Methods to functionalize a biopolymer and to obtain a solid biopolymer material, and, use

Description

/ 34 METHODS FOR FUNCTIONALIZING GELATIN AND FOR OBTAINING A SOLID BIOPOLYMER MATERIAL, AND USE FIELD OF THE INVENTION

[001] The invention relates to a method for functionalizing a biopolymer, in which 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 group that absorbs UV-Vis radiation directly adjacent to that bond. The invention also relates to a method for obtaining a solid biopolymer material, in which the functionalized biopolymer obtained by the method specified above undergoes reversible crosslinking when exposed to light in the UV-Vis range. The invention also relates to the use of a functionalized biopolymer as a support material for 3D bioprinting, as well as to the use of a functionalized biopolymer for the production of selected structures from spheroids, organoids, artificial organs, coatings, and tissue models. The invention is used in tissue engineering, transplantology, and medical and pharmacological research. Background of the art

[002] Chemical compounds that can participate in cycloaddition reactions are used in crosslinking reactions of natural polymers (Pellá MCG, 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, 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. GattásAsfura, Eric Weisman, Fotios M. Andreopoulos, Miodrag Micic, Bill Muller, Sanjeev Sirpal, Si M. Pham, and Roger M. Leblanc, NitrocinnamateFunctionalized Gelatin: Synthesis and “Smart” Hydrogel Formation via Petition 870250086086, dated 09 / 23 / 2025, page 63 / 99 / 34 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, Jürgen 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.)

[003] However, it is still necessary to offer new ways to functionalize biopolymers that can be crosslinked (either during 3D bioprinting or during other techniques for forming solid biopolymer structures, such as molding) only after exposure to light, that is, without the use of crosslinking initiator additives, such as photoinitiators, metal salts, transition metal complexes and hypervalent iodine compounds, which constitute an undesirable contaminant in the target structure. It is particularly desirable to eliminate photoinitiators that have the greatest cytotoxic effect on cells that may constitute a component of the biopolymer material subjected to crosslinking.

[004] Furthermore, there is a constant need to provide new functionalized biopolymers capable of crosslinking after exposure to longer wavelength light (i.e., lower energy) due not only to the reduction in energy consumption of the crosslinking process, but above all to limiting the negative impact of UV radiation on the viability of cells contained in the biopolymer material subjected to crosslinking. In addition, there is a constant need to provide new functionalized biopolymers capable of reversible crosslinking after exposure to light, which in certain applications can significantly facilitate the modification of the structures of formed biopolymers or a more detailed analysis of Petition 870250086086, dated 09 / 23 / 2025, p. 64 / 99 / 34 same. SUMMARY OF THE INVENTION

[005] The subject of the invention is a method of 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 chromophore group that absorbs UV-Vis radiation and a UV-Vis chromophore group that absorbs UV-Vis radiation immediately adjacent to that bond. Immediate proximity in this context means that the chromophore group is attached to the carbon atom directly adjacent to the carbon atom of the multiple bond. Preferably, the compound containing at least one C=C or C=C bond is a carboxylic acid derivative, preferably selected from an active ester, anhydride, or acid chloride. Preferably, the carboxylic acid derivative is selected from a coumarin-3-carboxylic acid derivative, an exo-5-nonbornene-carboxylic acid derivative, and a trans-cinnamic acid derivative.Preferably, the active ester is the N-hydroxysuccinimidinyl ester, which is obtained by reacting a carboxylic acid containing at least one C=C or C=C bond with N-hydroxysuccinimide in the presence of a coupling agent, preferably selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIC).

[006] 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, and is preferably selected from gelatin, chitosan and hyaluronic acid, and most preferably is gelatin.

[007] The object of the invention is also a method for obtaining a solid biopolymer material, in which the functionalized biopolymer obtained Petition 870250086086, dated 09 / 23 / 2025, page 65 / 99 / 34, by the method specified above, is subjected to reversible crosslinking after exposure to light in the UV-VIS range, preferably in the light range with a wavelength of 280-800 nm, and more preferably with a wavelength selected from between 365 nm and 405 nm.

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

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

[0010] Functionalized biopolymers obtained using the method according to the invention allow obtaining fully crosslinking solutions in the concentration range of 0.5% to 50% without the addition of a photoinitiator such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959). A functionalized biopolymer, such as gelatin, hyaluronic acid, alginate, and other polymers of natural or semi-synthetic origin, with a molecular weight ranging from 1000 kDa to 500000 kDa, after functionalization according to the method according to the invention, has a degree of substitution of 10 to 100%, wherein the substituent is a compound containing at least one C=C or C=C bond and a chromophore group that absorbs UV-Vis radiation directly adjacent to that bond.

[0011] The functionalized biopolymers obtained by the method according to the invention maintain total solubility (in volumes from 0.1 to 1000 ml) in aqueous environments, which include physiological saline solutions or cell media with pH from 4 to 8, or others, depending on the application.

[0012] The functionalized biopolymers obtained by the method according to the invention are suitable for use in extrusion, printing Petition 870250086086, dated 09 / 23 / 2025, page 66 / 99 / 34 volumetric and other 3D printing technologies that require the use of a material with adequate viscosity with a transparent gel point to obtain a uniform and coherent fiber to maintain print resolution. The printing temperature can vary from 5 °C to 50 °C, depending on the concentration of the prepolymer solution used, and in this case the prepolymer should be understood as the functionalized biopolymer according to the invention before the start of the crosslinking process. The printing pressure in extrusion printing typically ranges from 5 to 150 kPa. Printing speeds generally range from 1 to 100 mm / s. The needle diameter typically ranges from 50 to 900 μm. The printed model can include any dimensions expressed in mm, as well as an infill level from 5% to 100%, meaning that it is possible to obtain both solid and hollow prints.

[0013] The functionalized biopolymers obtained by the method according to the invention are fully crosslinked without the presence of a photoinitiator when exposed to light with a wavelength of 280 nm to 800 nm, with a luminous power in the range of 1 mW / cm2 to 1000 mW / cm2, for a time of 10 to 720 seconds.

[0014] The functionalized biopolymers obtained by the method according to the invention are fully suitable for use in 3D cultures, tissue engineering and other applications with living cells in which UV-Vis light is the crosslinking factor. The technology does not require the use of an external primer, which may have a potential cytotoxic effect.

