Preparation method and application of universal hydrogel microspheres

Abstract

The invention relates to the technical field of hydrogel microsphere preparation, and discloses a hydrogel microsphere preparation method and application, the hydrogel microsphere preparation method comprises the following steps: S1, adding a pore-forming agent into a hydrogel precursor to obtain a dispersed phase; s2, preparing hydrogel microspheres from the continuous phase and the dispersed phase through one of a microfluidic method, an electrostatic spraying method and an emulsion polymerization method; specifically, in the step S1, the pore-forming agent is a fluorinated solution; in the step S2, the continuous phase contains a fluorinated solution. The hydrogel microspheres obtained by the method are uniform in particle size distribution, uniform in pore size distribution, high in pore structure stability and good in biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel microsphere preparation, and more specifically, to a method for preparing hydrogel microspheres and applications thereof. Background Art

[0002] Hydrogel microspheres are a kind of macromolecular polymer with internal molecular cross-linking and have a three-dimensional network structure. Photosensitive hydrogel porous microspheres combine the water absorption, porosity, high specific surface area and biocompatibility of hydrogels, as well as the responsiveness of photosensitive materials to specific light wavelengths, and can be used as materials for devices / instruments such as microstructural units, microseparators, microreactors, and micromemories, and are applied in biomedical material fields such as drug release, cell culture, and tissue engineering.

[0003] Regarding the preparation process of hydrogel microspheres, patent document CN106947019 A discloses a method and device for preparing hydrogel microspheres, which uses an aqueous mixture of a water-soluble monomer, a cross-linking agent, an initiator and a pore-forming agent as the dispersed phase, and an oily mixture prepared from a water-insoluble liquid and a lipophilic and hydrophilic amphiphilic molecule as the continuous phase to generate micron-sized water-in-oil droplets in a microfluidic chip device. After the droplets are collected, they are solidified and demulsified to recover monodisperse micron-sized hydrogel microspheres. The main technical problems of this preparation method are as follows:

[0004] (1) The dispersed phase is not uniform, affecting the encapsulation of the continuous phase, resulting in different particle sizes of the obtained hydrogel microspheres;

[0005] (2) If the pore-forming agent is a water-soluble molecule such as polyethylene glycol, methanol or ethanol, it is miscible with the hydrogel, affecting the pore size distribution;

[0006] (3) If the pore-forming agent is water, the pore structure stability of the obtained hydrogel microspheres is poor, and the pore size may disappear after the microspheres are redissolved; if the pore-forming agent is polyethylene glycol, methanol or ethanol, the obtained hydrogel microspheres have biological toxicity, affecting cell activity and having poor biocompatibility. Summary of the Invention

[0007] The present invention provides a method for preparing hydrogel microspheres, and the obtained hydrogel microspheres (1) have a uniform particle size distribution, or (2) have a uniform pore size distribution, or (3) have high pore structure stability, or (4) have good biocompatibility.

[0008] Another object of the present invention is to provide hydrogel microspheres prepared by the method for preparing hydrogel microspheres.

[0009] Another object of the present invention is to provide the application of the hydrogel microspheres prepared by the method for preparing hydrogel microspheres in the preparation of biomedical materials.

[0010] To solve the above technical problems, the technical solution provided by the present invention is:

[0011] A method for preparing hydrogel microspheres, comprising

[0012] S1. Adding a pore-forming agent to a hydrogel precursor to obtain a dispersed phase;

[0013] S2. Obtaining hydrogel microspheres by using one of microfluidics, electrospray, and emulsion polymerization for the continuous phase and the dispersed phase;

[0014] Specifically: In S1, the pore-forming agent is a fluorinated liquid;

[0015] In S2, the continuous phase contains a fluorinated liquid.

[0016] The preparation method of the hydrogel provided by the present invention achieves the following technical effects:

[0017] (1) The prepared hydrogel microspheres have a uniform particle size distribution

[0018] In step S2, the continuous phase contains a fluorinated liquid and a surfactant, which can prevent droplet coalescence, clearly separate each droplet from each other and make them not easily adhere. In this way, each droplet can be photocured and crosslinked, and the particle size of the microspheres can be accurately controlled.

