Hydrogel particles having high reactivity, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are hydrogel particles having high reactivity, a preparation method therefor, and the use thereof. The preparation method comprises the following steps: (1) polymerizing double bond-modified polyethylene glycol, a modified natural polymer and an optional active functional monomer to obtain a gel; (2) crushing the gel to obtain gel particles; and (3) the gel particles and an enzyme undergoing an enzymolysis reaction so as to obtain the reactive hydrogel particles. The double bond-modified polyethylene glycol contains a double bond and a polyethylene glycol chain segment, the modified natural polymer contains a sulfydryl or a double bond, the active functional monomer is selected from a sulfydryl-functionalized bioadhesive peptide or a sulfydryl-functionalized cholesterol, and the enzyme is an enzyme corresponding to the natural polymer. The preparation method of the present invention involves a simple process and can achieve mass production; the obtained hydrogel particles have high reactivity and thus can be used for subsequent secondary crosslinking; in addition, the hydrogel particles further have good biocompatibility.

Description

Highly reactive hydrogel particles, preparation method and application thereof Technical Field

[0001] The present invention relates to the technical field of hydrogel particles, in particular to highly reactive hydrogel particles and a preparation method and application thereof. Background Art

[0002] Granular hydrogels are versatile materials with broad application prospects. Their properties, including injectability, self-healing, and modularity, give them significant potential for medical applications. First, their injectability makes them ideal vehicles for cell and drug delivery. Precise positioning and delivery via a syringe allows precise delivery of the granular hydrogel to the target site, enabling effective treatment and delivery. Second, granular hydrogels exhibit self-healing capabilities, capable of repairing cracks or breaks. This property increases the gel's stability and longevity, reducing the risk of material damage and failure. Furthermore, granular hydrogels are modular, allowing for customization and combination. This allows the gel's components to be flexibly adjusted to meet the individual requirements of specific applications, such as cell delivery, tissue engineering, and bioprinting. To further enhance the performance of granular hydrogels, surface modification of the hydrogel particles is crucial. By introducing specific bioactive molecules or surface modifiers, the gel's ability to interact with cells or tissues can be enhanced, improving biocompatibility and bioadhesion. Furthermore, customized mechanical properties are crucial. Different types of tissues and cells have different requirements for materials, so it is necessary to adjust factors such as the composition and degree of cross-linking of the gel particles to achieve customization of stiffness, elasticity, and deformability to meet the culture and growth requirements of different types of tissue cells. Therefore, the development of a large-scale preparation method for highly reactive hydrogel particles is of great practical significance. This method can easily perform surface modification and mutual cross-linking, meeting the cutting-edge requirements of the biomedical field for personalized needs of new material carriers. With the in-depth understanding and continuous innovation of the properties of particle gels, this material will bring more innovative applications to fields such as cell delivery, tissue engineering, and bioprinting.

[0003] Traditional methods for modifying active hydrogel particles include microdroplet self-assembly, layer-by-layer chemical assembly, and chemical grafting. While these methods offer high controllability and produce highly monodisperse hydrogel particles, they suffer from low throughput and are not suitable for large-scale production. Furthermore, the need for surfactants and oil-based dispersions necessitates complex purification steps and extremely high production costs.

[0004] For example, Chinese patent CN112458075A discloses a double-crosslinked particle gel, the preparation method of which includes the following steps: (1) preparing a precursor solution of polyvinyl polyethylene glycol polymer, thiolated sodium alginate, and microorganisms; (2) preparing hydrogel microspheres encapsulating the microorganisms, preparing the microspheres by oil-in-water emulsion dispersion and solidifying the microspheres; (3) preparing a column filled with the double-crosslinked particle gel for biocatalytic reaction to enhance the microbial catalytic process. This preparation method involves the oil-in-water emulsion dispersion method, which is relatively complex and cannot be produced on a large scale. In addition, the resulting double-crosslinked particle gel has very low reactivity and cannot be used for subsequent secondary crosslinking.