[0015] The functionalized biopolymers obtained using the method according to the invention can be used as a single material in a bioink or as an additive to a mixture of other printable materials. This means that in the 3D bioprinting process, during which the crosslinking of functionalized biopolymers occurs, the crosslinking reactions can occur not only between molecules of one type of Petition 870250086086, dated 09 / 23 / 2025, p. 67 / 99 / 34 functionalized biopolymer, but also between molecules of two or more different functionalized biopolymers. A functionalized biopolymer or a mixture of functionalized biopolymers with various compounds may constitute from 0.1% to 99.9% of the total mixture.

[0016] The functionalized biopolymers according to the invention or mixtures thereof have potential applications in biological research using the L929 reference cell line in the initial phase of research, or in accordance with ISO 10993-5 standard for replacement lines, provided that the same or similar MTT test results are obtained, i.e., CCL 1 (NCTC 929 clone), CCL 163 (Balb / 3T3 A31 clone), CCL 171 (MRC5) and CCL 75 (WI-38), CCL 81 (Vero) and CCL 10 [BHK-21 (C-13) and V-79 379A], and in subsequent phases of research using specific cell lines consistent with the application. Functionalized biopolymers according to the invention, their mixtures, and materials with added cell lines can be homogenized using the mixing technique between syringes or specialized cell mixers dedicated to biological applications. BRIEF DESCRIPTION OF THE FIGURES IN THE DRAWINGS

[0017] The subject of the invention in one embodiment is illustrated, without limiting the scope of the invention, in the accompanying drawing, in which: Figure 1 shows the 1H NMR spectrum of the material obtained in example 1 in the crosslinking process of gelatin functionalized with coumarin-3-carboxylic acid; Figure 2a shows the 1H NMR spectrum of exo-5-non-bornenecarboxylic acid; Figure 2b shows the 1H NMR spectrum of the material obtained in Example 2 in the crosslinking process of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 15% gelatin solution functionalized with exo-5-non-carboxylic acid. Petition 870250086086, dated 09 / 23 / 2025, pp. 68 / 99 / 34 bornenecarboxylic acid; Figure 3a shows the 1H NMR spectrum of trans-cinnamic acid; Figure 3b shows the 1H NMR spectrum of the material obtained in example 3 in the crosslinking process of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 10% gelatin solution functionalized with trans-cinnamic acid; Figure 3c shows the 1H NMR spectrum of the material obtained in example 3 in the crosslinking process of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 15% gelatin solution functionalized with trans-cinnamic acid; Figure 4 shows photos of flock-like frames printed with different reticulation times; Figure 5 shows the 1H NMR spectrum of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid (GelCM); Figure 6 shows the 1H NMR spectrum of a 12.5% ​​GelCM solution; Figure 7 shows the 1H NMR spectrum of a 10% GelCM solution; Figure 8 shows the layout of the g.code file [template.gcode]; Figure 9 shows a diagram of a fiber bending test platform; Figure 10 shows microscopic images of constructs in the fiber bonding test; Figure 11 shows the percentage of the Dfr fiber diffusion rate in the fiber bonding test; Figure 12 shows the printing capability of Pr in the test. Petition 870250086086, dated 09 / 23 / 2025, page 69 / 99 / 34 regarding fiber optic connection; Figure 13 shows the results of the Alamar blue test for cells printed in 10%, 12.5% ​​and 15% GelCM and 10% methacrylated gelatin construct (GelMA); Figure 14 shows microscopic images of constructs printed with L929 cells immediately after the printing process. Figure 15 shows microscopic images of constructs printed with L929 cells on day 3 of the experiment. Figure 16 shows microscopic images of constructs printed with L929 cells on day 7 of the experiment. Figure 17 shows photos of biomaterials with L-929 cells after transfer to 6-well plates with supplemented culture medium, where A - 10% GELMA, 12.5% ​​GelCM, 15% GelCM, B - 10% GelCM Figure 18 shows the percentage of lactate dehydrogenase (LDH) release from L-929 cells as a result of interaction with the tested biomaterials for 1, 5, 7, and 14 days. Figure 19 shows microscopic photographs of L-929 cell line cultures exposed to 10% GelCM, 12.5% ​​GelCM, 15% GelCM, and 10% GELMA biomaterials after a 14-day incubation. DETAILED DESCRIPTION OF THE INVENTION

[0018] The invention relates to a method of functionalizing a biopolymer in which a biopolymer containing -OH or -NH2 groups reacts with at least one compound containing at least one C=C or C=C bond (activated or non-activated), i.e., a bond capable of undergoing a 2+2 cycloaddition reaction, and a chromophore group that absorbs UV-Vis radiation.

[0019] The main objective of the method, according to the invention, is to obtain suitably functionalized derivatives of natural polymers (hereinafter described and illustrated as Helix / P) for use in engineering of Petition 870250086086, dated 09 / 23 / 2025, page 70 / 99 / 34 tissues, which include 3D bioprinting, classical and 3D cell culture, formation of spheroids / organoids and artificial (bionic) organs, formation of material coatings, printing of specific tissue models (normal and cancerous), which include vessels and the specific mass of artificial organs using cell lines, micro-organs (including pancreatic islets), organoids, spheroids and other three-dimensional cellular structures.

[0020] The proposed technology and the materials used for its implementation allow the crosslinking / hardening of materials, mainly soft polymers, using UV-VIS radiation without the use of additives that initiate crosslinking, i.e., photoinitiators, metal salts, transition metal complexes, and hypervalent iodine compounds. The crosslinking of the aforementioned materials, i.e., functionalized biopolymers obtained by the method according to the invention, occurs due to a [2+2] cycloaddition reaction of compounds containing a carbon-carbon multiple bond and activated double or triple bonds, which are used as a separate source of the copolymer or constitute an integral part of the polymer within the same molecule. Illustrative diagrams of possible pathways of the biopolymer functionalization process and its subsequent crosslinking are presented below.The paths presented are examples, and crosslinking with the materials used can occur in any possible way resulting from the chemical reaction mechanism that forms the basis of the proposed polymer crosslinking methods.

[0021] An important feature of the described methodology is the possibility of using light in the UV-Vis range with a wavelength of 280-800 nm, depending on the structure and absorption properties of the chromophore substituent located on the multiple bond undergoing 2+2 cycloaddition. Changing the wavelength range to higher wavelengths (lower energy) not only has a practical utility aspect (reducing the Petition 870250086086, dated 09 / 23 / 2025, pp. 71 / 99 10 / 34 energy consumption of the process), but it also has a significant impact on the use of materials created using this technology for use in widely understood tissue engineering. Radiation near the visible range has a much smaller impact on causing damage to cells that may be part of the hardened material.

