Hyaluronic acid / alginic acid double-network hydrogel as well as preparation method and application thereof

Abstract

The invention discloses hyaluronic acid / alginic acid dual-network hydrogel as well as a preparation method and application thereof. The hyaluronic acid / alginic acid double-network hydrogel comprises a first network and a second network which are not crosslinked with each other, and the first network and the second network are distributed in the hydrogel in an interpenetrating manner; the first network comprises an ionic cross-linked network of alginic acid with cell adhesion sites and calcium ions, and the second network comprises a chemical cross-linked network formed by covalent cross-linking of methacrylic anhydride modified hyaluronic acid. The hyaluronic acid / alginic acid double-network hydrogel disclosed by the invention is short in curing time, supports three-dimensional spreading of cells and formation of capillary lumens, has good mechanical stability, and has good application prospects in the aspects of three-dimensional culture of the cells, tissue engineering construction and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to a hyaluronic acid / alginate double network hydrogel and a preparation method and application thereof. Background Art

[0002] With the continuous development of biomedical technology, the types and application scope of biomedical materials are also expanding. Nowadays, biomedical materials can be applied to various fields, such as tissue engineering, biosensors, artificial organs, medical nanotechnology, etc. Biomaterials include various materials that can be used in medical and biological applications, such as metals, ceramics, polymer materials, etc. Among them, hydrogel, as a special polymer material, has been widely used in the biomedical field.

[0003] Hydrogel is a three-dimensional mesh-like water-containing structure with extracellular matrix-like properties, so it has excellent biocompatibility. In addition, hydrogel has high morphological adjustability and can be made into materials of different shapes and sizes, such as fibers and spheres. These hydrogels can be used in various medical applications, such as tissue engineering, cell culture, drug sustained release, etc.

[0004] Currently, the polymer materials commonly used to construct hydrogels include chitosan, polyethylene glycol, hyaluronic acid, alginate, etc. Since polymers have many active functional groups, they can be double-bonded to form hydrogels by free radical initiation; however, many double-bonded covalently cross-linked hydrogels are difficult to support cells to dynamically respond to the hydrogel matrix and reshape the microenvironment due to the stability of the covalent bonds, which limits the three-dimensional growth of cells. Therefore, it is particularly important to regulate the microstructure of the hydrogel to make it more conducive to the three-dimensional growth of cells.

[0005] Dynamically cross-linked hydrogels are more suitable for cell growth than covalently cross-linked hydrogel networks because the cross-linking points of the hydrogel network are reversible dynamic bonds such as ionic bonds, hydrogen bonds, hydrophilic and hydrophobic interactions, etc. During cell growth, the network around the cells in the hydrogel can undergo bond shearing and remodeling. However, due to the instability of dynamic bonds, it is often difficult to maintain the long-term stability of the hydrogel structure, limiting its use in applications such as cell culture or tissue engineering. Summary of the invention

[0006] The main purpose of the present invention is to provide a hyaluronic acid / alginate double network hydrogel and a preparation method and application thereof, so as to overcome the deficiencies of the prior art.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0008] An embodiment of the present invention provides a hyaluronic acid / alginate double-network hydrogel, which includes a first network and a second network that are not cross-linked to each other, and the first network and the second network are distributed in the hydrogel in an interpenetrating manner; the first network includes an ion-crosslinked network of alginate and calcium ions having cell adhesion sites, and the second network includes a chemically cross-linked network formed by covalent cross-linking of methacrylic anhydride-modified hyaluronic acid.

[0009] The present invention also provides a method for preparing the aforementioned hyaluronic acid / alginate double network hydrogel, which comprises:

[0010] Methacrylic anhydride is used to double-bond-modify hyaluronic acid to obtain methacrylic anhydride-modified hyaluronic acid;

[0011] reacting a mixed reaction system comprising alginic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, sodium salt of N-hydroxysuccinimide sulfonate, and trifluoroacetate of N-(2-aminoethyl)maleimide to obtain maleimide-modified alginic acid;

[0012] The maleimide-modified alginate is subjected to Michael addition reaction with cysteine-arginine-glycine-aspartic acid-serine at 20-25° C. to obtain alginate with cell adhesion sites;

[0013] Furthermore, the alginate with cell adhesion sites is mixed with calcium salt for reaction, and then a photoinitiator and the methacrylic anhydride-modified hyaluronic acid are added, mixed and reacted to prepare a hyaluronic acid / alginate double network hydrogel.

[0014] The embodiments of the present invention also provide the use of the aforementioned hyaluronic acid / alginate double network hydrogel in the field of cell culture or tissue engineering.

[0015] The embodiment of the present invention also provides the use of the aforementioned hyaluronic acid / alginate double network hydrogel in preparing cell culture materials.

[0016] The embodiment of the present invention further provides a three-dimensional cell culture carrier, which comprises the aforementioned hyaluronic acid / alginate double network hydrogel.

