Preparation method of high-strength multi-crosslinking hydrogel for organoid tissue engineering

Abstract

The invention discloses a preparation method of high-strength multi-crosslinking hydrogel for organoid tissue engineering, which comprises the following steps: S1, respectively dissolving modified sodium alginate and modified chitosan in water, and uniformly stirring and mixing to obtain a modified sodium alginate solution and a modified chitosan solution; s2, uniformly mixing the modified sodium alginate solution and the modified chitosan solution obtained in the step S1, adding an initiator 907, and defoaming after the initiator 907 is completely dissolved to obtain a hydrogel precursor solution; and S3, carrying out water bath culture on the hydrogel precursor solution obtained in the step S2 under ultraviolet irradiation to obtain the high-strength multi-crosslinked hydrogel for organoid tissue engineering. The prepared hydrogel has a triple network structure, the strength of the hydrogel is remarkably improved, and the hydrogel has good biocompatibility and no cytotoxicity and can be well suitable for culture of organoid tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of ceramic materials, and particularly relates to a preparation method of a high-toughness silicon carbide ceramic material. Background Art

[0002] The present invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering. Summary of the Invention

[0003] In order to solve the deficiencies existing in the prior art, the purpose of the present invention is to provide a preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering. The hydrogel obtained by the preparation method of the present application has a triple network structure, significantly improves the strength of the hydrogel, has good biocompatibility and no cytotoxicity, and can be well applied to the culture of organoid tissues.

[0004] In order to achieve the above purpose, the technical scheme adopted by the present invention is as follows: A preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering, comprising the following steps: Step S1: Dissolve modified sodium alginate and modified chitosan in water respectively, stir and mix evenly to obtain a modified sodium alginate solution and a modified chitosan solution; Step S2: After mixing the modified sodium alginate solution and the modified chitosan solution obtained in Step S1 evenly, add initiator 907, and after complete dissolution, defoam to obtain a hydrogel precursor solution; Step S3: Perform water bath culture on the hydrogel precursor solution obtained in Step S2 under ultraviolet light irradiation to obtain a high-strength multi-crosslinked hydrogel for organoid tissue engineering.

[0005] Further, the preparation method of the modified sodium alginate in Step S1 is specifically as follows: 1) Add sodium alginate to deionized water, fully dissolve it to make its mass percentage 2-4%, then add sodium periodate and stir and react in the dark at room temperature. After reacting for a period of time, add ethylene glycol to terminate the reaction. After dialysis treatment and freeze-drying, obtain oxidized sodium alginate and measure its oxidation degree; 2) Add 2-chlorobenzamide to an ethanol solution, ultrasonically disperse it to obtain a 2-chlorobenzamide solution, then slowly add an aqueous solution of 2-(dimethylamino)ethyl methacrylate to the 2-chlorobenzamide solution, stir and react at room temperature for 10-12 h. After the reaction is completed, evaporate and remove the solvent, wash the obtained product with acetone, and filter by suction to obtain an intermediate compound; 3) Add the sodium alginate oxide obtained in step 1) into deionized water to fully dissolve it to obtain a sodium alginate oxide solution. Then, add triethylamine to the sodium alginate oxide solution and stir to mix evenly. Next, add the intermediate compound obtained in step 2) thereto and stir and react at 60-70 °C for 8-10 h to obtain modified sodium alginate.

[0006] Further, in step 1), the molar ratio of sodium periodate to sodium alginate is 1:(1.5-1.8); the reaction time is 12-18 h.

[0007] Further, the specific method of dialysis treatment in step 1) is as follows: place the reaction solution in a regenerated cellulose dialysis bag with a molecular weight cut-off of 3000-4000 Da and dialyze for 2-4 days, changing the deionized water every 6-8 h; the method for measuring the oxidation degree of sodium alginate oxide is as follows: weigh 0.1-0.3 g of sodium alginate oxide, dissolve it in 40-60 mL of hydroxylamine hydrochloride solution, and titrate it with a potentiometric titrator. According to the amount of NaOH consumed in the titration, calculate the oxidation degree of sodium alginate oxide.

[0008] Further, in step 2), the molar ratio of 2-chlorobenzamide to 2-(dimethylamino)ethyl acrylate is 1:(1-2).

[0009] Further, in step 3), the molar ratio of sodium alginate oxide to the intermediate compound is 1:(1-2.5), and the dosage of triethylamine is 3-5% of the total weight of the sodium alginate solution and the intermediate compound.