[0022] The biopolymer functionalization method according to the invention utilizes 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 compounds containing a coumarin moiety in their structure, for example, coumarin-3-carboxylic acid and coumarin-6-carboxylic acid. When exposed to light of an appropriate wavelength, these compounds undergo [2+2] cycloaddition reactions.

[0023] The scheme for obtaining functionalized biopolymers according to the invention and then crosslinking them is presented below: (i) functionalization of the biopolymer with compounds containing multiple carbon-carbon bonds (ii) reversible crosslinking of the functionalized biopolymer - for variant (a) Petition 870250086086, dated 09 / 23 / 2025, page 72 / 99 / 34 P R4 R2R1 ,R3~\ UV-Vis _ x—R3A—R-R1 )=\P—-------- R1-J—I—R3—Xr2r4UV-Visr2r4p where Helix = P represents a biopolymer selected from oligopeptides, proteins, polysaccharides, such as gelatin, hyaluronic acid, alginate, chitosan, dextran, starch, cellulose, collagen, chitin, carrageenan, inulin, glycogen, heparin; X represents -OH or -NH2; R1, R2, R3, R4, R5, R6, each independently of the others, represent H, C1-C30-alkyl, C2-C30-alkenyl, C2-C30-alkynyl, C6-C10-aryl or C5-C10-heteroaryl containing 1 to 3 heteroatoms selected from N, S and O.

[0024] The crosslinking in step (ii) is performed using a light beam with a wavelength of 280-800 nm, with the preferred wavelength values ​​being 365 nm or 405 nm. The exposure time is 1-6000 s, preferably 10-360 s. The power of the light source used is 1-3000 mW / cm2.

[0025] An important advantage of the functionalization of biopolymers according to the invention, illustrated in point (i) of the scheme above, is the possibility of crosslinking the hydrogel based on a suitably functionalized water-soluble or buffer-soluble polymer without the need to use an initiator and the possibility of reversing the cyclization reaction at a suitably selected wavelength, as illustrated in point (ii) of the scheme above.

[0026] One of the preferred biopolymers for functionalization using the method according to the invention is gelatin. It is a natural polymer made of protein chains. In its structure, gelatin has amino acids such as lysine, hydroxylysine, proline, and hydroxyproline, which play a fundamental role in functionalization reactions, since the amino residues Petition 870250086086, dated 09 / 23 / 2025, pp. 73 / 99 12 / 34 and the hydroxyl groups of these amino acids participate in the reactions illustrated in the scheme below: Gelatin derivative / dECM 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 the cross-linking mechanism of gelatin] R = RI defined above NHS - N-hydroxysuccinimide EDC - l-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0027] Another preferred biopolymer for functionalization according to the invention is chitosan. This polysaccharide is a chitin derivative produced in the deacetylation process. Chitosan is composed of β-glucosamine molecules connected by 3-1,4-glycosidic linkages. The functionalization reactions of chitosan involve free hydroxyl groups present in deacetylated β-glucosamine units. The functionalization of chitosan, like the functionalization of gelatin and dECM, preferably occurs using active esters of the appropriate carboxylic acids, as illustrated in the scheme below: the Chitosan Derived from chitosan

[0028] Another preferred biopolymer for functionalization using the method according to the invention is hyaluronic acid. This polysaccharide Petition 870250086086, dated 09 / 23 / 2025, pp. 74 / 99 13 / 34 of the glycosaminoglycan group is composed of D-glucuronic acid and N-acetyl-D-glucosamine units, which are connected by β-1,4-glycosidic and 3-1,3-glycosidic linkages. Hyaluronic acid functionalization occurs using a coupling reagent, 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. Biopolymer functionalization can occur in two ways. The first involves the activation of carboxyl groups of D-glucuronic acid units and then the reaction of the activated carboxyl group with compounds containing amino or hydroxyl groups, as illustrated in the scheme below: HO HO Hyaluronic acid r—x DMTMM RX Derived from hyaluronic acid X - as defined above

[0029] The second method of functionalizing hyaluronic acid involves activating the carboxyl groups of any acids with DMTMM and then reacting the activated acid with the hydroxyl groups of N-acetyl-D-glucosamine units, as illustrated in the scheme below: Hyaluronic acid. Derivative of hyaluronic acid. X - as defined above

[0030] As mentioned above, one of the preferred examples of the biopolymer functionalization method according to the invention is a Petition 870250086086, dated 23 / 09 / 2025, page 75 / 99 / 34 two-step reaction using a carboxylic acid derivative that constitutes an active ester. For this purpose, the carboxylic acid is reacted with NHS and a condensation reagent, such as EDC, DCC or DIC. The active N-hydroxysuccinimidyl ester of the carboxylic acid used in this way is reacted with the biopolymer. The active ester reacts with the free amino and hydroxyl groups present in the structure of the natural polymer, resulting in an appropriate functionalized biopolymer. Instead of the active ester, the anhydride or acid chloride of the selected carboxylic acid may be used.

[0031] Controlling pH, temperature, concentration, and amount of substrates allows for control of the degree of substitution of derivatives in the range of 20-100%. The value of the degree of substitution affects the mechanical properties of the resulting material. EXEMPLARY MODALITIES EXAMPLE 1 - synthesis of materials Step 1. Synthesis of the active ester

[0032] 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 fitted with a stirrer. All were dissolved in 12.5 ml of DMF. N,N'-dicyclohexylcarbodiimide (DCC, 4 equivalents) was then added to the flask in portions while stirring (1000 rpm). The reaction was allowed to proceed for 4 hours at room temperature (25 °C, 1000 rpm). After this time, the post-reaction mixture containing the active ester was filtered through a Schott funnel (G5) and used for the next step of the synthesis. Step 2. Functionalization of gelatin