[0019] (2) The prepared hydrogel microspheres have a uniform pore size distribution

[0020] In step S1, the pore-forming agent added to the hydrogel precursor is a fluorinated liquid. The fluorinated liquid has a low surface tension, low viscosity, and high density, and can be uniformly dispersed in the hydrogel precursor. In this way, a hydrogel precursor is obtained, and a stable porous structure can be formed in the photocrosslinking and curing step of S2, and hydrogel microspheres with a uniform pore size distribution are prepared.

[0021] (3) The prepared hydrogel microspheres have high pore structure stability

[0022] In step S1, the pore-forming agent added to the hydrogel precursor is a fluorinated liquid. The fluorinated liquid has high chemical stability and is not easily chemically reacted with the hydrogel matrix. In this way, after the photocrosslinking reaction in step S2 is completed, the pore size structure of the obtained hydrogel microspheres is stable. During its use, even when it is full of water, the pores can maintain their original shape and will not change significantly or disappear.

[0023] (4) The prepared hydrogel microspheres have good biocompatibility

[0024] In step S1, the fluorinated liquid has high chemical inertness, is stable in a biological environment, has extremely low toxicity and good cell compatibility, and will not have an obvious negative impact on cell growth, proliferation, and metabolism. And the fluorinated liquid has the ability to dissolve and transport oxygen, providing a basis for cell survival in biomedical applications.

[0025] Preferably, the grafting amount of methacrylate groups in the hydrogel microspheres obtained in S2 is 20% to 80%.

[0026] Preferably, the grafting amount of methacrylate groups in the hydrogel microspheres obtained in S2 is 25% to 70%.

[0027] The hydrogel grafted with methacrylate groups in the present invention is a photocurable hydrogel.

[0028] Preferably, before the step S1, there is also S0. Preparation of a hydrogel precursor, and S0 includes the following steps: dissolving a water-soluble hydrogel, adding a photoinitiator, and preparing a hydrogel modified with methacrylate groups; the mass ratio of the water-soluble hydrogel to the photoinitiator is (1 to 30):(0.1 to 1.5).

[0029] Preferably, the mass ratio of the water-soluble hydrogel to the photoinitiator is (2 to 25):(0.25 to 1).

[0030] More preferably, a surfactant is also added in the preparation method of the S0 hydrogel precursor; the mass ratio of the water-soluble hydrogel, the photoinitiator and the surfactant is (1 to 30):(0.1 to 1.5):(0.5 to 5).

[0031] Preferably, the mass ratio of the water-soluble hydrogel, the photoinitiator and the surfactant is (2 to 25):(0.25 to 1):(0.5 to 5).

[0032] In the present invention, a surfactant is also added in the preparation method of S0 for the hydrogel precursor, which is beneficial to promoting the stability of the two-phase system of the dispersed phase and the continuous phase in the subsequent step S2, so as to obtain hydrogel microspheres with uniform particle size distribution, high pore structure stability and uniform pore size distribution.

[0033] The water-soluble hydrogel in S0 of the present invention is selected from photosensitive hydrogels.

[0034] The water-soluble hydrogel in S0 of the present invention is selected from one or more of hyaluronic acid, chondroitin sulfate, dextran, gelatin, chitosan, sodium alginate and polylysine.

[0035] In the present invention, the photoinitiator in S0 is selected from one or more of Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), and Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO).

[0036] In the present invention, the surfactant in S0 is selected from one or more of polyvinyl alcohol PVA, Tween-20, Tween-60, and Tween-80.

[0037] During the process of introducing methacrylate groups into the water-soluble hydrogel, there is a certain proportional range between the addition amounts of the initiator and the surfactant and the mass of the hydrogel. If the addition amount of the initiator is too much, the initiator is prone to agglomeration and has certain biological toxicity. If the addition amount is too little, the number of methacrylate groups incorporated into the hydrogel is small, affecting the photocuring effect of S2.

[0038] Preferably, the addition amount of the fluorinated liquid in S1 is 10% - 50% of the volume of the hydrogel precursor.

[0039] More preferably, the addition amount of the fluorinated liquid in S1 is 20% - 40% of the volume of the hydrogel precursor.

[0040] The addition amount of the fluorinated liquid as a pore-forming agent should be within a reasonable range. If too much is added, multiple micropores will aggregate, affecting the stability of the pore structure of the obtained microspheres. If too little is added, the number of pores is too small, affecting the water absorption and use effect of the obtained hydrogel microspheres.

[0041] Preferably, S2 specifically includes the following steps:

[0042] S21. Pump the continuous phase and the dispersed phase into a microfluidic device together to form droplets.