[0017] An embodiment of the present invention also provides a cell culture method, which is for non-medical diagnosis and treatment purposes, and includes: using the aforementioned hyaluronic acid / alginate double network hydrogel as a three-dimensional culture cell carrier to culture human umbilical vein endothelial cells, and promoting the proliferation, three-dimensional spreading and microvascular lumen formation of the endothelial cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention forms the first network of the hydrogel by ionically cross-linking alginate and calcium ions with cell adhesion sites, and uses covalently cross-linked methacrylic anhydride-modified hyaluronic acid to form the second network of the hydrogel. The double-network hydrogel obtained by the present invention has excellent macroscopic mechanical stability, can provide cells with good adhesion sites, a microscopic dynamic environment to support the three-dimensional growth and spreading of cells, has good biocompatibility, and has low toxicity. It can also be used as a three-dimensional culture cell carrier to culture human umbilical vein endothelial cells, thereby improving the adhesion and proliferation of endothelial cells on the hydrogel scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Schematic diagram of the preparation mechanism of hyaluronic acid / alginate double network hydrogel in a typical embodiment of the present invention;

[0021] Figure 2 This is a chemical synthesis step of a hyaluronic acid / alginate double network hydrogel in a typical embodiment of the present invention;

[0022] Figure 3 This is a graph showing the results of a hydrogen nuclear magnetic resonance spectrum of hyaluronic acid modified with methacrylic anhydride in a typical embodiment of the present invention;

[0023] Figure 4 is a graph showing the results of hydrogen nuclear magnetic resonance spectrum of maleimide-modified alginate in a typical embodiment of the present invention;

[0024] Figure 5 is a standard curve for determining different CRGDS contents using trinitrobenzenesulfonic acid in a typical embodiment of the present invention;

[0025] Figure 6 is a microscopic structure diagram of a hyaluronic acid / alginate double network hydrogel obtained in a typical embodiment of the present invention;

[0026] Figure 7 is a compression curve diagram of a hyaluronic acid / alginate double network hydrogel obtained in a typical embodiment of the present invention;

[0027] Figure 8 This is a confocal image of dead and live staining of human umbilical vein endothelium at different time points in a hyaluronic acid / alginate double network hydrogel obtained in a typical embodiment of the present invention;

[0028] Fig. 9 This is a graph showing the proliferation of human umbilical vein endothelial cells at different time points in a hyaluronic acid / alginate double network hydrogel obtained in a typical embodiment of the present invention;

[0029] Fig.10 This is a graph showing the mass change of the hyaluronic acid / alginate double network hydrogel obtained in a typical embodiment of the present invention after being immersed in PBS. DETAILED DESCRIPTION

[0030] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Specifically, as one aspect of the technical solution of the present invention, a hyaluronic acid / alginate double-network hydrogel involved therein comprises a first network and a second network that are not cross-linked to each other, and the first network and the second network are distributed in the hydrogel in an interpenetrating manner; the first network comprises an ion-crosslinked network of alginate and calcium ions having cell adhesion sites, and the second network comprises a chemically cross-linked network formed by covalent cross-linking of methacrylic anhydride-modified hyaluronic acid.

[0032] In some preferred embodiments, the hyaluronic acid / alginate double network hydrogel is composed of a first network and a second network that are not cross-linked to each other, wherein the first network is formed by cross-linking alginate having cell adhesion sites and calcium ions, and the second network is formed by covalent cross-linking of hyaluronic acid modified with methacrylic anhydride.

[0033] In some preferred embodiments, the hyaluronic acid / alginate double network hydrogel has a porous structure, and the pores contained in the hyaluronic acid / alginate double network hydrogel have a pore size of 30 to 70 μm.

[0034] In some preferred embodiments, the elastic modulus of the hyaluronic acid / alginate double network hydrogel is 1 to 3 kPa.

[0035] Specifically, the first network in the hyaluronic acid / alginate double network hydrogel of the present invention is to obtain alginate (Alg-RGD) with cell adhesion sites and calcium ions (Ca 2+) ion-crosslinked network; the second network is a chemical crosslinked network of hyaluronic acid (HA-MA) modified by photoinduced methacrylic anhydride. The first network has a crosslinked network of alginate and calcium ions with cell adhesion sites, which supports the spreading and growth of cells in a three-dimensional environment due to the microscopic reversibility of ionic crosslinking; the second network is a hyaluronic acid modified by photoinduced methacrylic anhydride, which provides stable macroscopic mechanical properties of the hydrogel due to the covalent crosslinking characteristics.

[0036] Another aspect of the embodiments of the present invention further provides a method for preparing the aforementioned hyaluronic acid / alginate double network hydrogel, which comprises:

[0037] Methacrylic anhydride is used to double-bond hyaluronic acid to prepare methacrylic anhydride-modified hyaluronic acid (HA-MA);

[0038] A mixed reaction system comprising alginic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS), and N-(2-aminoethyl)maleimide trifluoroacetate is reacted to prepare maleimide-modified alginic acid (Alg-Mal);

[0039] The maleimide-modified alginate is subjected to Michael addition reaction with cysteine-arginine-glycine-aspartic acid-serine (CRGDS) at 20-25° C. to obtain alginate with cell adhesion sites (Alg-RGD);

[0040] Furthermore, the alginate with cell adhesion sites is mixed with calcium salt for reaction, and then a photoinitiator and the methacrylic anhydride-modified hyaluronic acid are added, mixed and reacted to prepare a hyaluronic acid / alginate double network hydrogel.

[0041] In some preferred embodiments, the methacrylic anhydride-modified hyaluronic acid has a structure as shown in formula (I):

[0042]

[0043] Among them, the value of n is 500 to 1000.

[0044] In some preferred embodiments, the alginic acid having cell adhesion sites has a structure as shown in formula (II):

[0045]

[0046]

[0047] The value of n is 1000 to 5000, and CRGDS is cysteine-arginine-glycine-aspartic acid-serine.

[0048] In some preferred embodiments, the preparation method specifically includes: dissolving hyaluronic acid in water and adjusting the pH value of the obtained hyaluronic acid aqueous solution to 7-8, then adding methacrylic anhydride at 4-10°C and stirring the reaction for 18-48 hours, and then post-treating to obtain the methacrylic anhydride-modified hyaluronic acid.

[0049] Furthermore, the concentration of the hyaluronic acid aqueous solution is 0.5-2wt%.

[0050] Furthermore, the mass ratio of the hyaluronic acid to methacrylic anhydride is 1:1-10.