[0010] Specifically, due to the good biocompatibility, low toxicity, and low cost of alginic acid, it is widely used in the field of organoid tissue engineering. However, the hydrogel formed by sodium alginate has an uncontrollable degradation rate in vivo, and the degradation products have a high molecular weight and are difficult to remove from the body, which greatly limits its application in the field of organoid tissue engineering. Therefore, in this application, sodium alginate is modified. First, sodium alginate is partially oxidized by sodium periodate, an oxidizing agent, to generate oxidized sodium alginate containing aldehyde groups. The oxidized sodium alginate not only retains the advantages of good biocompatibility, degradability, and low toxicity of sodium alginate but also has highly active aldehyde groups that can bind to bioactive substances such as polypeptides and proteins, thereby enhancing the interaction between the hydrogel material and organoid tissue cells and facilitating the cultivation of organoid tissues. However, since sodium alginate with too high an aldehyde group content cannot form a gel, it is necessary to strictly control the degree of oxidation of oxidized sodium alginate. Therefore, in this application, the molar ratio of sodium periodate to sodium alginate is strictly controlled to be 1:(1.5 - 1.8) to prevent the over-oxidation of sodium alginate due to too high a molar ratio of sodium periodate to sodium alginate, resulting in too high an aldehyde group content in the generated oxidized sodium alginate. Second, an intermediate compound containing carbon-carbon double bonds, amino groups, and quaternary ammonium salt structures is synthesized from 2-chlorobenzamide and 2-(dimethylamino)ethyl methacrylate. Finally, grafting reactions are carried out between the amino groups in the intermediate compound and the hydroxyl or carboxyl groups of oxidized sodium alginate under the action of a catalyst, triethylamine, to obtain modified sodium alginate. Carbon-carbon double bonds and quaternary ammonium salt structures are grafted onto the surface of the modified sodium alginate. The carbon-carbon double bonds provide new reaction sites for subsequent cross-linking reactions, and the quaternary ammonium salt structures have excellent antibacterial properties and can synergistically interact with chitosan and the nano-titanium dioxide grafted onto the surface of the modified chitosan, significantly improving the antibacterial property of the hydrogel.

[0011] Further, the preparation method of the modified chitosan in step S1 is specifically as follows: (1) Add hydrophilic nano-titanium dioxide to absolute ethanol and ultrasonically disperse it for 1.5 - 2.5 h to form a titanium dioxide dispersion; (2) Add the silane coupling agent 3-mercaptopropyltriethoxysilane to the titanium dioxide dispersion obtained in step (1), react at a temperature of 60 - 70 °C for 6 - 8 h to obtain a reaction solution, and then add a chitosan acetic acid solution to the reaction solution and react under stirring conditions for 3 - 5 h to obtain modified chitosan.

[0012] Further, in step (1), the mass ratio of nano-titanium dioxide to absolute ethanol is (0.03 - 0.05):1; the mass percentage of titanium dioxide in the titanium dioxide dispersion is 3.5 - 7.5%.

[0013] Further, in the step (2), the molar ratio of titanium dioxide, silane coupling agent 3-mercaptopropyltriethoxysilane, and chitosan is 1:(2-2.5):(0.8-1.2); the mass percentage of chitosan in the chitosan acetic acid solution is 8-15%.

[0014] Specifically, in this application, nano-titanium dioxide is grafted onto the surface of chitosan through the silane coupling agent 3-mercaptopropyltriethoxysilane to obtain modified chitosan; on the one hand, the modified chitosan retains its own amino and hydroxyl groups. When the modified chitosan and modified sodium alginate are mixed, the cationic amino groups in chitosan and the anionic carboxyl groups of sodium alginate undergo electrostatic interaction to form a stable first cross-linked network structure; at the same time, the amino groups of the modified chitosan can also react with the aldehyde groups in the modified sodium alginate to form a dense cross-linked second chemical network structure; at the same time, under the action of initiator 907, the thiol groups grafted on the surface of the modified chitosan react with the carbon-carbon double bonds in the modified sodium alginate to form a third cross-linked network structure.

[0015] Further, in the step S2, the mass ratio of the modified sodium alginate solution, the modified chitosan solution, and the initiator 907 is 1:(1.2-1.8):(0.05-0.08); the defoaming method in the step S2 is static defoaming or vacuum defoaming.

[0016] Further, in the step S3, the temperature of the water bath culture is 25-50 °C, and the water bath culture time is 24-28 h.

