are used for subsequent material testing.

[0145] Example 6 - Printability

[0146] Solution preparation

[0147] (i) 15% (w / w) GELCM

[0148] A 2.5 ml 15% (w / w) GELCM solution (AZ-057-9, DS=50%) was prepared.

[0149] Weigh 378.6 mg of lyophilized GELCM onto a weighing container using an analytical balance. Then, transfer the weighed lyophilized GELCM to a 5 mL Falcon tube and add 2.145 mL of PBS. Incubate the GELCM solution in a hot block (50 °C, 400 rpm) for 30 minutes. After the GELCM has completely dissolved, check the pH of the solution (pH 0 = 6.55) and adjust it to pH 7.39 (by adding 5 μL of 5 M NaOH and 1.5 μL of 5 M HCl). Filter the prepared solution into a 5 mL sterile aluminum foil-wrapped Falcon tube using a 0.22 μm syringe filter.

[0150] (ii) 12.5% ​​(w / w) GELCM

[0151] A 2.5 ml solution of 12.5% ​​(w / w) GELCM (AZ-057-9, DS=50%) was prepared.

[0152] Weigh 318.8 mg of the lyophilized GELCM onto a weighing container using an analytical balance. Transfer the weighed lyophilized GELCM to a 5 ml Falcon tube and add 2.232 ml of PBS. Incubate the GELCM solution in a hot block (50°C, 400 rpm) for 30 minutes. After the GELCM has completely dissolved, check the pH of the solution (pH 0 = 6.58) and adjust it to pH 7.39 by adding 4 μl of 5M NaOH and 0.5 μl of 5M HCl. Filter the prepared solution through a 0.22 μm syringe filter into a 5 ml sterile aluminum foil-wrapped Falcon tube.

[0153] (iii) 10% (w / w) GELCM

[0154] A 2.5 ml 10% (w / w) GELCM solution (AZ-057-9, DS=50%) was prepared.

[0155] Weigh 254.3 mg of the lyophilized GELCM onto a weighing container using an analytical balance. Transfer the weighed lyophilized GELCM to a 5 ml Falcon tube and add 2.289 ml of PBS. Incubate the GELCM solution in a hot block (50°C, 400 rpm) for 30 minutes. After the GELCM has completely dissolved, check the pH of the solution (pH 0 = 6.66) and adjust it to pH 7.43 by adding 3 μl of 5M NaOH and 0.5 μl of 5M HCl. Filter the prepared solution through a 0.22 μm syringe filter into a 5 ml sterile aluminum foil-wrapped Falcon tube.

[0156] (iv)10%(w / w)GELMA+0.25%(w / w)LAP

[0157] A 2.5 ml solution of 10% (w / w) GELMA (P10-01, DS = 81 / 86%) was prepared.

[0158] Weigh 318.8 mg of lyophilized GELMA onto a weighing container using an analytical balance. Then, transfer the weighed lyophilized GELMA to a 5 ml Falcon tube and add 2.291 ml of PBS. Incubate the GELMA solution in a hot block (50°C, 400 rpm) for 30 minutes. After dissolving the GELMA, transfer 6.5 mg of LAP (weighed from the weighing flask) along with the solution to the Falcon tube. After completely dissolving the GELMA with LAP, check the pH of the solution (pH 0 = 7.33). Filter the prepared solution into a 5 ml sterile aluminum foil-wrapped Falcon tube using a 0.22 μm syringe filter.

[0159] Printability test

[0160] Printability tests were performed using a BioX CELLINK bioprinter. The printing parameters for each material are listed in Table 2 below. The parameters used allowed for the production of uniform, dense fibers. The procedures for printability testing were based on a literature review (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).

[0161] Table 2. Printing parameters in the printability test of pre-crosslinked materials

[0162]

[0163]

[0164] The proposed techniques include extrusion printing, volumetric printing, and other printing methods that require appropriate material viscosity to obtain uniform and coherent fibers, thereby maintaining print resolution. Printing temperatures can range from 5°C to 50°C, depending on the material used. Extrusion printing pressures range from 5 to 150 kPa. Printing speeds range from 1 to 100 mm / s. Needle diameters range from 50 to 900 μm. Printed models can include any size expressed in mm, and infill levels from 5% to 100%, resulting in stencil printing.

