Highly reactive hydrogel particles, methods of making and using the same

Abstract

The application discloses a kind of high reactive hydrogel particles and its preparation method and application.The preparation method includes the following steps: (1) double bond modified polyethylene glycol, modified natural polymer and optional active functional monomer are polymerized, and gel is obtained;(2) the gel is crushed, and gel particles are obtained;(3) the gel particles are enzymolysis with enzyme, and the hydrogel particles with reactivity are obtained.The double bond modified polyethylene glycol has double bond and polyethylene glycol segment, the modified natural polymer has sulfydryl or double bond, the active functional monomer is selected from sulfydryl functionalized bioadhesive peptide or sulfydryl functionalized cholesterol, and the enzyme is the corresponding enzyme of the natural polymer.The preparation method of the application is simple, and can be mass-produced.The obtained hydrogel particles have high reactivity, can be used for subsequent secondary crosslinking, and the hydrogel particles also have good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of hydrogel particle technology, and in particular to a highly reactive hydrogel particle, its preparation method, and its application. Background Technology

[0002] Particulate gels are multifunctional materials with broad application prospects. Their properties, including injectability, self-healing, and modularity, give them significant potential in the medical field. First, the injectability of particulate gels makes them ideal carriers for cell delivery and drug delivery. Precise positioning and delivery via syringes allow for accurate delivery of the particulate gel to the target location, achieving effective treatment and delivery. Second, particulate gels possess self-healing capabilities, able to repair cracks or fractures on their own. This property increases the stability and lifespan of the gel, reducing the risk of material damage and failure. Furthermore, particulate gels are modular, allowing for customization and combination as needed. This enables flexible adjustment of the gel's constituent units to meet the personalized requirements of cell delivery, tissue engineering, bioprinting, and other fields. To further improve the application performance of particulate gels, surface modification of the hydrogel particles is crucial. By introducing specific bioactive molecules or surface modifiers, the interaction between the gel and cells or tissues can be increased, improving biocompatibility and bioadhesion. In addition, customized mechanical properties are also key. Different types of tissues and cells have varying requirements for materials. Therefore, it is necessary to adjust factors such as the composition and cross-linking degree of gel particles to achieve customized stiffness, elasticity, and deformability to meet the culture and growth needs of different tissue cell types. Thus, developing a large-scale preparation method for highly reactive hydrogel particles is of significant practical importance. This method allows for convenient surface modification and cross-linking, meeting the cutting-edge requirements of the biomedical field for personalized new material carriers. With a deeper understanding and continuous innovation of the properties of particulate 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 well-dispersed hydrogel particles, they suffer from low throughput, hindering large-scale production. Furthermore, the need for surfactants and oil phases for dispersion makes purification steps cumbersome and production costs extremely high.

[0004] For example, Chinese patent CN112458075A discloses a double-crosslinked particulate gel, the preparation method of which includes the following steps: (1) preparing a polyvinyl polyethylene glycol polymer, thiolized sodium alginate, and a microbial precursor solution; (2) preparing hydrogel microspheres encapsulating microorganisms, preparing microspheres by water-in-oil emulsion dispersion and solidifying them; (3) preparing a double-crosslinked particulate gel packed column for biocatalytic reaction to enhance the microbial catalytic process. This preparation method involves water-in-oil emulsion dispersion, which is relatively complex and cannot be mass-produced. Furthermore, the obtained double-crosslinked particulate 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, which is prepared by blending and modifying branched macromolecules, thiolized hyaluronic acid, and solvents. Although the branched macromolecules can be grafted onto the thiolized hyaluronic acid backbone, the resulting modified hyaluronic acid has very low reactivity and cannot be used for subsequent secondary cross-linking. Moreover, the preparation process involves a dialysis step, requiring a large amount of dialysis fluid, which also makes large-scale production difficult. Summary of the Invention

[0006] The purpose of this invention is to prepare a hydrogel particle with high reactivity. The preparation process is simple and can be mass-produced. The prepared hydrogel particle has high reactivity and can be used for subsequent secondary crosslinking. The hydrogel particle also has good biocompatibility.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing reactive hydrogel particles, the method comprising the following steps:

[0009] (1) Polymerize double-bond modified polyethylene glycol, modified natural polymer and optional active functional monomer to obtain a gel;

[0010] (2) The gel is pulverized to obtain gel particles;

[0011] (3) The gel particles are subjected to an enzymatic hydrolysis reaction with an enzyme to obtain the hydrogel particles with reactive properties.