[0005] For example, Chinese patent CN114773608A discloses a long-acting hyaluronic acid for the treatment of osteoarthritis. It is prepared by blending and modifying branched macromolecules, thiolated hyaluronic acid, and a solvent. While the branched macromolecules can be grafted onto the thiolated hyaluronic acid backbone, the resulting modified hyaluronic acid has very low reactivity and cannot be used for subsequent secondary cross-linking. Furthermore, the preparation process involves a dialysis step, requiring the use of large amounts of dialysate, making large-scale production difficult.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to prepare hydrogel particles with high reactivity. The preparation process is simple and can be produced on a large scale. The prepared hydrogel particles have high reactivity and can be used for subsequent secondary cross-linking. The hydrogel particles also have good biocompatibility.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for preparing reactive hydrogel particles, comprising the following steps:

[0010] (1) polymerizing double-bond modified polyethylene glycol, modified natural polymer, and optional active functional monomer to obtain a gel;

[0011] (2) crushing the gel to obtain gel particles;

[0012] (3) subjecting the gel particles to an enzymatic hydrolysis reaction with an enzyme to obtain the reactive hydrogel particles;

[0013] The double-bond modified polyethylene glycol has a double bond and a polyethylene glycol segment, the modified natural polymer has a sulfhydryl group or a double bond, the active functional monomer is selected from a sulfhydryl- or double-bond functionalized bioadhesive peptide, a sulfhydryl- or double-bond functionalized cholesterol, and the enzyme is the enzyme corresponding to the natural polymer.

[0014] In the present invention, the optional active functional monomer means that the active functional monomer may or may not be added in step (1). When the active functional monomer is not added, the double bond-modified polyethylene glycol and the modified natural polymer undergo a polymerization reaction. When the active functional monomer is added, the double bond-modified polyethylene glycol, the modified natural polymer, and the active functional monomer undergo a polymerization reaction.

[0015] In the present invention, the gel obtained in step (1) has unreacted double bonds or other active groups, and the gel is in a cross-linked state. Most of the unreacted double bonds or other active groups are located inside the cross-linked structure of the gel. Step (3) uses enzymes corresponding to natural polymers to enzymatically hydrolyze and cut the natural polymer segments in the gel, so that the unreacted double bonds or other active groups in step (1) can be exposed to the surface of the hydrogel particles, so that the hydrogel particles have high reactivity. The high reactivity is beneficial to the subsequent application of the hydrogel particles, such as cell culture, or secondary cross-linking using the high reactivity.

[0016] In some embodiments, the double-bond modified polyethylene glycol is selected from one or more combinations of polyethylene glycol monoacrylate, polyethylene glycol diacrylate, polyethylene glycol monomethacrylate, polyethylene glycol dimethacrylate, hyperbranched polyethylene glycol diacrylate, and hyperbranched polyβ-hydrazide ester.

[0017] In some embodiments, the hyperbranched polyethylene glycol diacrylate is prepared from polyethylene glycol diacrylate by living polymerization.

[0018] In some embodiments, the hyperbranched polyethylene glycol diacrylate is prepared by RAFT living polymerization of polyethylene glycol diacrylate.

[0019] In some embodiments, the chain transfer agent used in the RAFT living polymerization is The structural formula of the polyethylene glycol diacrylate is

[0020] In some embodiments, the structural formula of the hyperbranched polyethylene glycol diacrylate is:

[0021] In some embodiments, the hyperbranched poly (β-hydrazide) is prepared by Michael addition reaction of 3,3'-dithiodipropionohydrazide with a primary amine and polyethylene glycol diacrylate.

[0022] In some embodiments, the hyperbranched poly (β-hydrazide) has the structural formula:

[0023] wherein n=5-20, preferably 10. The hyperbranched poly-β-hydrazide ester contains disulfide bonds and has a relatively small positive charge, while cells are slightly negatively charged. When it is used as a polymerization monomer in step (1), the resulting hydrogel particles will have a better cell adhesion effect when subsequently used in fields such as cell culture.

[0024] In some embodiments, the natural polymer is selected from one or more combinations of collagen, chitosan, gelatin, dextran, sodium alginate and hyaluronic acid.

[0025] In some embodiments, the bioadhesive peptide is a small molecule peptide containing sequence fragments such as arginine-glycine-aspartic acid-cysteine, mercaptopropionic acid-arginine-glycine-aspartic acid, or glycine-arginine-glycine-aspartic acid-serine-proline-cysteine.

[0026] In some embodiments, the thiol-functionalized bioadhesive peptide is thiol-functionalized RGD, whose amino acid sequence is GRGDSPC.