[0051] Furthermore, the post-treatment includes: washing the product obtained from the reaction with anhydrous ethanol precipitation for 3 to 5 times, and then dialyzing and freeze-drying; wherein the dialysis treatment time is 3 to 5 days, and the dialysis bag used in the dialysis treatment has a molecular weight cutoff of 3500 to 14000Da.

[0052] In some preferred embodiments, the preparation method specifically includes: dissolving alginate in water and adjusting the pH value of the obtained alginate aqueous solution to 4-6, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt and stirring at room temperature for 15-30 minutes, then adding N-(2-aminoethyl)maleimide trifluoroacetate and stirring for 8-12 seconds, and finally dialyzing and freeze-drying to obtain the maleimide-modified alginate.

[0053] Furthermore, the concentration of the alginate aqueous solution is 0.5-2 wt %.

[0054] Furthermore, the mass ratio of the alginic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt is 1:0.02-0.06:0.01-0.03.

[0055] Furthermore, the dialysis treatment lasts for 3 to 5 days, and the molecular weight cutoff of the dialysis bag used in the dialysis treatment is 3500 to 14000 Da.

[0056] In some preferred embodiments, the preparation method specifically includes: dissolving the maleimide-modified alginate in water and adjusting the pH value of the obtained maleimide-modified alginate aqueous solution to 8-9, then adding cysteine-arginine-glycine-aspartic acid-serine and performing a Michael addition reaction at 20-25°C, and then dialyzing and freeze-drying to obtain the alginate with cell adhesion sites.

[0057] Furthermore, the concentration of the maleimide-modified alginate aqueous solution is 0.5-2 wt %.

[0058] Furthermore, the mass ratio of the maleimide-modified alginate to cysteine-arginine-glycine-aspartic acid-serine is 1:0.01-0.05.

[0059] Furthermore, the dialysis treatment lasts for 3 to 5 days, and the molecular weight cutoff of the dialysis bag used in the dialysis treatment is 3500 to 14000 Da.

[0060] In some preferred embodiments, the preparation method specifically comprises:

[0061] Dissolving the alginate with cell adhesion sites in physiological saline to form an alginate solution with cell adhesion sites, slowly adding a calcium salt aqueous solution under stirring and stirring for 10 to 30 minutes, then adding a photoinitiator and the methacrylic anhydride-modified hyaluronic acid and mixing them evenly to form a gel precursor solution;

[0062] And, ultraviolet light is used to irradiate the gel precursor solution to react, so as to prepare the hyaluronic acid / alginate double network hydrogel.

[0063] Furthermore, the concentration of methacrylic anhydride-modified hyaluronic acid in the gel precursor solution is 0.6 to 3 g / 100 mL.

[0064] Furthermore, the concentration of alginate having cell adhesion sites in the gel precursor solution is 1-2 g / 100 mL.

[0065] Furthermore, the concentration of calcium salt in the gel precursor solution is 0.05-0.15 g / 100 mL.

[0066] Furthermore, the calcium salt aqueous solution includes calcium chloride aqueous solution, calcium sulfate aqueous solution and the like.

[0067] Furthermore, the volume ratio of the alginate solution having cell adhesion sites to the calcium salt aqueous solution is 1:0.8-1.

[0068] Furthermore, the concentration of the photoinitiator in the gel precursor solution is 0.05-0.25 g / 100 mL.

[0069] Furthermore, the photoinitiator includes phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0070] Furthermore, the wavelength of the ultraviolet light is 295-395 nm, and the intensity is 2-5 mW / cm 2 .

[0071] Furthermore, the ultraviolet light irradiation time is 20 to 60 seconds.

[0072] In some preferred embodiments, the schematic diagram of the preparation mechanism of the hyaluronic acid / alginate double network hydrogel of the present invention is as follows: Figure 1 shown.

[0073] In some more specific embodiments, the method for preparing the hyaluronic acid / alginate double network hydrogel comprises:

[0074] (1) Preparation of methacrylic anhydride-modified hyaluronic acid

[0075] A hyaluronic acid aqueous solution is prepared, the pH of the hyaluronic acid aqueous solution is adjusted to 7-8 with sodium hydroxide, methacrylic anhydride is slowly added dropwise at 4-10° C., and stirring is continued for 18-48 hours, the mixture is precipitated and washed with anhydrous ethanol for 3-5 times, dialyzed for 3-5 days, and freeze-dried to obtain a methacrylic anhydride-modified hyaluronic acid solid; preferably, the dialysis bag used for the dialysis has a molecular weight cutoff of 3500-14000Da.

[0076] (2) Preparation of maleimide-modified alginate

[0077] An alginate aqueous solution is prepared, and the pH of the alginate aqueous solution is adjusted to 4-6 with hydrochloric acid, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS) are added, and stirred at room temperature for 15-30 minutes, and then N-(2-aminoethyl) maleimide trifluoroacetate is added and stirred for 8-12 hours, dialyzed for 3-5 days, and freeze-dried to obtain maleimide-modified alginate solid; preferably, the dialysis bag used for the dialysis has a molecular weight cutoff of 3500-14000Da.

[0078] (3) Preparation of alginate with cell adhesion sites

[0079] The maleimide-modified alginate obtained in step (2) is prepared into an aqueous solution, the pH of the aqueous solution is adjusted to 8-9 with sodium hydroxide, the thiol group of cysteine-arginine-glycine-aspartic acid-serine (CRGDS) is subjected to Michael addition reaction with the maleimide-modified alginate at 20-25° C., dialyzed for 3-5 days, and freeze-dried to obtain alginate with cell adhesion sites; preferably, the dialysis bag used for the dialysis has a molecular weight cutoff of 3500-14000 Da.