[0017] Compared with the prior art, the positive and beneficial effects of the present invention are as follows: ​The present invention modifies sodium alginate and chitosan respectively. After modification, the modified sodium alginate not only retains the original carboxyl and hydroxyl groups, but also newly adds aldehyde groups and carbon-carbon double bond groups, laying a foundation for subsequent cross-linking reaction with modified chitosan to form a multiple hydrogel. On the one hand, the modified chitosan retains its own amino and hydroxyl groups. When the modified chitosan and the modified sodium alginate are mixed, the cationic amino groups in chitosan electrostatically interact with the anionic carboxyl groups of sodium alginate to form a stable first cross-linked network structure. The amino group of the modified chitosan can also react with the aldehyde group in the modified sodium alginate to form a dense cross-linked second chemical network structure. At the same time, under the action of initiator 907, the thiol groups grafted on the surface of the modified chitosan react with the carbon-carbon double bonds in the modified sodium alginate to form a third cross-linked network structure. The triple network structure formed by the cross-linking of the modified sodium alginate and chitosan not only significantly improves the mechanical properties of the hydrogel, but also the surface of the modified sodium alginate is grafted with a quaternary ammonium salt structure, and the quaternary ammonium salt structure can synergistically act with chitosan and the nano-titanium dioxide grafted on the surface of chitosan in terms of antibacterial performance and biocompatibility, thus significantly improving the antibacterial property and biocompatibility of the hydrogel, and being more conducive to the culture of organoids in the hydrogel. Description of the Drawings

[0018] Figure 1 It is a comparison chart of the cytotoxicity of the extracts of different hydrogels on cells, which is the biocompatibility of the hydrogels prepared in Example 2 and Comparative Examples 1-4 of the present invention; Figure 2 It is a result chart of the culture of the organoids (lung cancer tissue cells) of the present invention in the hydrogels prepared in Example 2 and Comparative Examples 1-4 on the 1st day and the 14th day respectively. Detailed Embodiments

[0019] The technical solutions of the present invention will be further specifically described below through examples and drawings. These examples are for the illustration of the present invention and are not intended to limit the present invention. All other examples obtained by those of ordinary skill in the art based on the examples in this application without creative efforts shall fall within the scope of protection of this application.

[0020] The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials described, unless otherwise specified, can all be obtained from commercial channels; other unpublicized reagents are all commercially available.

[0021] Example 1 A preparation method of a high-strength multiple cross-linked hydrogel for organoid tissue engineering, comprising the following steps: Step S1: Dissolve modified sodium alginate and modified chitosan in water respectively, stir and mix evenly to obtain a modified sodium alginate solution and a modified chitosan solution; Step S2: After mixing the modified sodium alginate solution and the modified chitosan solution obtained in Step S1 evenly, add initiator 907. After complete dissolution, let it stand to defoam to obtain a hydrogel precursor solution; wherein, the mass ratio of the modified sodium alginate solution, the modified chitosan solution to the initiator 907 is 1:1.2:0.05; Step S3: Place the hydrogel precursor solution obtained in Step S2 under ultraviolet light with a wavelength of 365 nm and a power of 1 mW / cm 2 Irradiate for 15 min, and then carry out water bath culture. The temperature of the water bath culture is 25 °C, and the water bath culture time is 28 h to obtain a high-strength multi-crosslinked hydrogel for organoid tissue engineering.

[0022] The preparation method of the modified sodium alginate is as follows: 1) Add sodium alginate to deionized water to make it fully dissolve, so that its mass percentage is 2%, then add sodium periodate and stir and react in the dark at room temperature for 12 h, add ethylene glycol to terminate the reaction, place the reaction solution in a 3000 Da regenerated cellulose dialysis bag, dialyze for 4 days, change deionized water every 6 h, and then freeze-dry the dialyzed reaction solution to obtain oxidized sodium alginate. Weigh 0.2 g of oxidized sodium alginate and dissolve it in 40 mL of a hydrochloric hydroxylamine solution with a mass percentage of 1.75%. Titrate it with a potentiometric titrator. According to the amount of NaOH consumed in the titration, calculate that the oxidation degree of oxidized sodium alginate is 25.32%; wherein, the molar ratio of sodium periodate to sodium alginate is 1:1.5; 2) Add 2-chlorobenzamide to an ethanol solution, ultrasonically disperse for 40 min to obtain a 2-chlorobenzamide solution, and then slowly add an aqueous solution of 2-(dimethylamino)ethyl methacrylate to the 2-chlorobenzamide solution, stir and react at room temperature for 10 h. After the reaction is completed, evaporate and remove the solvent, wash the obtained product with acetone and filter it to obtain an intermediate compound; wherein, the molar ratio of 2-chlorobenzamide to 2-(dimethylamino)ethyl methacrylate is 1:1; 3) Add the oxidized sodium alginate obtained in Step 1) to deionized water to make it fully dissolve to obtain an oxidized sodium alginate solution, then add triethylamine to the oxidized sodium alginate solution and stir and mix evenly, and then add the intermediate compound obtained in Step 2) thereto and stir and react at 60 °C for 8 h to obtain modified sodium alginate. Wherein, the molar ratio of oxidized sodium alginate to the intermediate compound is 1:1, and the dosage of the triethylamine is 3% of the total weight of the sodium alginate solution and the intermediate compound.