[0027] In some embodiments, the polymerization reaction in step (1) is carried out in water or PBS buffer, the mass volume concentration of the double bond-modified polyethylene glycol in water or PBS buffer is 2 to 20%, the mass volume concentration of the modified natural polymer in water or PBS buffer is 0.5 to 20%, and the mass volume concentration of the active functional monomer in water or PBS buffer is 0 to 20%.

[0028] In some embodiments, the mass ratio of the double-bond modified polyethylene glycol to the modified natural polymer active functional monomer is 5:1.5:0.5.

[0029] In some embodiments, the modified natural polymer has a thiol group, the polymerization reaction is Michael addition polymerization, and the polymerization reaction temperature is 0 to 30°C.

[0030] In some embodiments, the modified natural polymer has a double bond, the polymerization reaction is a free radical polymerization, and the polymerization reaction is carried out in the presence of a photoinitiator.

[0031] In some embodiments, the pulverization in step (2) is selected from extrusion crushing, grinding crushing or impact crushing.

[0032] Preferably, the pulverization in step (2) is grinding and crushing.

[0033] Furthermore, the grinding and crushing are performed 1 to 5 times. The more grinding times, the smaller the particle size of the hydrogel particles finally obtained.

[0034] Furthermore, the grinding and crushing time each time is 1-10 minutes, preferably 5 minutes.

[0035] In some embodiments, the reactive hydrogel particles have a particle size of 30 μm to 300 μm.

[0036] Preferably, the particle size of the reactive hydrogel particles is 100 μm to 150 μm.

[0037] In some embodiments, the polymerization reaction in step (1) is carried out at a pH of 7-8.

[0038] In some embodiments, the concentration of the enzyme in step (3) is 5U / mL-2000U / mL

[0039] Preferably, the polymerization reaction in step (1) is carried out at a pH of 7.2 to 7.4.

[0040] In some embodiments, the enzyme in step (3) is selected from one or more combinations of hyaluronidase, sodium alginate lyase, pancreatin, chitosanase or collagenase.

[0041] The present invention also provides reactive hydrogel particles prepared by the above-mentioned method for preparing reactive hydrogel particles. The hydrogel particles have high reactivity and can be used for subsequent secondary crosslinking. The hydrogel particles also have good biocompatibility.

[0042] The present invention also provides the use of the aforementioned reactive hydrogel particles for cell culture, tissue engineering, biological scaffolds, drug delivery or bioprinting.

[0043] The hydrogel particles of the present invention are primarily polymerized from double-bond-modified polyethylene glycol and modified natural polymers with thiol groups or double bonds. A gel block is first prepared by polymerization, which is then crushed to form gel particles. The resulting particles are then treated with enzymes to expose active sites, resulting in highly reactive hydrogel particles. Biomolecules, drugs, or cells can more easily interact with the active sites, enhancing their availability. The reactive sites can support cell adhesion, promoting cell attachment and proliferation. They can also provide binding sites or targeting receptors for more drugs, improving drug delivery and efficiency.

[0044] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0045] (1) High reactivity. The gel obtained in step (1) of the present invention has active double bonds that have not been completely reacted or other introduced active groups. The gel is in a cross-linked state. Most of the double bonds that have not been completely reacted or other active groups are located inside the cross-linked structure of the gel or are coated by natural polymers and cannot be effectively exposed to the surface of the hydrogel. Step (3) uses enzymes corresponding to natural polymers to enzymatically hydrolyze and cut the natural polymer segments in the gel, which can expose the double bonds or other active groups that have not been completely reacted in step (1) to the surface of the hydrogel particles, making the hydrogel particles have high chemical reactivity. At the same time, the exposure of a large number of active groups will greatly enhance the interaction between living cells and the material surface, promoting their early attachment and proliferation on the material surface. In addition, the above-mentioned active double bonds can subsequently be cross-linked with modified polymers for secondary cross-linking. The obtained secondary cross-linked gel particles can increase the cross-linking network density inside the particle gel, affect the pore structure and pore size distribution of the particle gel, and thus regulate the release rate of drugs or bioactive substances.

[0046] Double-bond-modified polyethylene glycol can effectively adjust the mechanical properties of hydrogel particles. Double-bond-modified polyethylene glycol can also avoid excessive degradation of gel particles during enzymatic hydrolysis in step (3), effectively promoting the exposure of double bonds and other active groups.