[0080] (4) dissolving the alginate with cell adhesion sites obtained in step (3) in physiological saline, slowly adding a calcium chloride aqueous solution or a calcium sulfate aqueous solution while continuously stirring, adding a photoinitiator and the methacrylic anhydride-modified hyaluronic acid obtained in step (1) after stirring for 10 to 30 minutes, and mixing them evenly to form a gel precursor solution; placing the gel precursor solution in a mold, and then placing the mold under an ultraviolet lamp for sufficient irradiation to obtain a hyaluronic acid / alginate double network hydrogel.

[0081] Furthermore, the wavelength of the ultraviolet light generated by the ultraviolet lamp is 295-395nm, and the intensity is 2-5mW / cm 2 ; The irradiation time is 20 to 60 seconds.

[0082] Another aspect of the embodiments of the present invention further provides the use of the aforementioned hyaluronic acid / alginate double network hydrogel in the field of cell culture or tissue engineering.

[0083] Another aspect of the embodiments of the present invention further provides use of the aforementioned hyaluronic acid / alginate double network hydrogel in preparing cell culture materials.

[0084] Another aspect of an embodiment of the present invention further provides a three-dimensional cell culture carrier, which comprises the aforementioned hyaluronic acid / alginate double network hydrogel.

[0085] Another aspect of an embodiment of the present invention further provides a cell culture method, which is for non-medical diagnosis and treatment purposes, and comprises: using the aforementioned hyaluronic acid / alginate double network hydrogel as a three-dimensional culture cell carrier to culture human umbilical vein endothelial cells, and promoting the proliferation, three-dimensional spreading and microvascular lumen formation of the endothelial cells.

[0086] Furthermore, the loading amount of the endothelial cells on the hyaluronic acid / alginate double network hydrogel is 1 to 5 million / mL.

[0087] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0088] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0089] Example 1

[0090] Step 1: First, prepare a 0.5% mass fraction hyaluronic acid aqueous solution, adjust the pH of the hyaluronic acid aqueous solution to 7 with sodium hydroxide, slowly add methacrylic anhydride at 4°C, the mass ratio of hyaluronic acid: methacrylic anhydride is 1:10, and continue stirring for 18 hours, wash with anhydrous ethanol precipitation 3 times, dialyze for 3 days, and freeze-dry to obtain methacrylic anhydride-modified hyaluronic acid solid (HA-MA), wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500Da. Figure 2 A is the synthesis step of methacrylic anhydride-modified hyaluronic acid prepared in Example 1. Figure 3 This is the result of hydrogen nuclear magnetic resonance spectrum of methacrylic anhydride modified hyaluronic acid prepared in Example 1.

[0091] Step 2: prepare an aqueous solution of alginic acid with a mass fraction of 0.5%, adjust the pH of the aqueous solution of alginic acid to 4 with hydrochloric acid, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS), stir at room temperature for 15 minutes, then add N-(2-aminoethyl)maleimide trifluoroacetate and continue stirring for 9 hours, the mass ratio of alginic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide sulfonic acid sodium salt is 1:0.02:0.01, dialyze for 3 days, freeze-dry, and obtain maleimide-modified alginate solid (Alg-Mal), the dialysis bag used for the dialysis has a molecular weight cutoff of 3500Da. Figure 2 B is the synthesis step of maleimide-modified alginate prepared in Example 1. Figure 4 This is the result of hydrogen nuclear magnetic resonance spectrum of maleimide-modified alginate prepared in Example 1.

[0092] Step 3: After step 2, the obtained maleimide-modified alginate is prepared into a 0.5% aqueous solution, the pH of the aqueous solution is adjusted to 8 with sodium hydroxide, and the thiol group of cysteine-arginine-glycine-aspartic acid-serine (CRGDS) is subjected to Michael addition reaction with maleimide-modified alginate at 20°C, the mass ratio of maleimide-modified alginate: cysteine-arginine-glycine-aspartic acid-serine is 1:0.01, dialyzed for 3 days, freeze-dried, and alginate (Alg-RGD) with cell adhesion sites is obtained; the dialysis bag used for the dialysis has a molecular weight cutoff of 3500Da. Figure 2 Figure C is the synthesis step of the alginate with cell adhesion sites prepared in Example 1. Figure 5 The standard curve for determining different CRGDS contents using trinitrobenzene sulfonic acid was used to calculate the CRGDS content in alginate with cell adhesion sites (3.1±0.4mg CRGDS / 1g Alg-RGD).

[0093] Step 4: Dissolve the obtained alginate with cell adhesion sites in physiological saline, slowly add calcium chloride aqueous solution during continuous stirring, add photoinitiator and methacrylic anhydride modified hyaluronic acid obtained in step 1 after stirring for 10 minutes, mix well to form a gel precursor solution; place the gel precursor solution in a mold, and then place the mold in a 365nm wavelength and 3.5mW / cm 2 The hyaluronic acid / alginate double network hydrogel was prepared by irradiating the mixture under ultraviolet light for 30 seconds.

[0094] Example 2

[0095] Step 1: First, prepare a hyaluronic acid aqueous solution with a mass fraction of 1%, adjust the pH of the hyaluronic acid aqueous solution to 7.2 with sodium hydroxide, slowly add methacrylic anhydride at 5°C, the mass ratio of hyaluronic acid: methacrylic anhydride is 1:3, and continue stirring for 24 hours, wash with anhydrous ethanol precipitation 4 times, dialyze for 4 days, and freeze-dry to obtain methacrylic anhydride-modified hyaluronic acid solid, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 7500Da.