[0023] The preparation method of modified chitosan is as follows: (1) Add hydrophilic nano-titanium dioxide into absolute ethanol and ultrasonically disperse it for 2.5 h at an ultrasonic frequency of 80 kHz to form a titanium dioxide dispersion; wherein, the mass ratio of nano-titanium dioxide to absolute ethanol is 0.03:1; the mass percentage of titanium dioxide in the titanium dioxide dispersion is 3.5%; (2) Add the silane coupling agent 3-mercaptopropyltriethoxysilane to the titanium dioxide dispersion obtained in step (1), react at 60 °C for 8 h to obtain a reaction solution, and then add a chitosan acetic acid solution to the reaction solution and react under stirring conditions for 3 h to obtain modified chitosan. Among them, the molar ratio of titanium dioxide to the silane coupling agent 3-mercaptopropyltriethoxysilane and chitosan is 1:2:0.8); the mass percentage of chitosan in the chitosan acetic acid solution is 8%.

[0024] Example 2 A preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering includes the following steps: Step S1: Dissolve modified sodium alginate and modified chitosan in water respectively, stir and mix evenly to obtain a modified sodium alginate solution and a modified chitosan solution; Step S2: After mixing the modified sodium alginate solution and the modified chitosan solution obtained in step S1 evenly, add initiator 907, and after complete dissolution, use vacuum defoaming to obtain a hydrogel precursor solution; wherein, the mass ratio of the modified sodium alginate solution, the modified chitosan solution to the initiator 907 is 1:1.5:0.06; Step S3: Place the hydrogel precursor solution obtained in step S2 under ultraviolet light with a wavelength of 365 nm and a power of 1 mW / cm 2 irradiate for 15 min, and then carry out water bath culture at a water bath culture temperature of 40 °C for 26 h to obtain a high-strength multi-crosslinked hydrogel for organoid tissue engineering.

[0025] The preparation method of modified sodium alginate is as follows: 1) Sodium alginate was added to deionized water and fully dissolved to make its mass percentage 3%. Then sodium periodate was added and the mixture was stirred and reacted in the dark at room temperature for 15 h. Ethylene glycol was added to terminate the reaction. The reaction solution was placed in a 3500 Da regenerated cellulose dialysis bag and dialyzed for 3 days, with deionized water changed every 7 h. Then the dialyzed reaction solution was freeze-dried to obtain oxidized sodium alginate. 0.2 g of oxidized sodium alginate was weighed and dissolved in 40 mL of a 1.75% by mass hydroxylamine hydrochloride solution. Titration was carried out using a potentiometric titrator. According to the amount of NaOH consumed in the titration, the oxidation degree of oxidized sodium alginate was calculated to be 28.83%; among them, the molar ratio of sodium periodate to sodium alginate was 1:1.6; 2) 2-Chlorobenzamide was added to an ethanol solution and ultrasonically dispersed for 50 min to obtain a 2-chlorobenzamide solution. Then an aqueous solution of 2-(dimethylamino)ethyl methacrylate was slowly added to the 2-chlorobenzamide solution, and the mixture was stirred and reacted at room temperature for 11 h. After the reaction was completed, the solvent was evaporated off, and the product was washed with acetone and filtered by suction to obtain an intermediate compound; among them, the molar ratio of 2-chlorobenzamide to 2-(dimethylamino)ethyl methacrylate was 1:1.5; 3) The oxidized sodium alginate obtained in step 1) was added to deionized water and fully dissolved to obtain an oxidized sodium alginate solution. Then triethylamine was added to the oxidized sodium alginate solution and stirred to mix evenly. Then the intermediate compound obtained in step 2) was added thereto, and the mixture was stirred and reacted at 65 °C for 9 h to obtain modified sodium alginate. Among them, the molar ratio of oxidized sodium alginate to the intermediate compound was 1:2, and the amount of triethylamine used was 4% of the total weight of the sodium alginate solution and the intermediate compound.