[0043] S22. Successively perform photocrosslinking curing, washing, and freeze-drying on the droplets obtained in S21 to obtain hydrogel microspheres.

[0044] Preferably, in S21, the pumping flow rate of the dispersed phase into the liquid phase is (1 - 1000) μL / min; the pumping flow rate of the continuous phase is (10 - 5000) μL / min.

[0045] More preferably, the pumping flow rate of the dispersed phase in the liquid phase in S21 is (20 - 300) μL / min; the pumping flow rate of the continuous phase is (300 - 4000) μL / min.

[0046] The pumping flow rates of the dispersed phase and the continuous phase should be within a reasonable range. If the pumping flow rate of the dispersed phase is too fast, the continuous phase cannot completely wrap the dispersed phase, resulting in droplet adhesion and inaccurate control of the microsphere particle size; if the pumping flow rate of the dispersed phase is too slow, the production efficiency will be reduced, causing waste of the continuous phase, and multiple encasements of individual dispersed phases will affect the photocuring effect; if the pumping speed of the continuous phase is too fast, some dispersed phases will be encased multiple times, which will cause waste of the continuous phase on the one hand and affect the subsequent photocuring effect on the other hand; if the pumping of the continuous phase is too slow, it cannot wrap each dispersed phase droplet, resulting in droplet adhesion and inaccurate control of the microsphere particle size.

[0047] Preferably, in S22, photocrosslinking curing uses ultraviolet light, the photocuring wavelength is 276 - 405 nm, and the time is 30 - 120 s.

[0048] Preferably, the continuous phase in S2 further contains a surfactant, and the addition amount of the surfactant is 0.5% - 5% of the volume of the fluorinated liquid.

[0049] The fluorinated liquid described in the present invention can specifically be selected from one or more of hydrofluoroethers (HFE) 7000, 7100, 7200, 7300, 7400 and 7500, perfluoropentanone (FK-5-1-12), hydrofluorocarbons (HFC), perfluoropolyether derivatives and perfluorocarbons (FC-72).

[0050] The fluorinated liquid described in the present invention is selected from one or more of hydrofluoroethers (HFE) 7000, 7100, 7200, 7300, 7400 and 7500.

[0051] The surfactant in the continuous phase described in the present invention is selected from one or more of perfluoropolyoxypropylene - polyethylene glycol - perfluoropolyoxypropylene PFPE - PEGPFPE "A - B - A" type high - molecular polymers, "Rf - L - Rh - L - Rf" and "Rh - L - Rf - L - Rh" type perfluorinated high - molecular polymers.

[0052] The particle size D50 of the hydrogel microspheres obtained in the present invention is 0.2 μm - 900 μm.

[0053] The present invention also provides hydrogel microspheres prepared by the hydrogel microsphere preparation method described in any one of the above.

[0054] The present invention also provides the application of the hydrogel microspheres prepared by the hydrogel microsphere preparation method described in any one of the above in the preparation of biomedical materials. Description of the Drawings

[0055] Figure 1 It is the morphology diagram of the hydrogel microspheres obtained in Example 1.

[0056] Figure 2 It is the morphology diagram of the hydrogel microspheres obtained in Comparative Example 2

[0057] Figure 3 It is the microscopic particle size distribution diagram of the hydrogel microspheres obtained in Example 1.

[0058] Figure 4 It is the cell viability state diagram after cells are inoculated on the surface of the hydrogel microspheres obtained in Example 1.

[0059] Figure 5 It is the cell viability state diagram after cells are inoculated on the surface of the hydrogel microspheres obtained in Comparative Example 2.

[0060] Figure 6 It is the morphology diagram of the hydrogel microspheres obtained in Example 1 after water absorption.

[0061] Figure 7 It is the morphology diagram of the hydrogel microspheres obtained in Comparative Example 1 after water absorption. Detailed implementation manners

[0062] The following further details the present invention in conjunction with specific implementation manners.

[0063] In the relevant examples and comparative examples, the grafting amount of the methacrylate group is calculated by integrating the characteristic peaks of nuclear magnetic resonance hydrogen spectrum.