[0012] The double-bond modified polyethylene glycol has double bonds and polyethylene glycol segments, the modified natural polymer has thiol groups or double bonds, the active functional monomer is selected from thiol- or double-bond functionalized bioadhesion peptides or cholesterol, and the enzyme is the enzyme corresponding to the natural polymer.

[0013] In this invention, the optional active functional monomer means that an active functional monomer may or may not be added in step (1). When no active functional monomer is added, the double-bond modified polyethylene glycol and the modified natural polymer undergo a polymerization reaction. When an active functional monomer is added, the double-bond modified polyethylene glycol, the modified natural polymer, and the active functional monomer undergo a polymerization reaction.

[0014] In this invention, the gel obtained in step (1) has unreacted double bonds or other active groups. The gel is in a cross-linked state, and most of the unreacted double bonds or other active groups are located inside the cross-linked structure of the gel. In step (3), the enzyme corresponding to the natural polymer is used to enzymatically digest and cut the natural polymer chain segments in the gel, which can expose the unreacted double bonds or other active groups in step (1) to the surface of the hydrogel particles, so that the hydrogel particles have high reactivity. This high reactivity is beneficial to the subsequent application of the hydrogel particles, such as for cell culture, or for secondary cross-linking using this high reactivity.

[0015] 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 poly(β-hydroxyhydrazine).

[0016] In some embodiments, the hyperbranched polyethylene glycol diacrylate is prepared from polyethylene glycol diacrylate by living polymerization.

[0017] In some embodiments, the structural formula of the hyperbranched polyethylene glycol diacrylate is:

[0018]

[0019] The structural formula of the hyperbranched polyβ-hydrazide ester is:

[0020] Wherein, n = 5-20, preferably 10. This hyperbranched polyβ-hydrazide contains disulfide bonds, which have a small positive charge, while cells are slightly negatively charged. When it is used as the polymerization monomer in step (1), the resulting hydrogel particles will have a better adhesion effect on cells when used in cell culture and other fields.

[0021] In some embodiments, the hyperbranched polyβ-hydrazide is prepared by a method comprising the following steps: the hyperbranched polyβ-hydrazide is prepared by Michael addition reaction of 3,3'-dithiodipropionylhydrazide with a primary amine and polyethylene glycol diacrylate (PEGDA).

[0022] In some embodiments, the natural polymer is selected from one or more combinations of collagen, chitosan, gelatin, dextran, sodium alginate, and hyaluronic acid.

[0023] In some embodiments, the bioadhesion 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.

[0024] In some embodiments, the thiol-functionalized bioadhesion peptide is a thiol-functionalized RGD, and its amino acid series is GRGDSPC.

[0025] In some embodiments, the polymerization reaction in step (1) is carried out in water or PBS buffer, the double bond modified polyethylene glycol has a mass-volume concentration of 2-20% in water or PBS buffer, the modified natural polymer has a mass-volume concentration of 0.5-20% in water or PBS buffer, and the active functional monomer has a mass-volume concentration of 0-20% in water or PBS buffer.

[0026] In some embodiments, the mass ratio of the double-bond modified polyethylene glycol to the modified natural polymeric active functional monomer is 5:1.5:0.5.

[0027] In some embodiments, the modified natural polymer has thiol groups, the polymerization reaction is Michael addition polymerization, and the polymerization temperature is 0–30°C.

[0028] In some embodiments, the modified natural polymer has double bonds, the polymerization reaction is a free radical polymerization, and the polymerization reaction is carried out in the presence of a photoinitiator.

[0029] In some embodiments, the crushing in step (2) is selected from extrusion crushing, grinding crushing or impact crushing.

[0030] Preferably, the crushing in step (2) is grinding and crushing.

[0031] Furthermore, the grinding and crushing process is performed 1-5 times. The more times the grinding is performed, the smaller the particle size of the final hydrogel particles.

[0032] Furthermore, the grinding and crushing time for each step is 1-10 minutes, preferably 5 minutes.

[0033] In some embodiments, the particle size of the reactive hydrogel particles is 30 μm to 300 μm.