[0026] The preparation method of modified chitosan is specifically as follows: (1) Hydrophilic nano-titanium dioxide was added to absolute ethanol and ultrasonically dispersed for 2 h with an ultrasonic frequency of 90 kHz to form a titanium dioxide dispersion; among them, the mass ratio of nano-titanium dioxide to absolute ethanol was 0.04:1; the mass percentage of titanium dioxide in the titanium dioxide dispersion was 5%; (2) 3-Mercaptopropyltriethoxysilane, a silane coupling agent, was added to the titanium dioxide dispersion obtained in step (1), and the mixture was reacted at 65 °C for 7 h to obtain a reaction solution. Then a chitosan acetic acid solution was added to the reaction solution, and the mixture was reacted under stirring conditions for 4 h to obtain modified chitosan. Among them, the molar ratio of titanium dioxide to 3-mercaptopropyltriethoxysilane and chitosan was 1:2.2:1; the mass percentage of chitosan in the chitosan acetic acid solution was 12%.

[0027] Example 3 A preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering includes the following steps: Step S1: Dissolve modified sodium alginate and modified chitosan in water respectively, stir and mix them evenly to obtain a modified sodium alginate solution and a modified chitosan solution; Step S2: After mixing the modified sodium alginate solution and the modified chitosan solution obtained in Step S1 evenly, add initiator 907. After complete dissolution, use static defoaming or vacuum defoaming to obtain a hydrogel precursor solution; wherein, the mass ratio of the modified sodium alginate solution, the modified chitosan solution to the initiator 907 is 1:1.8:0.08; Step S3: Place the hydrogel precursor solution obtained in Step S2 under ultraviolet light with a wavelength of 365 nm and a power of 1 mW / cm 2 Irradiate for 15 min, and then carry out water bath culture. The temperature of the water bath culture is 50 °C, and the water bath culture time is 24 h to obtain a high-strength multi-crosslinked hydrogel for organoid tissue engineering.

[0028] The preparation method of the modified sodium alginate is specifically as follows: 1) Add sodium alginate to deionized water to make it fully dissolve, so that its mass percentage is 4%, then add sodium periodate and stir and react in the dark at room temperature for 18 h, add ethylene glycol to terminate the reaction, place the reaction solution in a 4000 Da regenerated cellulose dialysis bag, dialyze for 4 days, change deionized water every 8 h, and then freeze-dry the dialyzed reaction solution to obtain oxidized sodium alginate. Weigh 0.2 g of oxidized sodium alginate, dissolve it in 40 mL of a hydrochloric hydroxylamine solution with a mass percentage of 1.75%, titrate it with a potentiometric titrator, and calculate the oxidation degree of oxidized sodium alginate to be 38.54% according to the amount of NaOH consumed in the titration; wherein, the molar ratio of sodium periodate to sodium alginate is 1:1.8; 2) Add 2-chlorobenzamide to an ethanol solution, ultrasonically disperse for 40 min to obtain a 2-chlorobenzamide solution, and then slowly add an aqueous solution of 2-(dimethylamino)ethyl methacrylate to the 2-chlorobenzamide solution, stir and react at room temperature for 12 h. After the reaction is completed, evaporate and remove the solvent, wash the obtained product with acetone and filter it to obtain an intermediate compound; wherein, the molar ratio of 2-chlorobenzamide to 2-(dimethylamino)ethyl methacrylate is 1:2; 3) Add the oxidized sodium alginate obtained in Step 1) to deionized water to make it fully dissolve to obtain an oxidized sodium alginate solution, then add triethylamine to the oxidized sodium alginate solution and stir and mix evenly, and then add the intermediate compound obtained in Step 2) thereto and stir and react at 70 °C for 10 h to obtain modified sodium alginate. Among them, the molar ratio of oxidized sodium alginate to the intermediate compound is 1:12.5, and the dosage of the triethylamine is 5% of the total weight of the sodium alginate solution and the intermediate compound.