[0033] A round-bottomed, three-necked flask fitted with a mixing element was placed on the heating block on a magnetic stirrer. 50 ml of carbonate buffer (CB, pH 9.55) were poured into it using a funnel and heated to 50 °C. Then, 5 Petition 870250086086, dated 09 / 23 / 2025, page 76 / 99 / 34 g of gelatin (1 equivalent) were added to the buffer, with continuous stirring (1000 rpm). The mixture was allowed to dissolve completely (50 °C, 1000 rpm). Then, a solution of the active ester in DMF was added dropwise to the solution. The mixture was left for 24 hours (50 °C, 1000 rpm). After this time, the post-reaction mixture was diluted with 200 ml of phosphate-buffered saline solution (PBSx1, pH 7.40) and poured into 50 ml falcon-type tubes. The mixture was centrifuged (10,000 rpm, 30 min) and the top layer was filtered through a 0.22 μm filter. The clear solution was dialyzed (12-14 kDa tubes) at 40 °C for 3 days, changing the water 3 times a day. The purified solution was frozen and lyophilized (10 °C, 48 h, 0.01 mbar). The finished product was subjected to Ή NMR analysis to determine the degree of substitution. Synthesis optimization:

[0034] In order to optimize the synthesis, a series of experiments were carried out, altering parameters such as: pH of the buffer, type of solvent in stage 1 (DMF, DMSO, CHCl3), type of condensation reagent (EDC and DCC), reaction time (2 h, 4 h, 8 h, 16 h, 24 h, 48 h), the proportion of reagents in the reaction. 1H NMR analysis:

[0035] The analytical solution was prepared by weighing an appropriate amount of sample (usually about 5 mg), dissolving it in 600 l of deuterated water with the addition of 0.0916 mmol of tetramethylsilylpropanoic acid (TMSP, quantitative chemical shift standard) and placing it in 5 mm NMR tubes. The samples were then placed in an NMR spectrometer (Agilent DirectDrive2 700 MHz). The temperature was set to 60 °C. After temperature stabilization, the samples were mixed, the probe was adjusted, the pulse was measured, and the lack of homogeneity of the magnetic field was corrected. Then, the 1H spectrum was measured (measurement parameters: number of Petition 870250086086, dated 09 / 23 / 2025, pp. 77 / 99 16 / 34 scans 8, repetition time 15 s, pulse time 45° 2.5 ps). The Ή NMR spectrum obtained from the material obtained in this example in the crosslinking process of gelatin functionalized with coumarin-3-carboxylic acid is shown in Figure 1. Degree of substitution:

[0036] The Ή NMR spectrum of the material was analyzed using the NMRGlue package in the Python environment. After importing the data, exponential weighting (line broadening: 2 Hz), Fourier transform, phasing, and baseline correction were performed for the regions of 8.85 ppm + 8.6 ppm, 1.05 ppm + 0.8 ppm, and 0.1 ppm + -0.1 ppm. Subsequently, the integrals of the peaks in the region of 8.85 ppm + 8.6 ppm (corresponding to the proton in the double bond) and the peak in the region of 1.05 ppm + 0.8 ppm (where the proton peak comes from the mer) were calculated and integrated. Based on these parameters, the DSnmr value (degree of substitution) was calculated using the formula: =1^(8.85-8.5,,^) . . f pico (1.05-0.a) '

[0037] The Ή NMR spectrum is shown in Figure 1.

[0038] The functionalization scheme of gelatin is as follows: Gelatin Petition 870250086086, dated 09 / 23 / 2025, pp. 78 / 99 17 / 34

[0039] Simplified crosslinking scheme using gelatin functionalized with coumarin-3-carboxylic acid (last step of the process described above, with PX- = Gelatin): EXAMPLE 2 - synthesis of materials

[0040] The procedure was analogous to example 1, but in step 1, independently of the synthesis of the active ester using coumarin-3-carboxylic acid, a parallel synthesis of the active ester was carried out using exo-5-nonbornenecarboxylic acid (CAS: 934-30-5) (in the same amount in terms of molar equivalent). As a result, two different active esters were obtained, which were then used, also in parallel processes in step 2 (functionalization of gelatin), to obtain suitably functionalized gelatin derivatives.

[0041] Next, the crosslinking process of both functionalized gelatin derivatives was carried out together.

[0042] A simplified crosslinking scheme of a mixture of gelatin functionalized with coumarin-3-carboxylic acid and solutions of gelatin functionalized with exo-5-nonbornenecarboxylic acid is shown below, with PX gelatin: Petition 870250086086, dated 09 / 23 / 2025, pp. 79 / 99 18 / 34

[0043] Figure 2a shows the Ή NMR spectrum of exo-5nonbornenecarboxylic acid, and Figure 2b, the Ή NMR spectrum of the material obtained in this example in the crosslinking process of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 15% gelatin solution functionalized with exo-5nonbornenecarboxylic acid.

[0044] In this example, as well as in Examples 3 and 4 below, functionalized gelatin solutions were prepared in PBS. EXAMPLE 3 - materials synthesis

[0045] The procedure was analogous to example 1, but in step 1, independently of the synthesis of the active ester using coumarin-3-carboxylic acid, a parallel synthesis of the active ester using trans-cinnamic acid (CAS: 140-10-3) was carried out (in the same amount in terms of molar equivalent). As a result, two different active esters were obtained, which were then used, also in parallel processes in step 2 (functionalization of gelatin), to obtain suitably functionalized gelatin derivatives.

[0046] Next, the crosslinking process of both functionalized gelatin derivatives was carried out together.

[0047] A simplified crosslinking scheme of a mixture of solutions of gelatin functionalized with coumarin-3-carboxylic acid and gelatin functionalized with trans-cinnamic acid is shown below, with gelatin PX-: Petition 870250086086, dated 09 / 23 / 2025, pp. 80 / 99 19 / 34

[0048] Figure 3a shows the Ή NMR spectrum of trans-cinnamic acid, and Figure 3b - the Ή NMR spectrum of the material obtained in this example in the crosslinking process of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 10% gelatin solution functionalized with trans-cinnamic acid. EXAMPLE 4 - synthesis of materials

[0049] The procedure was analogous to example 3, but instead of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 15% gelatin solution functionalized with trans-cinnamic acid, a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 10% gelatin solution functionalized with trans-cinnamic acid was crosslinked.