[0047] (2) The preparation process is simple, enabling the mass production of highly reactive hydrogel particles at low cost. The prior art requires the use of relatively complex processes, such as oil-in-water emulsification dispersion, to prepare similar hydrogel particles. These processes are not conducive to large-scale batch production. The present invention, however, utilizes a simple pulverization and enzymatic hydrolysis process to obtain highly reactive hydrogel particles. The preparation process is gentle and green, requiring no complex equipment or operations. The preparation method of the present invention does not involve the use of surfactants or oil phases, simplifying the process while significantly reducing subsequent purification costs.

[0048] (3) The obtained hydrogel particles have good biocompatibility. The raw materials used to prepare the hydrogel particles of the present invention are safe, non-toxic, and have high biocompatibility. The obtained hydrogel particles have application prospects in the fields of cell culture, tissue engineering, drug delivery, and bioprinting. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a H-NMR spectrum of hyperbranched polyethylene glycol diacrylate;

[0050] Figure 2 is a H-NMR spectrum of hyperbranched poly-β-hydrazide;

[0051] Figure 3 is a H-NMR spectrum of hyaluronic acid and thiolated hyaluronic acid, wherein HA is hyaluronic acid and HA-SH is thiolated hyaluronic acid;

[0052] FIG4 is a flow chart of the preparation of hydrogel particles of the present invention;

[0053] FIG5 is a SEM image of the hydrogel particles prepared in Example 1;

[0054] FIG6 is a microscopic image and size distribution of hydrogel particles of Examples 1-3 at different grinding times;

[0055] FIG7 is a diagram of cell-hydrogel adhesion growth after 12 h of hyaluronidase treatment in Example 1 and Comparative Example 1;

[0056] FIG8 is a diagram of cell adhesion and proliferation in Example 8 and Comparative Example 2;

[0057] Figure 9 is a photo of the hydrogel particles before and after secondary cross-linking after 12 hours of hyaluronidase treatment in Example 8;

[0058] Figure 10 is the strain-modulus curve of the hydrogel particles before and after secondary crosslinking after hyaluronidase treatment for 12 hours in Example 1;

[0059] FIG11 is a strain-modulus curve of the hydrogel particles of Example 10 and the comparative particles;

[0060] FIG12 is a degradation curve of the hydrogel particles of Example 11 and the comparative particles under 500 U / mL hyaluronidase conditions;

[0061] FIG13 is a live-dead staining graph showing the biocompatibility of a 5 mg / mL active hydrogel material extract of Example 1. DETAILED DESCRIPTION

[0062] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0063] Preparation Example 1

[0064] Preparation of hyperbranched polyethylene glycol diacrylate (HB-PEGDA):

[0065] DS (structural formula ) as RAFT agent, 2′-azobis(2-methylnitrile) (AIBN) as initiator, and homopolymerized polyethylene glycol diacrylate PEGDA (structural formula: )(Average Mn=575)(0.4mol·L -1 ) as monomers, RAFT polymerization was carried out in butanone at 70°C with the feedstock; the molar ratio of [PEGDA]: [DS]: [AIBN] was 25:1:1.4. The polymer product HB-PEGDA was obtained, and its structural formula is: Its NMR characterization is shown in Figure 1.

[0066] Preparation Example 2

[0067] Preparation of hyperbranched poly (β-hydrazide) ester (HB-PBHE):

[0068] 1) Polyethylene glycol diacrylate (PEGDA) and 3,3'-dithiodipropionylhydrazide (DTP) were dissolved in dimethyl sulfoxide (DMSO) at a molar ratio of 5:2 and reacted at 90°C. The resulting HB-PBHE has the following structural formula:

[0069] Its NMR characterization is shown in Figure 2.