[0029] The preparation method of modified chitosan is as follows: (1) Add hydrophilic nano-titanium dioxide into absolute ethanol and ultrasonically disperse it for 1.5 h at an ultrasonic frequency of 100 kHz to form a titanium dioxide dispersion; wherein, the mass ratio of nano-titanium dioxide to absolute ethanol is 0.05:1; the mass percentage of titanium dioxide in the titanium dioxide dispersion is 7.5%; (2) Add the silane coupling agent 3-mercaptopropyltriethoxysilane to the titanium dioxide dispersion obtained in step (1), react at 70 °C for 6 h to obtain a reaction solution, and then add a chitosan acetic acid solution to the reaction solution and react under stirring conditions for 5 h to obtain modified chitosan. Among them, the molar ratio of titanium dioxide to the silane coupling agent 3-mercaptopropyltriethoxysilane and chitosan is 1:2.5:1.2; the mass percentage of chitosan in the chitosan acetic acid solution is 15%.

[0030] Comparative Example 1 The steps of the preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering in this comparative example are exactly the same as those in Example 2, and the only difference is that: the molar ratio of sodium periodate to sodium alginate in the modified sodium alginate is 1.2:1.5.

[0031] Comparative Example 2 The steps of the preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering in this comparative example are exactly the same as those in Example 2, and the only difference is that: 2-(dimethylamino)ethyl methacrylate in step 2) of the modified sodium alginate is replaced by dodecyl dimethyl tertiary amine.

[0032] Comparative Example 3 The steps of the preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering in this comparative example are exactly the same as those in Example 2, and the only difference is that: 2-chlorobenzamide in step 2) of the modified sodium alginate is replaced by benzamide.

[0033] Comparative Example 4 The steps of the preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering in the comparative example are exactly the same as those in Example 2, and the only difference is that: nano-titanium dioxide is not introduced into the modified chitosan.

[0034] Performance Test (1) Compressive strength and tensile strength test of the multi-crosslinked hydrogel According to the preparation methods of the multi-crosslinked hydrogels in Examples 1 to 3 and Comparative Examples 1 to 4, cylindrical hydrogels (with a diameter of 7.5 mm and a height of 9 mm) and cuboid hydrogels (with a length of 20 mm, a width of 8 mm, and a height of 2 mm) were prepared in a mold, and a thin layer of vaseline was evenly coated on the surface of the hydrogels to prevent the hydrogels from dehydrating significantly during the experiment. The universal mechanical testing machine was used to perform compression tests and tensile tests on the cylindrical hydrogels and cuboid hydrogels at a speed of 1 mm / min, and the specific test results of the compression modulus and tensile strength are shown in Table 1.

[0035] Table 1 Test results of the mechanical properties of the multi-crosslinked hydrogels prepared in Examples 1 to 3 and Comparative Examples 1 to 4 It can be seen from the data in Table 1 that the mechanical strengths of the multi-crosslinked hydrogels prepared in Examples 1 to 3 are all superior to those in Comparative Examples 1 to 4, indicating that the multi-crosslinked hydrogels prepared by the preparation method of the present invention have good mechanical strength. By comparing Comparative Example 1 with Example 2, it can be seen that since the molar ratio of sodium periodate to sodium alginate in Comparative Example 1 (1.2:1.5) is higher than the molar ratio of sodium periodate to sodium alginate (1:(1.5 - 1.8)) defined in the present invention, the aldehyde group content in the oxidized sodium alginate is too high, resulting in difficulty in forming a hydrogel from sodium alginate, and thus reducing the mechanical strength of the multi-crosslinked hydrogel in the present invention. By comparing Comparative Example 2 with Example 2, it can be seen that since 2-(dimethylamino)ethyl methacrylate in Comparative Example 2 was replaced by dodecyl dimethyl tertiary amine, the intermediate compound formed by the reaction of dodecyl dimethyl tertiary amine and 2-chlorobenzamide did not introduce a carbon-carbon double bond, resulting in no carbon-carbon double bond in the modified sodium alginate and being unable to undergo a crosslinking reaction with the thiol groups grafted on the surface of the modified chitosan to form a third crosslinked network structure, thereby reducing the mechanical strength of the multi-crosslinked hydrogel. By comparing Comparative Example 3 with Example 2, it can be seen that since 2-chlorobenzamide in Comparative Example 3 was replaced by benzamide, the intermediate compound formed by the reaction of benzamide and 2-(dimethylamino)ethyl methacrylate did not introduce a quaternary ammonium salt structure, but the quaternary ammonium salt structure has little influence on the crosslinked network structure of the hydrogel, so the mechanical strength of the multi-crosslinked hydrogel prepared in Comparative Example 3 decreased not significantly compared with that in Example 2. By comparing Comparative Example 4 with Example 2, it can be seen that nano-titanium dioxide was not added to the modified chitosan in Comparative Example 4, but titanium dioxide has little influence on the crosslinked network structure of the hydrogel, so the mechanical strength of the multi-crosslinked hydrogel prepared in Comparative Example 4 decreased not significantly compared with that in Example 2.