[0050] Figure 3c shows the Ή NMR spectrum of the material obtained in this example in the crosslinking process of a mixture of a 15% gelatin solution functionalized with coumarin-3-carboxylic acid and a 15% gelatin solution functionalized with trans-cinnamic acid. EXAMPLE 5 - determination of the crosslinking profile

[0051] The experiment included material preparation, material digestion using a 0.5% type II collagenase solution, lyophilization, and Ή NMR analysis for the following variants: Variant 1: Printing of 10x10 mm flake-like frameworks of four layers with different crosslinking times (time: 60 s, 120 s, 240 s, 360 s, lamp power: 13.0 mW / cm2, wavelength: 365 nm) for a solution with a concentration of 15%. Variant 2: printing of frameworks similar to flakes of Petition 870250086086, dated 09 / 23 / 2025, page 81 / 99 / 34 single layer of 10x10 mm with different crosslinking times (time: 120 s, 240 s, 360 s, lamp power: 13.0 mW / cm2, wavelength: 365 nm) for solutions with concentrations of 10% and 12.5% ​​Solution preparation

[0052] 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 PBSx1 was added using an automatic pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a thermoblock (40 °C, 400 rpm) for approximately 30 minutes to dissolve the substance.

[0053] To prepare a 12.5% ​​GelCM solution, 250.0 mg of GelCM (AZ-049-9) were weighed into a 5 ml bottle using an analytical balance. Then, 1.750 ml of PBSx1 was added using an automatic pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a thermoblock (40 °C, 400 rpm) for approximately 30 minutes to dissolve the substance. The resulting solution was then adjusted to pH = 7.37 with 5 l / L of 5 M NaOH solution.

[0054] To prepare a 10% GelCM solution, 200.0 mg of GelCM (AZ-049-9) were weighed into a 5 ml bottle using an analytical balance. Then, 1800 ml of PBSx1 were added using an automatic pipette. The bottle containing the solution, wrapped in aluminum foil, was transferred to a thermoblock (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 5 M NaOH solution. Printing of frameworks resembling flakes

[0055] The BioX CELLINK bioprinter and the Polbionica UV-Vis lamp were used for testing. Petition 870250086086, dated 23 / 09 / 2025, page 82 / 99 / 34 (a) 15% GelCM Solution

[0056] Four 10x10 mm four-layer flake-like scaffolds were printed from the file 10x10x4_05_G4S10. Appropriate printing parameters (13.5-14 °C, 10 mm / s, 175 kPa) and material crosslinking were also selected: P1. 360s; 365nm; 13.0 mW / cm2; P2. 240s; 365nm; 13.0 mW / cm2; P3. 120s; 365nm; 13.0 mW / cm2; P4. 60s; 365nm; 13.0 mW / cm2 A 580 µm needle (pink, plastic needle) was used for printing.

[0057] Figure 4 shows photos of flock-like frames printed with different reticulation times. (b) 12.5% ​​GELCM solution

[0058] Three flake-like scaffolds measuring 10x10x5 mm were printed. Appropriate printing parameters (14 °C, 81-87 kPa, 20 mm / s and 15 °C, 63-68 kPa, 20 mm / s) and material crosslinking were also selected: Q5. 360 s; 365 nm; 13.0 mW / cm2; P6. 240 s; 365 nm; 13.0 mW / cm2; P7. 120 s; 365 nm; 13.0 mW / cm2(c) 10% GelCM solution

[0059] Three flake-like scaffolds measuring 10x10x5 mm were printed. Appropriate printing parameters (14 °C, 33-40 kPa, 20 mm / s and 15 °C, 21-27 kPa, 20 mm / s) and material crosslinking were also selected: P8. 360s; 365nm; 13.0 mW / cm2 P9. 240s; 365nm; 13.0 mW / cm2 P10. 120s; 365nm; 13.0 mW / cm2 Digestion of flake-like frameworks

[0060] The printed flake-like frameworks were placed on 24-well plates. Each flake-like framework was Petition 870250086086, dated 09 / 23 / 2025, p. 83 / 99 / 34 added with 1 ml of 0.5% type II collagenase solution and placed in an incubator for 48 hours at 37 °C. Freeze-drying

[0061] Digested flake-like scaffolds were frozen at -80 °C. They were then freeze-dried for 48 h (15% variant) and 24 h (12.5%, 10% variants). Freeze-drying conditions: 10 °C, 0.01 mbar. 1H NMR analysis

[0062] In order to perform the 1H NMR analysis, the lyophilized product was weighed into 10 2 ml Eppendorf tubes, 590 μl of deuterated water and 10 μl of TMSP standard solution with a concentration of 0.95 mg / ml were added. The samples were placed in a thermoblock (40 °C, 400 rpm) and heated for 10 minutes to dissolve the substance. Then, the samples were subjected to 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. RESULTS

[0063] Analytical signals from the digested flake-like scaffolds are found in the 1H NMR spectrum in the range of 8.85 ^ 8.6 ppm and 3.95 ^ 4.0 ppm. The peak at 8.85 ^ 8.6 ppm corresponds to protons in the double bond in the coumarin moiety. As the cyclization reaction of this moiety progresses, this signal disappears and, at the same time, a signal appears at 3.95 ^ 4.0 ppm, which in turn corresponds to protons in the cyclobutane ring formed in the cyclization reaction of the coumarin moiety. The presence of signals in the range of 3.95 ^ 4.0 ppm proves that the GelCM material crosslinks under the influence of UV-Vis radiation.

[0064] The results of the 1H NMR measurements were analyzed and the percentage of unreacted coumarin-3-carboxylic acid groups (DS of Petition 870250086086, dated 09 / 23 / 2025, p. 84 / 99 23 / 34 cross-linked material) was determined, considering the mer (lysine residue) content in the sample, according to the following formula: fP|CT(8.85-8.6PPP1).996.1M% J peak (1.05—0.8)

[0065] The percentage of unreacted species was converted to the degree of crosslinking of the material, considering the degree of substitution of the non-crosslinked material (DS of the starting material). The results are summarized in the table below: Table 1: Summary of the results of the NMR analysis of Ή - degree values Substitution (DS) of crosslinked material Concentration Sample No. DS of crosslinked material, [%] DS of starting material, [%] Degree of crosslinking, [%] 15% of GelCM PI 11.8 27 56.3 P2 21.9 18.9 P3 24.7 8.5 P4 27.8 ~0 12.5% ​​of GelCM P5 5.7 29 80.3 P6 12.4 57.2 P7 19.2 33.8 10% of GelCM P8 4.1 29 85.8 P9 7.4 74.5 P10 11.0 62

[0066] Figure 5 shows the Ή NMR spectrum for a 15% GelCM solution (a), Figure 6 - for a 12.5% ​​GelCM solution (b) and Figure 7 - for a 10% GelCM solution (c). Conclusions

[0067] Ή NMR analysis indicates that the longer the crosslinking time, the greater the degree of crosslinking of the material for each concentration variant. The highest degree of crosslinking was obtained for the following crosslinking parameters: 360 s, 13.0 mW / cm2, 365 nm. These parameters were used in subsequent material tests. EXAMPLE 6 - printing capacity Solution preparation Petition 870250086086, dated 09 / 23 / 2025, page 85 / 99 / 34 (i) 15% (p / p) of GELCM

[0068] 2.5 ml of a 15% (w / w) GELCM solution (AZ-057-9, DS = 50%) were prepared.