[0064] Example 1

[0065] A preparation method of hydrogel microspheres includes the following steps:

[0066] S0. Prepare a hydrogel precursor:

[0067] Dissolve the water-soluble hydrogel chondroitin sulfate (ChS) in deionized water, and sequentially add the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (lrgacure 2959) and the surfactant polyvinyl alcohol PVA to prepare a hydrogel precursor; the mass ratio of the water-soluble hydrogel, photoinitiator and surfactant is 15:0.6:2;

[0068] S1. Add the electronic fluorinated liquid 7100 to the hydrogel precursor ChSMA solution obtained in S0 and stir. When a milky white dispersion is formed, a dispersed phase is obtained; the addition amount of the electronic fluorinated liquid 7100 is 30% of the volume of the hydrogel precursor obtained in S0; the stirring process is: rotation speed 1000 rpm, stirring time 10 min;

[0069] S2. Use microfluidic technology to prepare hydrogel microspheres, which specifically includes the following steps:

[0070] S21. Pump the dispersed phase and continuous phase obtained in S1 into a co-flow microfluidic device to form droplets; the pumping flow rate of the dispersed phase in the pumped liquid phase is 150 μL / min; the pumping flow rate of the continuous phase is 1400 μL / min; the preparation method of the continuous phase is: add surfactant PFE-PEG to the fluorinated liquid to form the continuous phase; the addition amount of the surfactant is 2% of the volume of the fluorinated liquid.

[0071] S22. First, irradiate the droplets obtained in S21 at a wavelength of 405 nm for 60 s for ultraviolet crosslinking and curing to obtain ChSMA hydrogel porous microspheres; then, wash the ChSMA hydrogel porous microspheres with a microsphere cleaning solution and rinse twice to remove excess oil and surfactant; finally, collect the ChSMA hydrogel porous microspheres into pure water, pre-freeze at -80 °C and then put them into a freeze dryer for freeze-drying, and ChSMA hydrogel porous microspheres are obtained after freeze-drying; the access amount of methacrylate groups in the ChSMA hydrogel porous microspheres is 25%.

[0072] Example 2

[0073] This example is the second example of the present invention, which is different from Example 1 in that:

[0074] A method for preparing hydrogel microspheres, comprising the following steps:

[0075] S0. Prepare a hydrogel precursor:

[0076] Dissolve water-soluble hydrogel dextran (DeX) in deionized water, and sequentially add lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and surfactant polyvinyl alcohol PVA; obtain a hydrogel precursor; the mass ratio of the water-soluble hydrogel, photoinitiator and surfactant is 15:0.25:0.5.

[0077] S1. Add perfluorocyclohexanone (FK-5-1-12) to the hydrogel precursor DeXMA solution obtained in S0 and stir. When a milky white dispersion is formed, a dispersed phase is obtained; the addition amount of the electronic fluorinated liquid 7100 is 40% of the volume of the hydrogel precursor obtained in S0; the stirring process is: rotation speed 1000 rpm, stirring time 8 min.

[0078] S2. Prepare hydrogel microspheres by electrospray method, which specifically includes the following steps:

[0079] S21. Loading of the dispersed phase and setup of the apparatus: First, transfer the dispersed phase prepared in S1 to the syringe of the electrospray device. The inner diameter of the syringe needle is 0.5 mm. Then, fix the syringe on a three-dimensional moving platform. Adjust the vertical distance between the needle and the receiving device (a container containing the electronic fluorinated liquid 7100) to 8 cm. Turn on the high-voltage power supply, set the DC voltage between the needle and the receiving device to 8 kV, and the syringe propulsion rate to 1 mL / h. Finally, pre-inject the electronic fluorinated liquid 7100 into the receiving device as the continuous phase, and keep the liquid level 2 - 3 cm away from the top of the container.

[0080] S22. Injection of microspheres and photocuring: First, start the high-voltage power supply and the injection pump. Under the action of the electric field, the dispersed phase forms a stable jet from the tip of the needle. The dispersed phase droplets split into micron-sized droplets under the action of the electric field force and are uniformly dispersed into the electronic fluorinated liquid 7100 in the receiving device. Then, synchronously turn on the ultraviolet light source (wavelength 365 nm, power density 10 mW / cm 2 ), irradiate the dispersed droplets in the receiving device, and the photoinitiator Irgacure 2959 initiates the cross-linking reaction of ChSMA. The curing time is 3 min to form solid hydrogel microspheres.