[0036] (2) In vitro biocompatibility test Since the in vitro biocompatibility properties of the multi-crosslinked hydrogels prepared in Examples 1 to 3 of the present invention are similar, and Comparative Examples 1 to 4 are all set in comparison with Example 2, the multi-crosslinked hydrogels prepared from Example 2 with better mechanical strength and Comparative Examples 1 to 4 were selected for in vitro biocompatibility testing. The multi-crosslinked hydrogels prepared in Example 2 and Comparative Examples 1 to 4 were respectively immersed in complete medium and soaked at 37°C for 48 h to obtain 5 kinds of hydrogel extracts with a concentration of 0.1 g / mL; among them, the complete medium was Advanced DMEM / F12 (containing R-spondin 1, 250 ng / mL; fibroblast growth factor 7, 25 ng / ml; fibroblast growth factor 10, 20 ng / ml; follistatin, 100 ng / ml; small molecule inhibitor A83-01, 500 nM; small molecule inhibitor Y-27632, 5 μM; glutathione precursor N-acetylcysteine, 1.25 mM; nicotinamide, 10 mM; cell culture additive 50×, 1×; L-glutamine replacement additive 100×, 1×; pH buffer, 10 mM; antibiotics and antifungal agent - Primocin, 50 μg / mL; penicillin / streptomycin, 100 mg / mL). The biocompatibility of the multi-crosslinked hydrogel was verified by the cytotoxicity experiment of the multi-crosslinked hydrogel extract and the culture experiment of lung cancer tissue in the multi-crosslinked hydrogel. The results of the cytotoxicity experiment on the 5th day of the multi-crosslinked hydrogel extract prepared in Example 2 and the multi-crosslinked hydrogel extracts prepared in Comparative Examples 1 to 4 are as Figure 1 shown, from Figure 1 which it can be seen that the cell survival rate of the multi-crosslinked hydrogel prepared in Example 2 of the present invention was greater than 95% on the 5th day, and the cell survival rate of the multi-crosslinked hydrogels prepared in Comparative Examples 1 to 4 was greater than 85% on the 5th day, indicating that the multi-crosslinked hydrogels prepared in the present invention and Comparative Examples 1 to 4 were non-cytotoxic. By culturing lung cancer tissue cells in the multi-crosslinked hydrogels prepared in Example 2 and Comparative Examples 1 to 4, the results are as Figure 2As shown: In the multiple cross-linked hydrogels prepared in Example 2, Comparative Example 1, and Comparative Example 2 of the present invention, as the culture time of lung cancer tissue cells extended, the lung cancer tissue cells grew significantly larger and the density increased, and the multiple cross-linked hydrogels did not show an obvious inhibitory growth effect on lung cancer tissue; while for the surface cells of the multiple cross-linked hydrogels prepared in Comparative Example 3 and Comparative Example 4, as the culture time extended, the growth of lung cancer tissue cells was not obvious, and their density also decreased. The multiple cross-linked hydrogels showed an obvious inhibitory growth effect on lung cancer tissue. This is mainly because the quaternary ammonium salt structure and nano-titanium dioxide have good biocompatibility and antibacterial properties. The quaternary ammonium salt structure was not introduced in Comparative Example 3, and nano-titanium dioxide was not added in Comparative Example 4, thus significantly reducing the biocompatibility and antibacterial properties of the multiple cross-linked hydrogels prepared in Comparative Example 3 and Comparative Example 4. This also indirectly indicates that the multiple cross-linked hydrogel prepared by the present invention has good biocompatibility and no cytotoxicity and can be safely and non-toxicly applied to the culture of organoid tissues.

[0037] Finally, it should also be noted that although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention application shall still fall within the scope covered by the present invention.