[0096] Step 2: prepare an aqueous solution of alginic acid with a mass fraction of 1%, adjust the pH of the aqueous solution of alginic acid to 4.5 with hydrochloric acid, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS), stir at room temperature for 20 minutes, then add N-(2-aminoethyl)maleimide trifluoroacetate and continue stirring for 10 hours, the mass ratio of alginic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide sulfonic acid sodium salt is 1:0.03:0.02, dialyze for 4 days, freeze-dry, and obtain maleimide-modified alginate solid, the dialysis bag used for the dialysis has a molecular weight cutoff of 14000Da.

[0097] Step 3: After step 2, the obtained maleimide-modified alginate is prepared into a 1% aqueous solution, the pH of the aqueous solution is adjusted to 8.5 with sodium hydroxide, and the thiol group of cysteine-arginine-glycine-aspartic acid-serine (CRGDS) is subjected to Michael addition reaction with maleimide-modified alginate at 21°C, the mass ratio of maleimide-modified alginate:cysteine-arginine-glycine-aspartic acid-serine is 1:0.02, dialyzed for 3 days, freeze-dried, and alginate with cell adhesion sites was obtained; the dialysis bag used for the dialysis has a molecular weight cutoff of 7500Da.

[0098] Step 4: Dissolve the obtained alginate with cell adhesion sites in physiological saline, slowly add calcium chloride aqueous solution during continuous stirring, add photoinitiator and methacrylic anhydride modified hyaluronic acid obtained in step 1 after stirring for 15 minutes, mix well to form a gel precursor solution; place the gel precursor solution in a mold, and then place the mold in a 295nm wavelength and 5mW / cm 2 The hyaluronic acid / alginate double network hydrogel was prepared by irradiating the hyaluronic acid / alginate double network hydrogel under ultraviolet light for 20 seconds.

[0099] Example 3

[0100] Step 1: First, prepare a hyaluronic acid aqueous solution with a mass fraction of 1%, adjust the pH of the hyaluronic acid aqueous solution to 7.4 with sodium hydroxide, slowly add methacrylic anhydride at 6°C, the mass ratio of hyaluronic acid to methacrylic anhydride is 1:5, and continue stirring for 30 hours, wash with anhydrous ethanol precipitation 5 times, dialyze for 5 days, and freeze-dry to obtain a methacrylic anhydride-modified hyaluronic acid solid, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 14000Da.

[0101] Step 2: prepare an aqueous solution of alginic acid with a mass fraction of 0.5%, adjust the pH of the aqueous solution of alginic acid to 5 with hydrochloric acid, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS), stir at room temperature for 25 minutes, then add N-(2-aminoethyl)maleimide trifluoroacetate and continue stirring for 11 hours, the mass ratio of alginic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide sulfonic acid sodium salt is 1:0.04:0.03, dialyze for 5 days, freeze-dry, and obtain maleimide-modified alginate solid, the dialysis bag used for the dialysis has a molecular weight cutoff of 14000Da.

[0102] Step 3: After step 2, the obtained maleimide-modified alginate is prepared into a 0.5% aqueous solution, the pH of the aqueous solution is adjusted to 9 with sodium hydroxide, and the thiol group of cysteine-arginine-glycine-aspartic acid-serine (CRGDS) is subjected to Michael addition reaction with maleimide-modified alginate at 22°C, the mass ratio of maleimide-modified alginate: cysteine-arginine-glycine-aspartic acid-serine is 1:0.03, dialyzed for 5 days, freeze-dried, and alginate with cell adhesion sites is obtained; the dialysis bag used for the dialysis has a molecular weight cutoff of 14000Da.

[0103] Step 4: Dissolve the obtained alginate with cell adhesion sites in physiological saline, slowly add calcium chloride aqueous solution during continuous stirring, add photoinitiator and methacrylic anhydride modified hyaluronic acid obtained in step 1 after stirring for 20 minutes, mix well to form a gel precursor solution; place the gel precursor solution in a mold, and then place the mold in a 395nm wavelength and 2mW / cm 2 The hyaluronic acid / alginate double network hydrogel was prepared by irradiating the hyaluronic acid / alginate double network hydrogel under ultraviolet light for 60s.

[0104] Example 4

[0105] Step 1: First, prepare a hyaluronic acid aqueous solution with a mass fraction of 1.5%, adjust the pH of the hyaluronic acid aqueous solution to 7.8 with sodium hydroxide, slowly add methacrylic anhydride at 8°C, the mass ratio of hyaluronic acid to methacrylic anhydride is 1:7, and continue stirring for 40 hours, wash with anhydrous ethanol precipitation 3 times, dialyze for 3 days, and freeze-dry to obtain methacrylic anhydride-modified hyaluronic acid solid (HA-MA), wherein the dialysis bag used for dialysis has a molecular weight cutoff of 5000Da. Figure 2 A is the synthesis step of methacrylic anhydride-modified hyaluronic acid prepared in Example 1. Figure 3This is the result of hydrogen nuclear magnetic resonance spectrum of methacrylic anhydride modified hyaluronic acid prepared in Example 1.

[0106] Step 2: prepare an aqueous solution of alginic acid with a mass fraction of 1.5%, adjust the pH of the aqueous solution of alginic acid to 5.5 with hydrochloric acid, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS), stir at room temperature for 30 minutes, then add N-(2-aminoethyl)maleimide trifluoroacetate and continue stirring for 12 hours, the mass ratio of alginic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide sulfonic acid sodium salt is 1:0.05:0.01, dialyze for 3 days, freeze-dry, and obtain maleimide-modified alginate solid (Alg-Mal), the dialysis bag used for the dialysis has a molecular weight cutoff of 5000Da. Figure 2 B is the synthesis step of maleimide-modified alginate prepared in Example 1.