[0070] Example 1

[0071] 30 mg of thiolated hyaluronic acid (HA-SH, whose NMR spectrum is shown in Figure 3) was dissolved in 1 mL of deionized water to prepare a 3% (w / v) precursor solution A. 100 mg of the hyperbranched poly-β-hydrazide ester (HB-PBHE) prepared in Preparation Example 2 was dissolved in 1 mL of deionized water to prepare a 10% (w / v) precursor solution B. The pH of precursor solution A was adjusted to 7.2 with aqueous sodium hydroxide solution. Precursor solution A and precursor solution B were then mixed in a 1:1 volume ratio and allowed to stand to obtain a hydrogel. The prepared hydrogel was ground using a 100-mesh metal screen for 5 minutes and then treated with 500 U / mL of hyaluronidase for 12 hours to obtain active hydrogel particles with a particle size of 100 μm. The SEM image of the hydrogel is shown in Figure 5, and the particle size diagram is the image corresponding to the first grinding step in Figure 6.

[0072] Example 2

[0073] The method is basically the same as Example 1, except that the grinding is performed twice, with each grinding time being the same as Example 1. The particle size of the finally obtained active hydrogel particles is 70-80 μm. The particle size diagram is the diagram corresponding to the second grinding in Figure 6.

[0074] Example 3

[0075] The method is basically the same as Example 1, except that the grinding is performed three times, with each grinding time being the same as Example 1. The particle size of the finally obtained active hydrogel particles is 50-60 μm. The particle size diagram is the diagram corresponding to the third grinding in Figure 6.

[0076] It can be seen that with the increase of grinding times, the diameter of hydrogel particles continues to decrease.

[0077] Example 4

[0078] 20 mg of thiolated sodium alginate was dissolved in 1 mL of deionized water to prepare a 2% (w / v) precursor solution A. 100 mg of hyperbranched polyethylene glycol diacrylate (HB-PEGDA) prepared in Preparation Example 1 was dissolved in 1 mL of deionized water to prepare a 10% (w / v) precursor solution B. The pH of precursor solution A was adjusted to 7.3, and the adjusted precursor solution A and precursor solution B were mixed in a 2:1 volume ratio. The mixture was allowed to stand to obtain a hydrogel. The prepared hydrogel was ground using a 100-mesh metal screen for 5 minutes and then treated with 500 U / mL of sodium alginate lyase for 12 hours to obtain active hydrogel particles with a particle size distribution of 100-150 μm.

[0079] Example 5

[0080] 20 mg of thiolated gelatin (Gel-SH) was dissolved in 1 mL of deionized water to prepare a 2% (w / v) precursor solution A. 100 mg of hyperbranched polyethylene glycol diacrylate (HB-PEGDA) prepared in Preparation Example 1 was dissolved in 1 mL of deionized water to prepare a 10% (w / v) precursor solution B. The pH of precursor solution A was adjusted to 7.4, and the adjusted precursor solution A and precursor solution B were mixed in a 2:1 volume ratio and allowed to stand to obtain a hydrogel. The prepared hydrogel was ground using a 100-mesh metal screen for 5 minutes and then treated with 500 U / mL of collagenase for 12 hours to obtain active hydrogel particles with a particle size distribution of 100-150 μm.

[0081] Example 6

[0082] 20 mg of thiolated chitosan (SH-CS) was dissolved in 1 mL of deionized water to prepare a 2% (w / v) precursor solution A. 100 mg of the hyperbranched poly-β-hydrazide ester (HB-PBHE) prepared in Preparation Example 2 was dissolved in 1 mL of deionized water to prepare a 10% (w / v) precursor solution B. The pH of precursor solution A was adjusted to 7.4, and then the adjusted precursor solution A and precursor solution B were mixed in a 2:1 volume ratio and allowed to stand to obtain a hydrogel. The prepared hydrogel was ground using a 100-mesh metal screen for 5 minutes and then treated with 500 U / mL of chitosanase for 12 hours to obtain active hydrogel particles with a particle size distribution of 100-150 μm.

[0083] Comparative Example 1

[0084] The method is basically the same as Example 1, except that the step of treating the mixture with 500 U / mL hyaluronidase for 12 hours is omitted. That is, the hydrogel particles are directly obtained after grinding.