Claims

1. A preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering, characterized in that, It includes the following steps: Step S1: Dissolve modified sodium alginate and modified chitosan in water respectively, stir and mix evenly to obtain a modified sodium alginate solution and a modified chitosan solution; Step S2: After mixing the modified sodium alginate solution and the modified chitosan solution obtained in Step S1 evenly, add initiator 907, and after complete dissolution, defoam to obtain a hydrogel precursor solution; Step S3: Carry out water bath culture on the hydrogel precursor solution obtained in Step S2 under ultraviolet light irradiation to obtain a high-strength multi-crosslinked hydrogel for organ tissue engineering.

2. The preparation method of a high-strength multiple cross-linked hydrogel for organoid tissue engineering according to claim 1, wherein, The preparation method of the modified sodium alginate in Step S1 is specifically as follows: 1) Add sodium alginate to deionized water to make it fully dissolve, so that its mass percentage is 2-4%, then add sodium periodate and stir and react in the dark at room temperature. After reaction for a period of time, add ethylene glycol to terminate the reaction. After dialysis treatment and freeze-drying, obtain oxidized sodium alginate and measure its oxidation degree; 1) Add 2-chlorobenzamide to an ethanol solution, disperse it by ultrasound to obtain a 2-chlorobenzamide solution, and then slowly add an aqueous solution of 2-(dimethylamino)ethyl methacrylate to the 2-chlorobenzamide solution, stir and react at room temperature for 10-12 h. After the reaction is completed, evaporate and remove the solvent, wash the obtained product with hot acetone and filter it to obtain an intermediate compound; 3) Add the oxidized sodium alginate obtained in Step 1 to deionized water to make it fully dissolve to obtain an oxidized sodium alginate solution, then add triethylamine to the oxidized sodium alginate solution and stir and mix evenly, and then add the intermediate compound obtained in Step 2 thereto and stir and react at 60-70 °C for 8-10 h to obtain modified sodium alginate.

3. The preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering according to claim 2, wherein In Step 2), the molar ratio of sodium periodate to sodium alginate is 1:(1.5-1.8); the reaction time is 12-18 h.

4. The preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering according to claim 2, characterized in that, In Step 2), the molar ratio of 2-chlorobenzamide to 2-(dimethylamino)ethyl methacrylate is 1:(1-2).

5. The preparation method of a high-strength multiple cross-linked hydrogel for organoid tissue engineering according to claim 2, characterized in that, In Step 3), the molar ratio of oxidized sodium alginate to the intermediate compound is 1:(1-2.5), and the dosage of triethylamine is 3-5% of the total weight of the sodium alginate solution and the intermediate compound.

6. The preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering according to claim 1, characterized in that, The preparation method of the modified chitosan in Step S1 is specifically as follows: (1) Add hydrophilic nano-titanium dioxide to anhydrous ethanol and disperse it by ultrasound for 1.5-2.5 h to form a titanium dioxide dispersion; (2) Add a silane coupling agent 3-mercaptopropyltriethoxysilane to the titanium dioxide dispersion obtained in Step (1), react at a temperature of 60-70 °C for 6-8 h to obtain a reaction solution, and then add a chitosan acetic acid solution to the reaction solution and react under stirring conditions for 3-5 h to obtain modified chitosan.

7. The preparation method of a high-strength multiple cross-linked hydrogel for organoid tissue engineering according to claim 6, characterized in that, In Step (1), the mass ratio of nano-titanium dioxide to anhydrous ethanol is (0.03-0.05):1; the mass percentage of titanium dioxide in the titanium dioxide dispersion is 3.5-7.5%.

8. The preparation method of a high-strength multiple cross-linked hydrogel for organoid tissue engineering according to claim 6, wherein, In the step (2), the molar ratio of titanium dioxide to the silane coupling agent 3-mercaptopropyltriethoxysilane and chitosan is 1:(2-2.5):(0.8-1.2); the mass percentage of chitosan in the chitosan acetic acid solution is 8-15%.

9. The preparation method of a high-strength multiple-crosslinked hydrogel for organoid tissue engineering according to claim 1, characterized in that, In the step S2, the mass ratio of the modified sodium alginate solution, the modified chitosan solution to the initiator 907 is 1:(1.2-1.8):(0.05-0.08); In the step S2, the defoaming method is static defoaming or vacuum defoaming.

10. The preparation method of a high-strength multi-crosslinked hydrogel for organoid tissue engineering according to claim 1, wherein, In the step S3, the temperature of the water bath culture is 25-50 °C, and the water bath culture time is 24-28 h.

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