[0069] Using an analytical balance, 378.6 mg of lyophilized GELCM were weighed into a weighing container. The weighed lyophilized product was then transferred to a 5 ml falcon-type tube and supplemented with 2.145 ml of PBSx1. The GELCM solution was left in the thermoblock (50 °C, 400 rpm) for 30 min. After complete dissolution of GELCM, the pH of the solution was checked (pH0 = 6.55), adjusting its value to pH = 7.39 (5 ​​L / L of 5M NaOH and 1.5 L / L of 5M HCl were added). The prepared solution was filtered using a 0.22 μm syringe filter into a sterile 5 ml falcon-type tube wrapped in aluminum foil. (ii) 12.5% ​​(w / w) of GELCM

[0070] 2.5 ml of a 12.5% ​​(w / w) GELCM solution (AZ-057-9, DS = 50%) were prepared.

[0071] Using an analytical balance, 318.8 mg of lyophilized GELCM were weighed into a weighing container. The weighed lyophilized product was then transferred to a 5 ml falcon-type tube and supplemented with 2.232 ml of PBSx1. The GELCM solution was left in the thermoblock (50 °C, 400 rpm) for 30 min. After complete dissolution of the GELCM, the pH of the solution was checked (pH0 = 6.58), adjusting its value to pH = 7.39 (4 ml of 5M NaOH and 0.5 ml of 5M HCl were added). The prepared solution was filtered using a 0.22 μm syringe filter into a sterile 5 ml falcon-type tube wrapped in aluminum foil. (iii) 10% (w / w) of GELCM

[0072] 2.5 ml of a 10% (w / w) solution of GELCM (AZ-057-9, DS = 50%) were prepared. Petition 870250086086, dated 09 / 23 / 2025, pages 86 / 99 / 34

[0073] Using an analytical balance, 254.3 mg of lyophilized GELCM were weighed into a weighing container. The weighed lyophilized product was then transferred to a 5 ml falcon-type tube and supplemented with 2.289 ml of PBSx1. The GELCM solution was left in the thermoblock (50 °C, 400 rpm) for 30 min. After complete dissolution of the GELCM, the pH of the solution was checked (pH0 = 6.66), adjusting its value to pH = 7.43 (3 gl of 5M NaOH and 0.5 gl of 5 M HCl were added). The prepared solution was filtered using a 0.22 μm syringe filter into a sterile 5 ml falcon-type tube wrapped in aluminum foil. (iv) 10% (w / w) GELMA + 0.25% (w / w) LAP

[0074] 2.5 ml of a 10% (w / w) GELMA solution (P10-01, DS = 81 / 86%) were prepared.

[0075] Using an analytical balance, 318.8 mg of GELMA lyophilized powder were weighed into a weighing container. The weighed lyophilized powder was then transferred to a 5 ml falcon tube and supplemented with 2.291 ml of PBSx1. The GELMA solution was left in the thermoblock (50 °C, 400 rpm) for 30 min. Then, after dissolving GELMA, 6.5 mg of LAP weighed into a weighing bottle were transferred to the falcon tube containing the solution. After complete dissolution of GELMA with LAP, the pH of the solution was checked (pH0 = 7.33). The prepared solution was filtered using a 0.22 μm syringe filter into a sterile 5 ml falcon tube wrapped in aluminum foil. Printability tests

[0076] Printability tests were performed using a BioX CELLINK bioprinter. The printing parameters for each material are included in Table 2 below. The parameters used allowed for obtaining a uniform and compact fiber. The procedure for the Petition 870250086086, dated 09 / 23 / 2025, page 87 / 99 / 34, the performance of printing tests was 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). Table 2. Printing parameters in printability tests of pre-screened materials____________________________________________________ Variant Pressure range [kPa] Temperature [°C] Velocity [mm / s] 15% (w / w) of GELCM 60-70 17 10 12.5% ​​(w / w) of GELCM 40-50 17 10 10% (w / w) of GELCM 30-40 17 10 10% (w / w) of GELMA + 0.25% (w / w) of LAP 30-40 17 10

[0077] The technology presented includes extrusion printing, volumetric printing, and other printing technologies that require adequate material viscosity to obtain a uniform and coherent fiber to maintain print resolution. The printing temperature can vary from 5 °C to 50 °C, depending on the material used. The printing pressure for extrusion printing varies from 5 to 150 kPa. Printing speed ranges from 1 to 100 mm / s. The needle diameter varies from 50 to 900 μm. The printed model can include any dimensions expressed in mm, as well as an infill degree from 5% to 100%, in order to obtain hollow prints. Fiber bonding test

[0078] An appropriate g-code was prepared for the fiber bonding test: template.gcode, which assumes printing two layers one after the other using the tested material without the use of crosslinking with an external lamp between them. The prints were made using a BioX CELLINK printer. The printing follows the pattern in a 0°-90° pattern, which gives a 2D effect and increases the distance between the fibers. The distance between the fibers was in the range of 1-5 mm with 1 mm increments. The printing speed, needle diameter, and extrusion width were also considered. Petition 870250086086, dated 09 / 23 / 2025, pp. 88 / 99 The 27 / 34 particles used in the test were 10 mm / s, 25G (0.250 mm), and 0.3 mm, respectively. During the test, the material was dosed within the appropriate pressure and temperature range indicated in Table 2 above. The print was crosslinked with an external UV-Vis lamp, Polbionic, using the following parameters: wavelength 365 nm, time 360 ​​s with a power of 13 mW / cm² in the case of GELCM and wavelength 405 nm, crosslinking time 30 s, power 28.5 mW / cm² in the case of GELMA. After printing, microscopic photographs were taken. The photo processing was performed using ImageJ software. Based on the results, two parameters were determined and described by the following equations: the percentage of the diffusion rate Dfr (material spreading rate) and the printability Pr. The diffusion rate of the pores without material spreading is 0 (i.e., At = Aa), and for a perfect representation of the model, the printability is 1.0. A — Λ„ Dfr= 100% L2P -----r16·ΛΠ At - theoretical surface area of ​​the pores, Aa - actual surface area of ​​the pores, L - actual circumference of the pore.