[0081] S23. Collection and post-treatment of microspheres. After stopping the electrospray, centrifuge the mixed liquid in the receiving device (rotation speed 2000 rpm, time 5 min) to separate the hydrogel microspheres. Then wash the microspheres with deionized water and ethanol 3 - 5 times in sequence to remove residues. Finally, place the washed microspheres in a vacuum drying oven and dry for 24 h to obtain DeXMA hydrogel microspheres with uniform particle size and smooth surface.

[0082] The grafting amount of methacrylate groups in the DeXMA hydrogel porous microspheres is 40%.

[0083] Example 3

[0084] This example is the 3rd example of the present invention. The difference from Example 1 is:

[0085] A method for preparing hydrogel microspheres, comprising the following steps:

[0086] S0. Prepare the hydrogel precursor:

[0087] Dissolve the water-soluble hydrogel hyaluronic acid (HA) in deionized water, and sequentially add the photoinitiator diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) and the surfactant polyvinyl alcohol PVA to prepare the hydrogel precursor. The mass ratio of the water-soluble hydrogel, photoinitiator, and surfactant is 2.5:0.25:1;

[0088] S1. Add the electronic fluorinated liquid 7100 to the hydrogel precursor HAMA solution obtained in S0 and stir. When a milky white dispersion is formed, a dispersed phase is obtained. The addition amount of the electronic fluorinated liquid 7100 is 30% of the volume of the hydrogel precursor obtained in S0. The stirring process is as follows: rotation speed 1000 rpm, stirring time 8 min.

[0089] S2. Prepare hydrogel microspheres by emulsion polymerization, which specifically includes the following steps:

[0090] S21. Emulsification process: First, transfer the dispersed phase prepared in S1 to a high-speed homogenizer, and add the electronic fluorinated liquid 7100 with the same volume as the dispersed phase as the continuous phase. Then, start the homogenizer, set the rotation speed to 10000 rpm, and the homogenization time to 10 min to form stable water-in-fluorinated liquid (W / O) droplets. The system temperature during the homogenization process is 20 - 30 °C.

[0091] S22. Photo-curing crosslinking: First, transfer the emulsified mixture to an ultraviolet light-curing device. Under the protection of an inert gas (such as nitrogen), turn on the ultraviolet light source (wavelength 405 nm, power density 15 mW / cm 2 ); Then, use the photoinitiator TPO to initiate the crosslinking of the methacrylate groups of HAMA, and the curing time is 2 min to form solid hydrogel microspheres. During the curing process, continuously stir the mixture with a magnetic stirrer at 500 rpm.

[0092] S23. Microsphere separation and purification: First, pour the cured mixture into a centrifuge tube, centrifuge at 1000 rpm for 5 min to separate the hydrogel microspheres; Then, wash the microspheres alternately with deionized water and ethanol 3 - 5 times to remove residues, and dry the washed microspheres to obtain hyaluronic acid-based hydrogel microspheres with uniform particle size.

[0093] The incorporation amount of methacrylate groups in the HAMA hydrogel porous microspheres is 70%.

[0094] Example 4

[0095] This example is the 4th example of the present invention. Different from Example 1, the mass ratio of the water-soluble hydrogel, photoinitiator, and surfactant in S0 is 1:1.5:0.5; the incorporation amount of methacrylate groups in the DeXMA hydrogel porous microspheres obtained in S22 is 30%.

[0096] Example 5

[0097] This example is the 5th example of the present invention. Different from Example 1, the mass ratio of the water-soluble hydrogel, photoinitiator, and surfactant in S0 is 30:0.1:5; the incorporation amount of methacrylate groups in the DeXMA hydrogel porous microspheres obtained in S22 is 30%.

[0098] Example 6

[0099] This example is the 4th example of the present invention. Different from Example 1, in the process of preparing the methacrylate group-modified hydrogel in S0, no surfactant is added; the incorporation amount of the methacrylate group in the obtained DeXMA hydrogel porous microspheres in S22 is 35%.

[0100] Example 7

[0101] This example is the 5th example of the present invention. Different from Example 1, the addition amount of the fluorinated liquid in S1 is 10% of the volume of the hydrogel precursor obtained by adding to S0.

[0102] Example 8

[0103] This example is the 8th example of the present invention. Different from Example 1, the addition amount of the fluorinated liquid in S1 is 50% of the volume of the hydrogel precursor obtained by adding to S0.

[0104] Example 9

[0105] This example is the 9th example of the present invention. Different from Example 1, the addition amount of the surfactant in the continuous phase of S2 is 0.5% of the volume of the fluorinated liquid.