[0034] Preferably, the particle size of the reactive hydrogel particles is 100 μm to 150 μm.

[0035] In some embodiments, the polymerization reaction described in step (1) is carried out at a pH of 7 to 8.

[0036] In some embodiments, the concentration of the enzyme in step (3) is 5 U / mL to 2000 U / mL.

[0037] Preferably, the polymerization reaction in step (1) is carried out at a pH of 7.2 to 7.4.

[0038] In some embodiments, the enzyme in step (3) is selected from one or more combinations of hyaluronidase, sodium alginate lyase, trypsin, chitosanase or collagenase.

[0039] This invention also provides reactive hydrogel particles prepared by the above-described method for preparing reactive hydrogel particles. These hydrogel particles exhibit high reactivity and can be used for subsequent secondary crosslinking. Furthermore, these hydrogel particles demonstrate good biocompatibility.

[0040] The present invention also provides the use of the aforementioned reactive hydrogel particles for cell culture, tissue engineering, bioscaffolds, drug delivery or bioprinting.

[0041] The hydrogel particles of this invention are made primarily from double-bond modified polyethylene glycol and natural polymers modified with thiol groups or double bonds. A gel mass is first prepared through polymerization, then pulverized to obtain gel particles. Enzymatic treatment is then used to expose active sites, resulting in highly reactive hydrogel particles. Biomolecules, drugs, or cells can more easily interact with these active sites, thereby enhancing the availability of activity. Reactive sites can provide support for cell adhesion, promoting cell attachment and proliferation. Furthermore, reactive sites can provide binding sites or target receptors for more drugs, improving drug delivery efficacy and efficiency.

[0042] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0043] (1) High reactivity. The gel obtained in step (1) of this invention has unreacted double bonds or other active groups. The gel is in a cross-linked state, and most of the unreacted double bonds or other active groups are located inside the cross-linked structure of the gel. In step (3), the enzyme corresponding to the natural polymer is used to enzymatically hydrolyze and cut the natural polymer chain segments in the gel, which exposes the unreacted double bonds or other active groups in step (1) to the surface of the hydrogel particles, making the hydrogel particles highly reactive and exposing a large number of active sites. The above-mentioned active sites can subsequently undergo secondary cross-linking with the modified polymer. The resulting secondary cross-linked gel particles can increase the density of the cross-linked network inside the particle gel, affect the pore structure and pore size distribution of the particle gel, thereby regulating the release rate of drugs or bioactive substances.

[0044] Double-bond modified polyethylene glycol can effectively regulate the mechanical properties of hydrogel particles. Double-bond modified polyethylene glycol can also prevent the gel particles from degrading too quickly during enzymatic hydrolysis in step (3), effectively promoting the exposure of double bonds and other active groups.

[0045] (2) The preparation process is simple, enabling the mass production and low-cost preparation of highly reactive hydrogel particles. Existing technologies require complex processes such as water-in-oil emulsion dispersion to prepare similar hydrogel particles, which are not conducive to large-scale mass production. However, this invention uses a simple pulverization and enzymatic hydrolysis process to obtain highly reactive hydrogel particles. The preparation process is mild and environmentally friendly, requiring no complex equipment or operations. The preparation method of this invention does not involve the use of surfactants or oil phases, which greatly reduces the cost of subsequent purification while simplifying the process.

[0046] (3) The obtained hydrogel particles have good biocompatibility. The raw materials used in the preparation of hydrogel particles by this invention are safe and non-toxic, with high biocompatibility. The obtained hydrogel particles have promising applications in cell culture, tissue engineering, drug delivery, bioprinting and other fields. Attached Figure Description

[0047] Figure 1 The 1H NMR spectrum of hyperbranched polyethylene glycol diacrylate;

[0048] Figure 2 The 1H NMR spectrum of hyperbranched polyβ-hydrazide ester;

[0049] Figure 3 The images show the 1H NMR spectra of hyaluronic acid and thiolated hyaluronic acid, where HA represents hyaluronic acid and HA-SH represents thiolated hyaluronic acid.

[0050] Figure 4 This is a flowchart illustrating the preparation process of the hydrogel particles of the present invention.