[0079] Figure 8 shows the schema of the g.code file [template, geodeJ Fiber bending test

[0080] The bending of the average span of the suspended fiber was analyzed to determine the collapse of the material. For the experiment, a special platform was designed, composed of seven pillars spaced apart by known distances of 1, 2, 3, 4, 5, 6 mm. The dimensions of the five posts placed inside the structure are 2 χ 10 * 6 mm3, and the dimensions of the two edge posts are 5 χ 10 χ 6 mm3. A single fiber of the tested material Petition 870250086086, dated 09 / 23 / 2025, pp. 89 / 99 28 / 34 was deposited onto the platform according to the g-code: MR testl .gcode, and then a photo of the print was immediately taken. Photo processing was performed using ImageJ software. During the process, temperature and pressure conditions were adjusted depending on the material being tested, and the actual print was made at a speed of 10 mm / s using a 25 G (0.250 mm) needle. Collapse area coefficient Cf, that is, the percentage of the actual area after bending the suspended fiber in relation to the theoretical area. A' cf= -£·100% Aac - theoretical area under the curve, Atc - actual area under the curve.

[0081] Figure 9 shows a diagram of the fiber bending test platform. Results

[0082] For each material variant, 3 fiber bonding tests were performed. Microscopic photographs of the resulting constructs were then taken – shown in Figure 10. Based on the measurements obtained, the percentage of the fiber diffusion rate Dfre and the printability Pr were calculated; the results are presented in the graphs shown in Figures 11 and 12, respectively. Conclusions

[0083] All tested materials exhibited continuous and compact fibers, allowing for high-resolution prints. It was not possible to print 1x1 mm pores in any of the tested materials (the exception being the second attempt to print 15% (w / w) of GELCM, which produced a small pore). For each material variant, a printability above 0.8 was achieved, as well as a diffusion rate percentage below 50%. The diffusion rate decreases with increasing pore size. The printability for each pore size, except for Petition 870250086086, dated 09 / 23 / 2025, p. 90 / 99 / 34 1x1 pores, is at the level of 0.8-0.9 for all materials. The best printability was shown by 15% (w / w) GELCM and 10% (w / w) GELMA + 0.25% (w / w) LAP. The lowest diffusion percentage for 4x4 and 5x5 pores was presented by 12.5% ​​GELCM, while the lowest diffusion percentage for 2x2 and 3x3 pores was presented by the reference material 10% (w / w) GELMA + 0.25% (w / w) LAP. EXAMPLE 4 - Study of proliferation rate and toxic effects on the cell. Alamar blue test.

[0084] This method is based on the conversion of a compound called Alamar blue (resazurin) into the compound resorufin along with living cells. Resazurin is known as an oxidative redox blue dye that freely passes through the cell membrane to enter the cell, where it is reduced and converted into fluorescent pink resorufin. Dead cells cannot reduce resazurin and are unable to generate a fluorescence signal due to the loss of metabolic activity. The resulting signal is detected using fluorometers, and the intensity increases as the number of viable cells increases.

[0085] The results of the Alamar blue test for cells printed on Gelcm 10%, 12.5% ​​and 15% and Gelma 10% constructs are shown in Figure 13. The control consisted of L929 cells seeded directly onto the plate, without biomaterial. Measurement was performed at 4 time points: on the day of printing and on the 3rd, 7th and 14th day after printing. Conclusions

[0086] Cells in 10% and 12% Gelcm biomaterials proliferated at a similar rate. The degree of cell proliferation (decrease in population growth rate) for 15% Gelcm was much lower than for cells in other biomaterials. The results indicate that the degree of cell proliferation in 10% Gelcm was highest in the first hours of the experiment; over time, the cells divided less. Petition 870250086086, dated 09 / 23 / 2025, pp. 91 / 99 / 34 easily compared with K or 10% and 12.5% ​​Gelcm. Microscopic observations I. Image of biomaterials with cells in the bright field.

[0087] After printing, the constructions were transferred to a multi-well plate and photographed under an Olympus bright-field microscope using a 10x objective. II. Image of biomaterials with cells after FDA / Pi staining.

[0088] Cell viability was assessed based on FDA / Pi staining according to a developed procedure in which cell staining (to distinguish dead from live cells) is performed using two fluorescent dyes: propidium iodide (PI) and fluorescein diacetate (FDA). Fluorescein diacetate can penetrate the cell membrane. After entering the cell, FDA is hydrolyzed by intracellular esterases into fluorescein, which exhibits fluorescent properties. Live cells can accumulate this compound, allowing them to emit intense green fluorescence. Propidium iodide has an electrical charge and does not penetrate intact cell membranes. It stains cells with necrotic red or late apoptosis. Samples were suspended in 1 x 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).

[0089] Figure 14 shows microscopic images of constructs printed with L929 cells immediately after the printing process, Figure 15 - microscopic images of constructs printed with L929 cells on the 3rd day of the experiment and Figure 16 - microscopic images of constructs printed with L929 cells on the 7th day of the experiment. Summary

[0090] In the tested groups: 10% Gelcm, 12.5% ​​Gelcm and 10% Gelcm, L929 cells showed a high level of viability. On days Petition 870250086086, dated 09 / 23 / 2025, pp. 92 / 99 / 34 following, observed a high level of viability, above 90%, and adequate development of the cells printed on the biomaterial: 10% Gelcm, 12.5% ​​Gelcm and 10% Gelma (as reference material). Evaluation of the cytotoxicity of GelCM biomaterials using the LDH release assay.