[0085] Example 7

[0086] The active hydrogel particles prepared in Example 1 and the hydrogel particles prepared in Comparative Example 1 were respectively subjected to secondary crosslinking with 1 mg / mL mercaptopolyethylene glycol rhodamine B, with the mass ratio of the hydrogel particles to mercaptopolyethylene glycol rhodamine B being 500:1, and incubated for 30 minutes. After the incubation, the particles were washed with PBS and observed under a fluorescence microscope. The results are shown in FIG7 , wherein the enzymatic hydrolysis in the left figure of FIG7 and the enzymatic hydrolysis for 3 h in the right figure represent the results corresponding to Example 1, and the untreated hydrogel particles in the left figure of FIG7 and the enzymatic hydrolysis for 0 h in the right figure represent the results corresponding to Comparative Example 1. It can be seen that the hydrogel particles treated with the enzyme in step (3) of the present invention can effectively expose the active functional groups, and the prepared hydrogel particles have high reactivity and can subsequently react with small molecules or macromolecules containing sulfhydryl groups, etc., while the gel particles that are not treated with the enzyme have extremely low reactivity.

[0087] Example 8

[0088] 30 mg of thiolated hyaluronic acid was dissolved in 1 mL of deionized water to prepare a 3% (w / v) precursor solution A. 100 mg of the hyperbranched poly-β-hydrazide prepared in Preparation Example 2 and 10 mg of the thiolated adhesion peptide RGD-SH (amino acid sequence: GRGDSPC) were dissolved in 1 mL of deionized water to prepare a precursor solution B. The pH of precursor solution A was adjusted to 7.4, and then the adjusted precursor solution A and precursor solution B were mixed in a 1:1 volume ratio. The mixture was allowed to stand to obtain a hydrogel. The prepared hydrogel was ground using a 100-mesh metal screen for 5 minutes, then treated with 500 U / mL hyaluronidase for 12 hours, and washed three times with PBS to obtain active hydrogel particles. The active hydrogel particles were then soaked in MEM complete medium for 1 hour, after which 5,000 L929 cells were added. The culture plate was placed in an incubator for culture and the cell growth was observed. The results of culturing for 48 h, 72 h, and 96 h are shown in the enzyme treatment pictures in FIG8 . It can be seen that the active groups of the active hydrogel particles obtained after enzyme treatment are exposed, which can effectively enable cell adhesion and proliferation.

[0089] Comparative Example 2

[0090] The method was essentially the same as Example 8, differing only in that the additional 12-hour treatment with 500 U / mL hyaluronidase was omitted. Instead, the hydrogel particles were obtained directly after grinding. The results of culturing cells for 24, 36, and 48 hours are shown in the untreated images in Figure 8 . It can be seen that the enzymatic treatment step of the present invention resulted in higher reactivity of the hydrogel particles, resulting in better cell adhesion and proliferation when used in cell culture.

[0091] Example 9

[0092] The active hydrogel particles prepared in Example 1 are mixed with thiolated hyaluronic acid and subjected to secondary cross-linking. As shown in the left figure of Figure 9, the strength of the hydrogel particles is greater after the secondary cross-linking, because the active functional groups exposed on the surface of the thiolated hyaluronic acid and the active hydrogel particles can react again. The right figure in Figure 9 shows the active hydrogel particles prepared in Example 1, which have not undergone secondary cross-linking. It can be seen that the strength of the hydrogel particles is slightly lower. Before and after the secondary cross-linking, the storage modulus of the hydrogel particles is shown in Figure 10, and it can be seen that the storage modulus increases by two orders of magnitude after the secondary cross-linking. Therefore, the highly reactive hydrogel particles prepared by the present invention can subsequently improve the mechanical properties through secondary cross-linking, and have broad application prospects in the fields of tissue engineering, biological scaffolds, drug delivery carriers, and biological printing.

[0093] Example 10

[0094] The method is basically the same as Example 4, except that the mass volume concentration (w / v) of the precursor solution B is replaced with 3.33%. The mechanical properties are shown in FIG11 for 2% HA-SH & 3.33% HB-PEGDA.

[0095] In contrast, when 2% HA-SH was used alone without adding HB-PEGDA, the mechanical properties of the corresponding gel particles were shown in FIG11 for 2% HA-SH.

[0096] It can be seen that HB-PEGDA played the role of a cross-linking agent and effectively improved the mechanical properties of the hydrogel particles.

[0097] Example 11

[0098] The process is basically the same as Example 1, except that the w / v concentration of precursor solution A is changed to 1.5%, and the w / v concentration of precursor solution B is changed to 5%. The degradation curves are shown in FIG12 for 1.5% HA-SH & 5% HB-PBHE.