[0165] Fiber bonding test

[0166] A suitable g-code, template.gcode, was prepared for the fiber bonding test. This code assumes two layers of test material are printed one after another without crosslinking between them using an external lamp. Printing was performed using a BioX CELLINK printer. The print followed a pattern of 0°–90°, which produced a 2D effect and increased the distance between fibers. The fiber distance ranged from 1–5 mm in 1 mm increments. The printing speed, needle diameter, and extrusion width used in the test were 10 mm / s, 25 G (0.250 mm), and 0.3 mm, respectively. During the test, the material was metered within the appropriate pressure and temperature ranges given in Table 2 above. The prints were crosslinked using an external UV-Vis lamp, Polybionica, with the following parameters: wavelength 365 nm, time 360 ​​seconds, and power 13 mW / cm² in the case of GELCM. 2In the case of GELMA, the wavelength is 405 nm, the crosslinking time is 30 seconds, and the power is 28.5 mW / cm. 2 After printing, photomicrographs were taken. The images were processed using ImageJ software. Based on the results, two parameters described by the following equation were determined: the percentage of diffusion rate, Df. r (Material diffusion rate) and printability P r The diffusion rate of a pore with no material diffusion is 0 (i.e., At = Aa), and for a perfect model representation, the printability is 1.0.

[0167]

[0168] A t —Theoretical pore surface area

[0169] A a —Actual orifice surface area

[0170] L—The actual circumference of the hole.

[0171] Figure 8 The scheme shown is for the g.code file [template.gcode].

[0172] Fiber bending test

[0173] The mid-span bending of the suspended fibers was analyzed to determine the material's collapse. For the experiment, a special platform consisting of seven columns spaced apart from each other at known intervals of 1, 2, 3, 4, 5, and 6 mm was designed. The five columns located within the structure have dimensions of 2 × 10 × 6 mm. 3 The dimensions of the two edge pillars are 5×10×6mm. 3 According to g-code: MR_test1.geode, a single fiber of the test material was placed on the platform, and the print was immediately photographed. The photograph was processed using ImageJ software. During this 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 / s. The collapse area coefficient C... f This refers to the percentage of the actual area to the theoretical area after the fiber is bent and suspended.

[0174]

[0175] A a c —The theoretical area under the curve

[0176] A t c —The actual area under the curve.

[0177] Figure 9 A schematic diagram of a fiber bending test platform is shown.

[0178] result

[0179] For each material variant, three fiber bonding tests were performed. Micrographs of the resulting constructs were then taken, such as... Figure 10 As shown. Based on the obtained measurements, the percentage Df of the fiber diffusion rate was calculated. r and printability P r The results were respectively Figure 11 and Figure 12 The curve shown in the figure represents this.

[0180] in conclusion

[0181] All test materials exhibited continuous, dense fibers, allowing for prints with good resolution. A 1×1 mm hole could not be printed in any of the test materials (the exception was a second attempt to print with 15% (w / w) GELCM, which produced a small hole). For each material variant, printability above 0.8 and diffusion rate percentage below 50% were achieved. The diffusion rate decreased with increasing hole size. Printability for each hole size was at the 0.8–0.9 level for all materials except for 1×1 holes. 15% (w / w) GELCM and 10% (w / w) GELMA + 0.25% (w / w) LAP showed the best printability. 12.5% ​​GELCM showed the lowest diffusion percentage for 4×4 and 5×5 holes, while the reference material 10% (w / w) GELMA + 0.25% (w / w) LAP showed the lowest diffusion percentage for 2×2 and 3×3 holes.

[0182] Example 4 - Study on cell proliferation rate and toxic effects

[0183] Alma Blue Test

[0184] This method is based on converting a compound called resazurin (almar blue) into a fluorescein compound (resorufin) along with living cells. Resazurin is a redox blue dye that freely crosses the cell membrane, enters the cell, and is reduced to the fluorescent pink fluorescein. Dead cells cannot reduce resazurin and therefore cannot produce a fluorescent signal due to loss of metabolic activity. The signal, detected using a fluorometer, shows an intensity that increases with the number of living cells.