[0051] Figure 5 Here is a SEM image of the hydrogel particles prepared in Example 1;

[0052] Figure 6 Microscopic images and size distribution of hydrogel particles after different grinding cycles in Examples 1-3;

[0053] Figure 7 The images show the cell-hydrogel adhesion and growth after 12 hours of hyaluronidase treatment in Example 1 and Comparative Example 1.

[0054] Figure 8 This is a diagram of cell adhesion and proliferation, shown in Example 8 and Comparative Example 2.

[0055] Figure 9 This is a photograph of the hydrogel particles before and after secondary cross-linking following hyaluronidase treatment for 12 hours in Example 8.

[0056] Figure 10 The strain-modulus curves of hydrogel particles before and after secondary crosslinking after 12 hours of hyaluronidase treatment in Example 1 are shown.

[0057] Figure 11 Strain-modulus curves of the hydrogel particles and control particles in Example 10;

[0058] Figure 12 Degradation curves of the hydrogel particles and control particles in Example 11 under 500 U / mL hyaluronidase conditions;

[0059] Figure 13 This is a biocompatibility-live / dead staining diagram of the 5 mg / mL active hydrogel material extract from Example 1. Detailed Implementation

[0060] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0061] Preparation Example 1

[0062] Preparation of hyperbranched polyethylene glycol diacrylate (HB-PEGDA):

[0063] Using DS as the RAFT reagent, 2′-azobis(2-methylnitrile) (AIBN) as the initiator, and homopolymer polyethylene glycol diacrylate PEGDA (average Mn = 575) (0.4 mol·L⁻¹) as the initiator... -1 The monomer [PEGDA] was subjected to RAFT polymerization in butanone at 70°C using a feedstock; the molar ratio of [PEGDA]:[DS]:[AIBN] was 25:1:1.4. The resulting HB-PEGDA structure is as follows: Its NMR characterization diagram is as follows Figure 1 As shown.

[0064] Preparation Example 2

[0065] Preparation of hyperbranched polyβ-hydroxyhydrazide ester (HB-PBHE):

[0066] 1) Polyethylene glycol diacrylate (PEGDA) and 3,3'-dithiodipropionyl hydrazide (DTP) were dissolved in dimethyl sulfoxide (DMSO) at a molar ratio of 5:2 and reacted at 90°C. The structural formula of the obtained HB-PBHE is as follows:

[0067] Its NMR characterization diagram is as follows Figure 2 As shown.

[0068] Example 1

[0069] 30 mg of thiolized hyaluronic acid (HA-SH) was used, and its NMR spectrum is shown below. Figure 3 The precursor solution A was prepared by dissolving 100 mg of the hyperbranched poly(β-hydroxyhydrazine) (HB-PBHE) prepared in 1 mL of deionized water to prepare a 3% (w / v) precursor solution. The pH of precursor solution A was adjusted to 7.2 with sodium hydroxide aqueous solution. The adjusted precursor solution A and precursor solution B were then mixed thoroughly at a 1:1 volume ratio and allowed to stand to obtain a hydrogel. The prepared hydrogel was ground using a 100-mesh metal sieve for 5 min, and then treated with 500 U / mL hyaluronidase for 12 h to obtain active hydrogel particles with a particle size of 100 μm. The SEM image is shown below. Figure 5 Its particle size diagram is as follows Figure 6 The diagram corresponding to the first grinding in the middle.

[0070] Example 2

[0071] The process is essentially the same as in Example 1, except that grinding is performed twice, with the same grinding time each time as in Example 1. The final active hydrogel particles have a particle size of 70-80 μm. The particle size distribution is shown in the diagram. Figure 6 The diagram corresponding to the second grinding.

[0072] Example 3

[0073] The process is basically the same as in Example 1, except that the grinding was performed three times, with the same grinding time each time as in Example 1. The final active hydrogel particles had a particle size of 50-60 μm. The particle size distribution is shown in the diagram. Figure 6 The diagram corresponding to the third grinding process.

[0074] It is evident that the diameter of the hydrogel particles decreases continuously with the increase of the number of grinding cycles.

[0075] Example 4

[0076] 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 thoroughly at a 2:1 volume ratio. After standing, a hydrogel was obtained. The prepared hydrogel was ground using a 100-mesh metal sieve for 5 min, and then treated with 500 U / mL sodium alginate lyase for 12 h to obtain active hydrogel particles with a particle size distribution of 100-150 μm.