[0099] In contrast, when 1.5% HA-SH was used alone without adding HB-PBHE, the degradation curve of the corresponding gel particles was shown in FIG12 for 1.5% HA-SH.

[0100] It can be seen that the hydrogel particles without PBHE degraded faster. Therefore, the addition of PBHE not only provides additional reaction sites, but also prevents the hydrogel particles from degrading too quickly during enzymatic hydrolysis, effectively promoting the exposure of double bonds and active groups.

[0101] Figure 13 shows the biocompatibility test results of a 5 mg / mL hydrogel extract prepared after freeze-drying the hydrogel particles prepared in Example 1 for 3 days. As shown in Figure 13, almost all cells appear green (viable cells), demonstrating the good biocompatibility of the hydrogel particles of the present invention.

Claims

1. A method for preparing reactive hydrogel particles, characterized in that: The preparation method comprises the following steps: (1) polymerizing a double-bond modified polyethylene glycol, a modified natural polymer, and an optional active functional monomer to obtain a gel; (2) crushing the gel to obtain gel particles; (3) subjecting the gel particles to an enzymatic hydrolysis reaction with an enzyme to obtain the reactive hydrogel particles; The double-bond modified polyethylene glycol has double bonds and polyethylene glycol segments, the modified natural polymer has sulfhydryl groups or double bonds, the active functional monomer is selected from sulfhydryl or double-bond functionalized bioadhesive peptides, sulfhydryl or double-bond functionalized cholesterol, and the enzyme is the enzyme corresponding to the natural polymer.

2. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The double-bond modified polyethylene glycol is selected from one or more combinations of polyethylene glycol monoacrylate, polyethylene glycol diacrylate, polyethylene glycol monomethacrylate, polyethylene glycol dimethacrylate, hyperbranched polyethylene glycol diacrylate, and hyperbranched polyβ-hydrazide ester. The hyperbranched polyethylene glycol diacrylate is prepared from polyethylene glycol diacrylate by living polymerization; and the hyperbranched polyβ-hydrazide ester is prepared from 3,3'-dithiodipropionyl hydrazide with primary amine and polyethylene glycol diacrylate by Michael addition reaction.

3. The method for preparing reactive hydrogel particles according to claim 2, characterized in that: The hyperbranched polyethylene glycol diacrylate is prepared from polyethylene glycol diacrylate through RAFT active polymerization.

4. The method for preparing reactive hydrogel particles according to claim 3, characterized in that: The chain transfer agent used in the RAFT active polymerization is The structural formula of the polyethylene glycol diacrylate is 5. The method for preparing reactive hydrogel particles according to claim 2, characterized in that: The structural formula of the hyperbranched polyethylene glycol diacrylate is: And / or, the structural formula of the hyperbranched poly-β-hydrazide ester is: Wherein, n is 5-20.

6. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The natural polymer is selected from one or more combinations of collagen, chitosan, gelatin, dextran, sodium alginate and hyaluronic acid.

7. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The polymerization reaction in step (1) is carried out in water or PBS buffer, the mass volume concentration of the double bond modified polyethylene glycol in water or PBS buffer is 2-20%, the mass volume concentration of the modified natural polymer in water or PBS buffer is 0.5-20%, and the mass volume concentration of the active functional monomer in water or PBS buffer is 0-20%.

8. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The modified natural polymer has a thiol group, the polymerization reaction is Michael addition polymerization, and the polymerization reaction temperature is 0 to 30° C.; or, the modified natural polymer has a double bond, the polymerization reaction is free radical polymerization, and the polymerization reaction is carried out in the presence of a photoinitiator.

9. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The pulverization in step (2) is selected from extrusion crushing, grinding crushing or impact crushing; and / or, the particle size of the reactive hydrogel particles is 30 μm to 300 μm.

10. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The polymerization reaction in step (1) is carried out at a pH of 7 to 8; and / or, the concentration of the enzyme in step (3) is 5U / mL-2000U / mL; and / or, the enzyme in step (3) is selected from a combination of one or more of hyaluronidase, sodium alginate lyase, pancreatin, chitosanase or collagenase.

11. The reactive hydrogel particles prepared by the method for preparing reactive hydrogel particles according to any one of claims 1 to 10.

12. Use of the reactive hydrogel particles according to claim 11 for cell culture, tissue engineering, biological scaffolds, drug delivery or bioprinting.

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