[0185] Figure 13The Almar Blue test results for cells printed using Gelcm 10%, 12.5%, and 15% and Gelma 10% constructs are shown. The control group consisted of L929 cells directly seeded onto the plate without any biological material. Measurements were performed at four time points: the day of printing and days 3, 7, and 14 post-printing.

[0186] in conclusion

[0187] Cell proliferation levels were similar in the 10% and 12% Gelcm biomaterials. Cell proliferation (a decrease in population growth rate) in 15% Gelcm was significantly lower than in the other biomaterials. The results indicate that cell proliferation was highest in 10% Gelcm during the first few hours of the experiment, and cells became less prone to division over time compared to K or 10% and 12.5% ​​Gelcm.

[0188] Microscopic observation

[0189] I. Imaging of biomaterials with cells in a bright field

[0190] After printing, the construct was transferred to a multi-well plate and imaged under a bright-field Olympus microscope using a 10x objective lens.

[0191] II. Imaging of cell-bearing biological materials after FDA / Pi staining

[0192] According to the developed procedure, cell viability was assessed based on FDA / Pi staining, which uses two fluorescent dyes: propidium iodide (PI) and fluorescein diacetate (FDA) for cell staining (to distinguish between dead and live cells). FDA can penetrate the cell membrane. Once inside the cell, FDA is hydrolyzed by intracellular esterases into fluorescein, which exhibits fluorescent properties. Live cells can accumulate this compound, enabling them to emit strong green fluorescence. Propidium iodide carries a charge and does not penetrate intact cell membranes. It stains necrotic or late-stage apoptotic cells red. Samples were suspended in 1x PBS (stained with FDA / PI) and observed immediately under a fluorescence microscope. Two staining solutions were prepared: FDA (5 mg / ml acetone solution) and PI (2 mg / ml PBS solution).

[0193] Figure 14 This shows a microscopic image of a printed construct with L929 cells immediately after the printing process. Figure 15 This shows a microscopic image of the printed construct containing L929 cells on day 3 of the experiment. Figure 16 This shows a microscopic image of the printed construct with L929 cells on day 7 of the experiment.

[0194] Summarize

[0195] In the test groups: 10% Gelcm, 12.5% ​​Gelcm, and 10% Gelcm, L929 cells showed high levels of survival. High levels of survival, exceeding 90%, were observed over the following days, and cells printed in the following biomaterials developed normally: 10% Gelcm, 12.5% ​​Gelcm, and 10% Gelma (as reference materials).

[0196] Cytotoxicity of GelCM biomaterials was assessed using an LDH release assay.

[0197] The objective of this study was to evaluate the cytotoxicity of the biomaterials GelCM 10%, GelCM 12.5%, and GelCM 15% (batch GelCM AZ-050-9). The study used a mouse fibroblast cell line based on L-929 (…). cat no: CCL-1 TM The assay of lactate dehydrogenase (LDH) activity was used to test the cytotoxicity of the biological construct.

[0198] Lactate dehydrogenase (LDH) activity assay - method principle

[0199] Cytotoxicity (cytotoxic activity) refers to the ability of a specific factor (chemical, physical, or biological) to disrupt cellular function. It also involves inhibiting cell growth, proliferation, or inducing 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):325-9.]. Currently, there are many tests on the market to determine cell viability after exposure to test substances, including the MTT assay (MTT is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), the neutral red assay (NR), or the measurement of the activity 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):325-9; 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):153-65;Wang S.,Yu H.,Wickliffe JK,Limitation of the MTT and XTT assays for measuring cellviability due to superoxide formation induced by nano-scale TiO2,ToxicolVitr.2011;25(8):2147-51.].

[0200] Lactate dehydrogenase (LDH) is a soluble, stable intracellular enzyme that is released into the culture medium when the cell membrane integrity is compromised. The released enzyme can be detected by various methods, such as colorimetry, fluorescence, or luminescence (the most sensitive method). In our own study, we used Promega's commercial LDH-Glo... TM A cytotoxicity assay kit that uses a bioluminescence method to quantitatively determine the release of LDH.