[0107] Step 3: After step 2, the obtained maleimide-modified alginate is prepared into a 1.5% aqueous solution by mass, and the pH of the aqueous solution is adjusted to 8.5 with sodium hydroxide, and the thiol group of cysteine-arginine-glycine-aspartic acid-serine (CRGDS) is subjected to Michael addition reaction with maleimide-modified alginate at 23°C, and the mass ratio of maleimide-modified alginate to cysteine-arginine-glycine-aspartic acid-serine is 1:0.04. The alginate is dialyzed for 3 days and freeze-dried to obtain alginate (Alg-RGD) with cell adhesion sites; the molecular weight cutoff of the dialysis bag used for the dialysis is 5000Da. Figure 2 Figure C is the synthesis step of the alginate with cell adhesion sites prepared in Example 1.

[0108] Step 4: Dissolve the obtained alginate with cell adhesion sites in physiological saline, slowly add calcium chloride aqueous solution during continuous stirring, add photoinitiator and methacrylic anhydride modified hyaluronic acid obtained in step 1 after stirring for 25 minutes, mix well to form a gel precursor solution; place the gel precursor solution in a mold, and then place the mold in a 365nm wavelength and 5mW / cm 2 The hyaluronic acid / alginate double network hydrogel was prepared by irradiating the sample under ultraviolet light for 40 s.

[0109] Example 5

[0110] Step 1: First, prepare a 2% mass fraction hyaluronic acid aqueous solution, adjust the pH of the hyaluronic acid aqueous solution to 8 with sodium hydroxide, slowly add methacrylic anhydride at 10°C, the mass ratio of hyaluronic acid to methacrylic anhydride is 1:10, and continue stirring for 48 hours, wash with anhydrous ethanol precipitation 5 times, dialyze for 5 days, and freeze-dry to obtain methacrylic anhydride-modified hyaluronic acid solid (HA-MA), wherein the dialysis bag used for dialysis has a molecular weight cutoff of 50,000 Da. Figure 2 A is the synthesis step of methacrylic anhydride-modified hyaluronic acid prepared in Example 1. Figure 3 This is the result of hydrogen nuclear magnetic resonance spectrum of methacrylic anhydride modified hyaluronic acid prepared in Example 1.

[0111] Step 2: prepare an aqueous solution of alginic acid with a mass fraction of 2%, adjust the pH of the aqueous solution of alginic acid to 6 with hydrochloric acid, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS), stir at room temperature for 20 minutes, then add N-(2-aminoethyl)maleimide trifluoroacetate and continue stirring for 12 hours, the mass ratio of alginic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide sulfonic acid sodium salt is 1:0.06:0.03, dialyze for 5 days, freeze-dry, and obtain maleimide-modified alginate solid (Alg-Mal), the dialysis bag used for the dialysis has a molecular weight cutoff of 50000Da. Figure 2 B is the synthesis step of maleimide-modified alginate prepared in Example 1.

[0112] Step 3: After step 2, the obtained maleimide-modified alginate is prepared into a 2% aqueous solution by mass, and the pH of the aqueous solution is adjusted to 9 with sodium hydroxide. The thiol group of cysteine-arginine-glycine-aspartic acid-serine (CRGDS) is subjected to Michael addition reaction with maleimide-modified alginate at 25°C, and the mass ratio of maleimide-modified alginate to cysteine-arginine-glycine-aspartic acid-serine is 1:0.05. The alginate is dialyzed for 3 days and freeze-dried to obtain alginate (Alg-RGD) with cell adhesion sites; the molecular weight cutoff of the dialysis bag used for the dialysis is 50,000 Da. Figure 2 Figure C is the synthesis step of the alginate with cell adhesion sites prepared in Example 1.

[0113] Step 4: Dissolve the obtained alginate with cell adhesion sites in physiological saline, slowly add calcium sulfate aqueous solution during continuous stirring, add photoinitiator and methacrylic anhydride modified hyaluronic acid obtained in step 1 after stirring for 30 minutes, mix well to form a gel precursor solution; place the gel precursor solution in a mold, and then place the mold in a 395nm wavelength and 4mW / cm 2 The hyaluronic acid / alginate double network hydrogel was prepared by irradiating the hyaluronic acid / alginate double network hydrogel under ultraviolet light for 60s.

[0114] Performance Test 1

[0115] The internal structure and pore size of the hyaluronic acid / alginate double network hydrogel obtained in Example 1 were tested on a field ring scanning electron microscope tester, and the operation method thereof includes:

[0116] The prepared hydrogel was placed in liquid nitrogen, then freeze-dried, sprayed with gold at 25 mA for 3.5 min, and the microscopic morphology of the hydrogel was observed using a scanning electron microscope (eg Figure 6 As shown in FIG. 1 ). Scanning electron microscopy shows that the photocurable hydrogel has a porous microstructure with a pore size of about 30 to 70 μm.

[0117] Performance Test 2

[0118] The mechanical properties of the hyaluronic acid / alginate double network hydrogel obtained in Example 1 were tested on a universal material tester. Figure 7 It can be seen that the hydrogel has good compression cycle performance, and the hydrogel structure can still be maintained intact after being compressed 50 times.

[0119] Performance Test 3

[0120] The cell survival and cell proliferation of the hyaluronic acid / alginate double network hydrogel in Example 1 in human umbilical vein endothelial cells (HUVEC) were determined using a dead / live cell staining method and a cell technology kit (CCK-8), and the operation method includes:

[0121] The hydrogel precursor solution described in Example 1 was prepared; the fifth generation HUVEC cultured in full culture medium was washed three times with PBS buffer, digested with trypsin for 3 minutes, counted after adding full culture medium to terminate digestion, and centrifuged at 1200rpm for 3 minutes to discard the supernatant; the above hydrogel precursor solution was mixed with cells to ensure that the cell concentration was 100,000 / mL; 100 μL of the above mixed solution was taken in each 24-well plate, and ultraviolet light was applied for 30 seconds to obtain HUVEC and the hyaluronic acid / alginate double network hydrogel in Example 1, and complete culture medium was added, and cultured in a 5% CO2, 37°C incubator.