[0106] Example 10

[0107] This example is the 10th example of the present invention. Different from Example 1, the addition amount of the surfactant in the continuous phase of S2 is 5% of the volume of the fluorinated liquid.

[0108] Example 11

[0109] This example is the 11th example of the present invention. Different from Example 1, the pumping flow rate of the dispersed phase in the pumped liquid phase in S21 is 1 μL / min; the pumping flow rate of the continuous phase is 10 μL / min.

[0110] Example 12

[0111] This example is the 12th example of the present invention. Different from Example 1, the pumping flow rate of the dispersed phase in the pumped liquid phase in S21 is 1000 μL / min; the pumping flow rate of the continuous phase is 5000 μL / min.

[0112] Example 13

[0113] This example is the 13th example of the present invention. Different from Example 1, the hydrogel microspheres are crosslinked by non-photocuring. The specific preparation method includes the following steps:

[0114] S0. Dissolve gelatin in deionized water, and then add surfactant polyvinyl alcohol (PVA) in sequence to obtain a hydrogel precursor; the mass ratio of the water-soluble hydrogel to the surfactant is 15:0.5;

[0115] S1. Add 7500 of electronic fluorinated liquid to the above-obtained gelatin solution of hydrogel precursor and stir. When a milky white dispersion is formed, a dispersed phase is obtained; the addition amount of the electronic fluorinated liquid 7500 is 40% of the volume of the hydrogel precursor obtained in S0; the stirring process is: rotation speed 1000 rpm, stirring time 8 min;

[0116] S2. Prepare hydrogel microspheres by microfluidic technology, which specifically includes the following steps:

[0117] Pump the above-obtained dispersed phase and continuous phase into a co-flow microfluidic device to form droplets; the pumping flow rate of the dispersed phase in the pumped liquid phase is 30 μL / min; the pumping flow rate of the continuous phase is 400 μL / min; the preparation method of the continuous phase is: add surfactant PFE-PEG to the fluorinated liquid to form a continuous phase; the addition amount of the surfactant is 1% of the volume of the fluorinated liquid; slowly add glutaraldehyde solution (1%) to the droplet system and react at room temperature for 0.5 hours to form gelatin microspheres. Then, wash the gelatin hydrogel porous microspheres with a microsphere cleaning solution, rinse twice to remove excess oil and surfactant; finally, collect the gelatin hydrogel porous microspheres into pure water, pre-freeze at -80 °C and then put them into a freeze dryer for freeze-drying, and gelatin hydrogel porous microspheres are obtained after freeze-drying.

[0118] Example 14

[0119] The difference between this example and Example 2 is that in S21, the inner diameter of the syringe needle is 0.2 mm; the vertical distance between the needle and the receiving device (a container containing electronic fluorinated liquid 7100) is adjusted to 20 cm, the DC voltage between the needle and the receiving device is set to 20 kV, and the syringe propulsion rate is 1 mL / h.

[0120] Example 15

[0121] The difference between this example and Example 3 is that in S21, the rotation speed is set to 5000 rpm and the homogenization time is 5 min.

[0122] Comparative Example 1

[0123] This comparative example is the first comparative example of the present invention. The difference from Example 1 is that in S1, the pore-forming agent is selected as water.

[0124] Comparative Example 2

[0125] This comparative example is the 2nd comparative example of the present invention. The difference from Example 1 is that methanol is selected as the pore-forming agent in S1.

[0126] Comparative Example 3

[0127] This comparative example is the 3rd comparative example of the present invention. The difference from Example 1 is that ethanol is selected as the pore-forming agent in S1.

[0128] Comparative Example 4

[0129] This comparative example is the 4th comparative example of the present invention. The difference from Example 1 is that the mass ratio of the water-soluble hydrogel, photoinitiator and surfactant in S0 is 35:2:6; the grafting amount of methacrylate group MA in the DeXMA hydrogel porous microspheres obtained in S22 is 50%.

[0130] Comparative Example 5

[0131] This comparative example is the 6th example of the present invention. The difference from Example 1 is that the addition amount of the fluorinated liquid in S1 is 8% of the volume of the hydrogel precursor obtained by adding to S0.

[0132] Comparative Example 6

[0133] This comparative example is the 7th example of the present invention. The difference from Example 1 is that the addition amount of the fluorinated liquid in S1 is 55% of the volume of the hydrogel precursor obtained by adding to S0.