[0201] LDH released from damaged cells catalyzes the oxidation of lactate to pyruvate, while NAD+ is reduced to NADH. The reductase uses NADH and its substrate to produce luciferin, which is then converted to luciferase by rLuciferase Ultra-Glo. TM It is converted into a bioluminescent signal. The generated luminescent signal is proportional to the amount of LDH present [Holmes RS, Goldberg E. Computational analysis of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs. Comput Biol Chem. 2009 Oct; 33(5):379-85; 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]. The determination of LDH activity in the supernatant is a measure of the toxicity of the test substance to cultured cells. This method allows us to clearly determine whether a given substance will damage the cell membrane, thereby leading to cell death.

[0202] Preparation of biomaterials for LDH testing

[0203] We received four biomaterials from the bioprinting team: 10% GELMA (Gelma batch P10-01), 10% GelCM, 12.5% ​​GelCM, and 15% GelCM (GelCM batch AZ-050-9), each containing printed mouse fibroblasts. Each biomaterial was printed three times. (5 × 10⁻⁶) 6One cell was used for printing with each 1 ml of given bio-ink. The bioconstruct was placed in a 6-well plate in DMEM medium (total volume 4 ml). Throughout the experiment, the construct was stored under standard culture conditions: 37°C and 5% CO2. Controls in the experiment included: L-929 cells alone as a negative control, L-929 cells treated with 0.1% Triton X-100 as a positive control, and cell-free printouts. Figure 17 The image shows a photograph of the biological material with L-929 cells after transfer to a 6-well plate containing supplemental culture medium: A-GELMA 10%, GelCM 12.5%, GelCM 15%, and B-GelCM 10%.

[0204] LDH Testing Procedure

[0205] LDH assays were used to evaluate the toxicity of the tested biomaterials GELMA 10%, GelCM 10%, GelCM 12.5%, and GelCM 15% to L-929 cells. Assays were performed at four time points—days 1, 5, 7, and 14. For the assays, culture medium samples were collected (on days 1, 5, 7, and 14) and diluted 1:100 in sample storage buffer (LDH storage buffer). Each study consisted of three replicates (each replicate printed with one biomaterial). Samples were stored at -20°C prior to the assays. The reaction mixture was prepared according to the manufacturer's recommendations before starting the assays: 50 μl of the LD assay enzyme mixture and 0.25 μl of reductase substrate / sample. A series of dilutions of LDH standards were also performed. The reaction mixture was spotted 1:1 with the test samples onto white 96-well plates. The plates were incubated at room temperature for 60 minutes, and then the luminescence was read using a microplate reader.

[0206] LDH release analysis

[0207] Figure 18 The percentage of lactate dehydrogenase (LDH) released from L-929 cells is shown as a result of interaction with the test biological material for 1, 5, 7, and 14 days. LDH enzyme activity results are expressed as a percentage relative to the positive control group (K+).

[0208] Figure 19 These are micrographs showing L-929 cell lines cultured after 14 days of incubation with biological materials containing 10% GelCM, 12.5% ​​GelCM, 15% GelCM, and 10% GELMA. These images were taken in bright field (BF) using an Olympus IX83 microscope at magnifications of 4x, 10x, and 20x.

[0209] Observations and conclusions:

[0210] All biological materials exhibit increased cytotoxicity over time; that is, the level of LDH in the culture medium increases with prolonged incubation of cells in a given biological material. Exposure to cells with 10%, 12.5%, and 15% GelCM materials for 7 days resulted in LDH release levels below 30%. Microscopic observation showed that until day 7, cell morphology remained normal (spindle-shaped, non-granular, and normally confluent).

Claims

1. A method for functionalizing biopolymers, characterized in that, A biopolymer having a -OH or -NH2 group reacts with at least one compound containing at least one C=C or C≡C bond and a chromophore that absorbs UV-Vis radiation directly adjacent to the bond.

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

5. The method according to any one of claims 1-4, 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, alginate, 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 solid biopolymer materials, characterized in that, The functionalized biopolymer obtained by the method of any one of claims 1-6 undergoes reversible crosslinking when exposed to light in the UV-VIS range, preferably in the light range of 280-800 nm, and most preferably in the light range of 365 nm and 405 nm.

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

9. Use of the functionalized biopolymer obtained by the method of any one of claims 1-6 for preparing structures selected from spheres, organoids, artificial organs, membranes, and tissue models.

Images