[0122] After culturing for 1, 3 and 7 days, the culture medium was removed, the cells were washed 3 times with PBS, and the cells were measured using a Live / dead kit. The cell activity was observed under a laser confocal microscope at an excitation wavelength of 488 / 561 nm; live cells were stained with calcein to emit green fluorescence, and dead cells were stained with propidium iodide to emit red fluorescence.

[0123] like Figure 8 The human umbilical vein endothelial cells survived well in the hyaluronic acid / alginate double network hydrogel obtained in Example 1 and showed three-dimensional growth, proliferation and microvascular lumen formation, indicating that the present invention can provide an excellent three-dimensional growth environment for cells and promote cell proliferation.

[0124] After 1, 3, and 7 days of culture, the culture medium was removed, 450 mL of fresh culture medium was added to each well, 50 μL of CCK-8 was added and mixed thoroughly, and the plate was placed in a 5% CO2, 37°C incubator for 4 h. 100 μL was taken out and the OD value was tested at 450 nm by a microplate reader in a 96-well plate.

[0125] like Fig. 9 After the HUVECs were blended with the hyaluronic acid / alginate double network hydrogel in Example 1, the cells survived well after 1 day of culture and showed obvious proliferation after 7 days of culture, indicating that the hyaluronic acid / alginate double network hydrogel obtained in Example 1 has low toxicity, good biocompatibility, and supports cell proliferation.

[0126] Performance Test 4

[0127] The hyaluronic acid / alginate double network hydrogel obtained in Example 1 was placed in a 37°C environment, immersed in PBS, and the solution was changed every 2 days. The mass was freeze-dried and weighed at different time points, and the percentage of the remaining freeze-dried sample mass to the initial freeze-dried sample mass was calculated.

[0128] like Fig.10 The quality of the hyaluronic acid / alginate double network hydrogel in Example 1 hardly decreases after being immersed in PBS for 7 days, and it can still remain stable after 21 days, which proves that the hydrogel has good long-term stability.

[0129] Comparative Example 1:

[0130] Generally speaking, conventional hydrogel networks cross-linked by covalent bonds are very stable after gelation and are difficult to support the morphogenesis of cells during growth, which limits their application in biomedicine.

[0131] Compared with Control Example 1, the hydrogel obtained in Example 1 of the present invention provides a hyaluronic acid / alginate double network hydrogel. During cell growth, due to the ionic cross-linked network formed by alginate and calcium ions with cell adhesion sites, the network around the cells in the hydrogel can undergo bond shearing and remodeling, and is therefore more suitable for cell growth than a purely covalently cross-linked hydrogel network.

[0132] Comparative Example 2:

[0133] Generally speaking, conventional hydrogel network gels cross-linked by dynamic bonds cannot maintain shape stability for a long time, making it difficult to carry out long-term cell culture and tissue engineering applications.

[0134] Compared with Control Example 2, the hydrogel obtained in Example 1 of the present invention provides a hyaluronic acid / alginate double network hydrogel. During the cell growth process, the covalently cross-linked network formed by hyaluronic acid modified with methacrylic anhydride is stable, and the hydrogel can be cultured stably for a long time. After 7 days of culture, the hydrogel can still maintain good mechanical properties.

[0135] Comparative Example 3:

[0136] Generally speaking, hydrogels formed by polysaccharides are difficult to support the adhesion of cells for culture in hydrogels due to the lack of corresponding cell adhesion sites.

[0137] Compared with Control Example 3, in the hydrogel obtained in Example 1 of the present invention, the cell adhesion sites (CRGDS) of alginate are modified, so that the cultured cells can interact with the hydrogel matrix, and the cells can grow and spread well in the three-dimensional cultured hydrogel.

[0138] Comparative Example 4:

[0139] If the first network and the second network of the hydrogel in the present application are cross-linked with each other, the covalent hyaluronic acid hydrogel network will limit the dynamic changes of the dynamic alginate hydrogel network, making it difficult to support the morphological changes of cells used for culture in the hydrogel.

[0140] Compared with Control Example 4, the hydrogel obtained in Example 1 of the present invention not only maintains the mechanical properties of the hydrogel by constructing two non-crosslinked networks, but also enables the cultured cells to interact with the hydrogel matrix, and the cells can grow and spread well in the three-dimensionally cultured hydrogel.

[0141] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0142] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A hyaluronic acid / alginate double network hydrogel, characterized in that: The invention comprises a first network and a second network which are not cross-linked to each other, and the first network and the second network are distributed in the hydrogel in an interpenetrating manner; the first network comprises an ion cross-linked network of alginate and calcium ions having cell adhesion sites, and the second network comprises a chemical cross-linked network formed by covalent cross-linking of hyaluronic acid modified with methacrylic anhydride.

2. The hyaluronic acid / alginate double network hydrogel according to claim 1, characterized in that: The hyaluronic acid / alginate double network hydrogel has a porous structure, and the pores contained in the hyaluronic acid / alginate double network hydrogel have a pore size of 30 to 70 μm; And / or, the elastic modulus of the hyaluronic acid / alginate double network hydrogel is 1 to 3 kPa.

3. The method for preparing the hyaluronic acid / alginate double network hydrogel according to claim 1 or 2, characterized in that: include: Methacrylic anhydride is used to double-bond-modify hyaluronic acid to obtain methacrylic anhydride-modified hyaluronic acid; reacting a mixed reaction system comprising alginic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, sodium salt of N-hydroxysuccinimide sulfonate, and trifluoroacetate of N-(2-aminoethyl)maleimide to obtain maleimide-modified alginic acid; The maleimide-modified alginate is subjected to a Michael addition reaction with cysteine-arginine-glycine-aspartic acid-serine to obtain alginate with cell adhesion sites; Furthermore, the alginate with cell adhesion sites is mixed with calcium salt for reaction, and then a photoinitiator and the methacrylic anhydride-modified hyaluronic acid are added, mixed and reacted to prepare a hyaluronic acid / alginate double network hydrogel.