[0091] The aim of the experiments was to evaluate the cytotoxicity of the biomaterials GelCM 10%, GelCM 12.5%, GelCM 15% (batch GelCM AZ-050-9). The cytotoxicity of the bioconstructions was tested using an assay based on the analysis of lactate dehydrogenase (LDH) activity of the mouse fibroblast cell line: L-929 (ATCC®, cat n°: CCL-1™). Lactate dehydrogenase (LDH) activity test - principle of the method

[0092] Cytotoxicity (cytotoxic activity) is the ability of a specific factor (chemical, physical or biological) to disrupt cell function. It involves, among other things, the inhibition of cell growth, proliferation or 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): 325-9.]. Currently, there are many tests used on the market to determine cell viability after exposure to the test substance, including the MTT test (MTT - 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), neutral red (NR) test, or measurement of cytoplasmic lactate dehydrogenase (LDH) enzyme activity [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 L.M., Kirkwood J.M., Whiteside T.L., Improved short- and long-term XTTbased colorimetric cellular cytotoxicity assay for melanoma and other tumor cells, J Immunol Methods. 1992; 147(2): 153-65; Wang S., Yu H., Wickliffe. Petição 870250086086, de 23 / 09 / 2025, pág. 93 / 99 / 34 J.K., Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2, Toxicol Vitr. 2011; 25(8):2147-51.

[0093] Lactate dehydrogenase (LDH) is a soluble and stable cytosolic enzyme that is released into the culture medium after the disruption of the cell's plasma membrane integrity. The released enzyme can be detected by various methods, for example, colorimetric, fluorometric, or luminescence (the most sensitive). In our own research, we used the commercial LDH-Glo™ Cytotoxicity Assay kit from Promega, based on a bioluminescent method for the quantitative determination of released LDH.

[0094] The LDH released by damaged cells catalyzes the oxidation of lactate to pyruvate, with the simultaneous reduction of NAD+ to NADH. Reductase uses NADH and the reductase substrate to produce luciferin, which is converted into a bioluminescent signal by rLuciferase Ultra-Glo™. The luminescent signal generated is proportional to the amount of LDH present [Holmes RS, Goldberg E. Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs. Comput Biol Chem.October 2009; 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]. Determining LDH activity in the supernatant is a measure of the toxicity of the tested substance to cultured cells. This method allows for a clear determination of whether a given substance causes damage to the plasma membrane of cells and, consequently, their death. Preparation of biomaterials for the LDH test.

[0095] Four biomaterials were received from the bioprinting team: 10% GELMA (Gelma batch P10-01), 10% GelCM, 12.5% ​​GelCM, 15% GelCM (GelCM batch AZ-050-9) with cells from Petition 870250086086, dated 09 / 23 / 2025, pp. 94 / 99 / 34 mouse fibroblasts printed. Each biomaterial was printed in three replicates. 5x10⁶ cells were used for printing per 1 ml of a given bioink. The bioconstructions were placed in a 6-well plate in DMEM culture medium (the total volume of the medium was 4 ml). Throughout the experiment, the constructions were stored under standard culture conditions, i.e., 37 °C and 5% CO₂. The controls in the experiment were: Isolated L-929 cells - negative control, L-929 cells treated with 0.1% Triton X-100 - positive control, and a cell-free print. Figure 17 shows photos of biomaterials with L-929 cells after transfer to 6-well plates with supplemented culture medium, A - GelCM 10%, GelCM 12.5%, GelCM 15%, B - GelCM 10%. LDH testing procedure

[0096] Evaluation of the toxicity level of the tested biomaterials GELMA 10%, GelCM 10%, GelCM 12.5%, GelCM 15% against L929 cells was performed using the LDH assay. The test was performed at four time points – on days 1, 5, 7, and 14. For the test, samples of the culture medium were collected (on days 1, 5, 7, and 14) and diluted in a 1:100 ratio in sample storage buffer (LDH Storage Buffer). A single study consisted of 3 replicates (1 biomaterial was printed in 3 replicates). Samples were stored at -20 °C until testing. Before starting the test, a reaction mixture was prepared according to the manufacturer's recommendations: 50 μl of LD detection enzyme mixture and 0.25 μl of substrate / sample reductase. A series of dilutions of the LDH standard was also performed. The reaction mixture was distributed in a 96-well white plate in a 1:1 ratio with the test samples.It was incubated at room temperature for 60 min, and then the luminescence was read using a microplate reader. LDH release analysis

[0097] Figure 18 shows the percentage of lactate dehydrogenase Petition 870250086086, dated 09 / 23 / 2025, pp. 95 / 99 / 34 (LDH) released from L-929 cells as a result of interaction with the biomaterials tested for 1, 5, 7, and 14 days. The results of LDH enzymatic activity were expressed as a percentage relative to the positive control (K+).

[0098] Figure 19 - Microscopic photographs of L-929 cell line cultures exposed to GelCM 10%, GelCM 12.5%, GelCM 15%, and GELMA 10% biomaterials after 14 days of incubation. Photographs were taken using an Olympus IX83 microscope in bright field (BF), at 4x, 10x, and 20x magnification. Observations and conclusions:

[0099] All biomaterials cause an increase in cytotoxic effect over time, that is, with the incubation time of cells in a given biomaterial, the LDH level in the medium increases. Exposure of 10% GelCM, 12.5% ​​GelCM, and 15% GelCM materials to cells for 7 days causes LDH release at a level below 30%. Microscopic observation shows that up to day 7 the cell morphology was normal (spindle shape, no granules, normal confluence). Petition 870250086086, dated 09 / 23 / 2025, pages 96 / 99

Claims

CLAIMS 1. A method for functionalizing gelatin, characterized by reacting it with a carboxylic acid derivative selected from a coumarin-3-carboxylic acid derivative and a trans-cinnamic acid derivative.

2. Method according to claim 1, characterized in that the carboxylic acid derivative is selected from an active ester, an anhydride and an acid chloride.

3. Method according to claim 2, characterized in that the active ester is the N-hydroxysuccinimidinyl ester, which is obtained by the respective reaction of a carboxylic acid with N-hydroxysuccinimide in the presence of a coupling agent, preferably selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIG).

4. Method for obtaining a solid biopolymer material, characterized in that the functionalized gelatin obtained by the method, as defined in any one of claims 1 to 3, is subjected to reversible crosslinking after exposure to light in the UV-VIS range, preferably in the light range with a wavelength of 280-800 nm, and more preferably with a wavelength selected from between 365 nm and 405 nm, in the absence of any additives selected from photoinitiators, metal salts, transition metal complexes and hypervalent iodine compounds.

5. Use, characterized by the fact that it is a functionalized biopolymer obtained by the method as defined in any one of claims 1-3, as a support material for 3D bioprinting.

6. Use, characterized by the fact that it is a functionalized biopolymer obtained by the method as defined in any one of claims 1-3, for the production of selected structures from spheroids, organoids, artificial organs, coatings, tissue models. Petition 870250086086, dated 09 / 23 / 2025, pp. 97 / 99

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