[0077] Example 5

[0078] 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 thoroughly at a 2:1 volume ratio. After standing, a hydrogel was obtained. The prepared hydrogel was ground using a 100-mesh metal sieve for 5 min, and then treated with 500 U / mL collagenase for 12 h to obtain active hydrogel particles with a particle size distribution of 100-150 μm.

[0079] Example 6

[0080] 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 hyperbranched poly(β-hydroxyhydrazine) (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 the adjusted precursor solution A and precursor solution B were mixed thoroughly at a 2:1 volume ratio. After standing, a hydrogel was obtained. The prepared hydrogel was ground using a 100-mesh metal sieve for 5 min, and then treated with 500 U / mL chitosanase for 12 h to obtain active hydrogel particles with a particle size distribution of 100-150 μm.

[0081] Comparative Example 1

[0082] The process is basically the same as in Example 1, except that the step of treating with 500 U / mL hyaluronidase for 12 hours is omitted. That is, hydrogel particles are obtained directly after grinding.

[0083] Example 7

[0084] The active hydrogel particles prepared in Example 1 and the hydrogel particles prepared in Comparative Example 1 were subjected to secondary crosslinking with 1 mg / mL mercaptopolyethylene glycol rhodamine B. The mass ratio of hydrogel particles to mercaptopolyethylene glycol rhodamine B was 500:1, and the mixture was incubated for 30 min. After incubation, the particles were washed with PBS and observed under a fluorescence microscope. The results are as follows. Figure 7 As shown, where Figure 7 The enzymatic hydrolysis in the left figure and the enzymatic hydrolysis for 3 hours in the right figure represent the results corresponding to Example 1. Figure 7 The untreated hydrogel particles in the left figure and the enzymatic hydrolysis 0h in the right figure represent the results corresponding to Comparative Example 1. It can be seen that the hydrogel particles treated with enzymes in step (3) of the present invention can effectively expose active functional groups, and the prepared hydrogel particles have high reactivity. They can then react with small or large molecules containing thiol groups, while the hydrogel particles without enzyme treatment have extremely low reactivity.

[0085] Example 8

[0086] 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 hyperbranched polyβ-hydroxyhydrazine (prepared in Example 2) and 10 mg of thiolated adhesion peptide RGD-SH (amino acid series 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 the adjusted precursor solutions A and B were mixed evenly at a 1:1 volume ratio. After standing, a hydrogel was obtained. The prepared hydrogel was ground using a 100-mesh metal sieve for 5 min, then treated with 500 U / mL hyaluronidase for 12 h, and washed three times with PBS to obtain active hydrogel particles. The active hydrogel particles were then soaked in MEM complete medium for 1 h, followed by the addition of 5000 L929 cells. The culture plate was placed in an incubator for culture, and cell growth was observed. The results after 48 h, 72 h, and 96 h of culture are as follows: Figure 8 The images show that after enzyme treatment, the active groups of the resulting active hydrogel particles are exposed, which can effectively promote cell adhesion and proliferation.

[0087] Comparative Example 2

[0088] The procedure is essentially the same as in Example 8, except that the step of treating the cells with 500 U / mL hyaluronidase for 12 hours is omitted. Hydrogel particles are obtained directly after grinding. The results of cell culture for 24, 36, and 48 hours are as follows... Figure 8As can be seen from the unprocessed image, when the enzyme treatment step of the present invention is used, the hydrogel particles have higher reactivity and better cell adhesion and proliferation effects when used for cell culture.

[0089] Example 9

[0090] The active hydrogel particles prepared in Example 1 were mixed with thiolized hyaluronic acid and subjected to secondary crosslinking. The results are as follows: Figure 9 As shown in the left figure, the hydrogel particles exhibit greater strength after secondary cross-linking because the thiolized hyaluronic acid can react again with the active functional groups exposed on the surface of the active hydrogel particles. Figure 9 The right-hand image shows the active hydrogel particles prepared in Example 1, without secondary crosslinking; the strength of the hydrogel particles is slightly lower. The storage modulus of the hydrogel particles before and after secondary crosslinking is as follows: Figure 10 As shown, the storage modulus increases by two orders of magnitude after secondary crosslinking. Therefore, the highly reactive hydrogel particles prepared by this invention can have their mechanical properties improved through secondary crosslinking, and have broad application prospects in tissue engineering, bioscaffolds, drug delivery carriers, bioprinting, and other fields.