4. The preparation method according to claim 3, characterized in that: The methacrylic anhydride-modified hyaluronic acid has a structure as shown in formula (I): Among them, the value of n is 500 to 1000; And / or, the alginate having cell adhesion sites has a structure as shown in formula (II): The value of n is 1000 to 5000, and CRGDS is cysteine-arginine-glycine-aspartic acid-serine.

5. The preparation method according to claim 3, characterized in that: Specifically include: Dissolving hyaluronic acid in water and adjusting the pH value of the obtained hyaluronic acid aqueous solution to 7-8, then adding methacrylic anhydride at 4-10° C. and stirring for reaction for 18-48 hours, and then post-treating to obtain the methacrylic anhydride-modified hyaluronic acid; Preferably, the concentration of the hyaluronic acid aqueous solution is 0.5 to 2 wt %; preferably, the mass ratio of the hyaluronic acid to methacrylic anhydride is 1:1 to 10; Preferably, the post-treatment comprises: washing the product obtained from the reaction with anhydrous ethanol precipitation for 3 to 5 times, and then dialyzing and freeze-drying the product; wherein the dialysis treatment time is 3 to 5 days, and the dialysis bag used in the dialysis treatment has a molecular weight cutoff of 3500 to 14000Da.

6. The preparation method according to claim 3, characterized in that: Specifically include: Dissolve alginic acid in water and adjust the pH value of the obtained alginic acid aqueous solution to 4-6, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt and stir at room temperature for 15-30 minutes, then add N-(2-aminoethyl)maleimide trifluoroacetate and stir for 8-12 seconds, and finally dialyze and freeze-dry to obtain the maleimide-modified alginate; Preferably, the concentration of the alginic acid aqueous solution is 0.5-2 wt %; preferably, the mass ratio of alginic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and sodium salt of N-hydroxysuccinimide sulfonate is 1:0.02-0.06:0.01-0.03; Preferably, the dialysis treatment lasts for 3 to 5 days, and the molecular weight cutoff of the dialysis bag used in the dialysis treatment is 3500 to 14000 Da.

7. The preparation method according to claim 3, characterized in that: Specifically include: The maleimide-modified alginate is dissolved in water and the pH value of the obtained maleimide-modified alginate aqueous solution is adjusted to 8-9, and then cysteine-arginine-glycine-aspartic acid-serine is added to perform Michael addition reaction at 20-25° C., and then dialyzed and freeze-dried to obtain the alginate with cell adhesion sites; Preferably, the concentration of the maleimide-modified alginate aqueous solution is 0.5-2 wt %; preferably, the mass ratio of the maleimide-modified alginate to cysteine-arginine-glycine-aspartic acid-serine is 1:0.01-0.05; Preferably, the dialysis treatment lasts for 3 to 5 days, and the molecular weight cutoff of the dialysis bag used in the dialysis treatment is 3500 to 14000 Da.

8. The preparation method according to claim 3, characterized in that: Specifically include: Dissolving the alginate with cell adhesion sites in physiological saline to form an alginate solution with cell adhesion sites, slowly adding a calcium salt aqueous solution under stirring and stirring for 10 to 30 minutes, then adding a photoinitiator and the methacrylic anhydride-modified hyaluronic acid and mixing them evenly to form a gel precursor solution; And, ultraviolet light is used to irradiate the gel precursor solution to react, so as to prepare the hyaluronic acid / alginate double network hydrogel.

9. The preparation method according to claim 8, characterized in that: The concentration of methacrylic anhydride-modified hyaluronic acid in the gel precursor solution is 0.6 to 3 g / 100 mL; and / or, the concentration of alginate having cell adhesion sites in the gel precursor solution is 1 to 2 g / 100 mL; and / or, the concentration of calcium salt in the gel precursor solution is 0.05-0.15 g / 100 mL; and / or, the calcium salt aqueous solution includes calcium chloride aqueous solution and / or calcium sulfate aqueous solution; and / or, the volume ratio of the alginate solution having cell adhesion sites to the calcium salt aqueous solution is 1:0.8-1; and / or, the concentration of the photoinitiator in the gel precursor solution is 0.05 to 0.25 g / 100 mL; and / or, the photoinitiator comprises phenyl-2,4,6-trimethylbenzoyl lithium phosphite; And / or, the wavelength of the ultraviolet light is 295-395nm, and the intensity is 2-5mW / cm 2 ; And / or, the ultraviolet light irradiation time is 20 to 60 seconds.

10. Use of the hyaluronic acid / alginate double network hydrogel according to claim 1 or 2 in the field of cell culture or tissue engineering.

11. Use of the hyaluronic acid / alginate double network hydrogel according to claim 1 or 2 in preparing cell culture materials.

12. A three-dimensional cell culture carrier, characterized in that The invention comprises the hyaluronic acid / alginate double network hydrogel according to claim 1 or 2.

13. A cell culture method for non-medical diagnosis and treatment purposes, characterized in that: include: Using the hyaluronic acid / alginate double network hydrogel according to claim 1 or 2 as a three-dimensional culture cell carrier to culture human umbilical vein endothelial cells, and promoting the proliferation, three-dimensional spreading and microvascular lumen formation of the endothelial cells; Preferably, the loading amount of the endothelial cells on the hyaluronic acid / alginate double network hydrogel is 1 to 5 million / mL.

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