[0091] Example 10

[0092] The process is basically the same as in Example 4, except that the mass-volume concentration (w / v) of precursor solution B is replaced with 3.33%. Its mechanical properties are as follows: Figure 11 The sample contains 2% HA-SH and 3.33% HB-PEGDA.

[0093] In contrast, when 2% HA-SH is used alone without the addition of HB-PEGDA, the mechanical properties of the corresponding gel particles are as follows: Figure 11 As shown in the figure, 2% HA-SH.

[0094] It is evident that HB-PEGDA acts as a crosslinking agent, effectively improving the mechanical properties of the hydrogel particles.

[0095] Example 11

[0096] The process is basically the same as in Example 1, except that the w / v concentration of precursor solution A is replaced with 1.5%, and the w / v concentration of precursor solution B is replaced with 5%. The degradation curves are as follows: Figure 12 The sample contains 1.5% HA-SH and 5% HB-PBHE.

[0097] In contrast, when 1.5% HA-SH is used alone without the addition of HB-PBHE, the degradation curve of the corresponding gel particles is as follows: Figure 12 As shown in the figure, 1.5% HA-SH.

[0098] It is evident that hydrogel particles without PBHE degrade 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.

[0099] Figure 13 The results are for testing the biocompatibility of a 5 mg / mL hydrogel extract prepared after freeze-drying the hydrogel particles prepared in Example 1 for 3 days. Figure 13 As can be seen, almost all the cells are green (living cells), which proves that the hydrogel particles of the present invention have good biocompatibility.

Claims

1. A method for preparing reactive hydrogel particles, characterized in that: The preparation method includes the following steps: (1) Polymerize double-bond modified polyethylene glycol, modified natural polymer and optional active functional monomer to obtain a gel; (2) The gel is pulverized to obtain gel particles; (3) The gel particles are subjected to an enzymatic hydrolysis reaction with an enzyme to obtain the hydrogel particles with reactive properties. The double-bond modified polyethylene glycol has double bonds and polyethylene glycol segments, the modified natural polymer has thiol groups, the active functional monomer is selected from thiol or double-bond functionalized bioadhesion peptides or cholesterol, and the enzyme is the enzyme corresponding to the natural polymer. The double-bond modified polyethylene glycol is selected from one or a combination of two of hyperbranched polyethylene glycol diacrylate and hyperbranched polyβ-hydrazide ester. The hyperbranched polyethylene glycol diacrylate is prepared by living polymerization of polyethylene glycol diacrylate; the structural formula of the hyperbranched polyethylene glycol diacrylate is: The structural formula of the hyperbranched polyβ-hydrazide ester is: Where n is 5-20; The natural polymer is selected from one or more combinations of collagen, chitosan, gelatin, dextran, sodium alginate, and hyaluronic acid.

2. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The hyperbranched polyβ-hydrazide ester is prepared by Michael addition reaction of 3,3'-dithiodipropionylhydrazide with a primary amine and polyethylene glycol diacrylate (PEGDA).

3. 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 double bond modified polyethylene glycol has a mass-volume concentration of 2-20% in water or PBS buffer, the modified natural polymer has a mass-volume concentration of 0.5-20% in water or PBS buffer, and the active functional monomer has a mass-volume concentration of 0-20% in water or PBS buffer.

4. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The crushing process described in step (2) is selected from extrusion crushing, grinding crushing or impact crushing.

5. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The reactive hydrogel particles have a particle size of 30 μm to 300 μm.

6. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The polymerization reaction described in step (1) is carried out at a pH of 7 to 8.

7. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The concentration of the enzyme in step (3) is 5 U / mL-2000 U / mL.

8. The method for preparing reactive hydrogel particles according to claim 1, characterized in that: The enzyme in step (3) is selected from one or more combinations of hyaluronidase, sodium alginate lyase, pancreatin, chitosanase or collagenase.

9. The reactive hydrogel particles prepared by the method for preparing reactive hydrogel particles according to any one of claims 1-8.

10. Use of the reactive hydrogel particles of claim 9 for cell culture, tissue engineering, bioscaffolds, drug delivery, or bioprinting.

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