[0134] Performance detection:

[0135] Morphology of hydrogel microspheres: The hydrogel microspheres obtained in the examples and comparative examples were placed under an electron microscope for observation. Figure 1 It is the morphology diagram of the hydrogel microspheres obtained in Example 1. It can be seen that the microspheres have regular shapes, uniform surface micropore distributions, and no adhesion of the microspheres occurs. Figure 2 It is the morphology diagram of the hydrogel microspheres obtained in Comparative Example 2. It can be seen that the microspheres are damaged in shape, the surface micropore distributions are uneven, and adhesion of some microspheres occurs.

[0136] Hydrogel microsphere particle size test:

[0137] (I) Preparation before testing

[0138] 1. Sample preparation: Take a small amount of the hydrogel microsphere dispersion obtained in any example or comparative example and drop it on a glass slide, and gently cover it with a coverslip. If the microsphere concentration is high, it can be appropriately diluted with phosphate buffer solution.

[0139] 2. Instrument preparation: Select a microscope according to the particle size of the microspheres. Use an optical microscope for micron-level, and an electron microscope for nano-level; adjust the light source, focal length, and select the objective lens for the optical microscope, and perform sample fixation, dehydration, and high-vacuum operation for the electron microscope.

[0140] (2) Test steps

[0141] 1. Place the glass slide on the microscope stage for fixation.

[0142] 2. First, find the uniform area of the microspheres under the low-power microscope, and then switch to the high-power microscope.

[0143] 3. Use the built-in software to take photos of multiple fields of view (at least 20).

[0144] 4. Use the image analysis software Image J to set the threshold to distinguish the microspheres from the background and measure the diameter of the microspheres.

[0145] The test results of the particle size distribution of the hydrogel microspheres obtained in Examples 1-15 and Comparative Examples 1-6 are shown in Table 1. The calculation method of the coefficient of variation CV is as follows: first calculate the average value and standard deviation of the data, then divide the standard deviation by the average value and multiply by 100%, and the resulting value is the CV value. The smaller the CV value, the more uniform the particle size distribution of the obtained hydrogel microspheres.

[0146] Test of the pore size distribution of hydrogel microspheres:

[0147] 1. Measurement: Observe the cross-section or surface of the microspheres with an optical or electron microscope. For regular pores, measure the longest and shortest diameters and take the average. For irregular pores, measure the diameters at different positions multiple times and then take the average. After measuring multiple pores of multiple microspheres, count the number or proportion of pores in different pore size ranges to obtain the pore size distribution.

[0148] 2. Image analysis: Take images of the microspheres with a microscope. Use the image analysis software Image J to preprocess and adjust the contrast and brightness. Set the threshold to distinguish the pores from the background. The software automatically identifies and measures the pore area, perimeter, etc., and calculates the equivalent pore diameter using the equivalent circular area formula to obtain the pore size distribution data.

[0149] 3. Model fitting: Select a mathematical model such as normal or lognormal according to the pore structure observed by the microscope. First, statistically analyze a large amount of pore measurement data to determine model parameters such as the mean value and standard deviation. Use Origin to calculate the probability density or cumulative distribution function in different pore size intervals according to the model, and obtain the pore size distribution curve and data.

[0150] The test results of the particle size and pore size distribution of the hydrogel microspheres obtained in Examples 1-15 and Comparative Examples 1-6 are shown in Table 1. The particle size distribution of the microspheres obtained in Example 1 is shown in Figure 3 .

[0151] Table 1 Particle size and pore size distribution of hydrogel microspheres obtained in Examples 1-15 and Comparative Examples 1-6

[0152]

[0153]

[0154] Test of the biocompatibility of hydrogel microspheres:

[0155] 1. Preparation stage

[0156] Material preparation: Ensure the purity and uniformity of the hydrogel microspheres, and prepare an appropriate amount of microsphere samples as well as necessary experimental equipment and reagents.

[0157] Cell preparation: Select appropriate cell lines, such as fibroblasts, endothelial cells, etc., for cell culture to ensure that the cells are in the logarithmic growth phase and in good condition.

[0158] 2. Cell adhesion and proliferation test:

[0159] Inoculate cells on the surface of the hydrogel microspheres obtained in the examples or comparative examples, and observe the adhesion and proliferation of cells on the surface of the hydrogel microspheres at different time points. After calcein staining, observe under a microscope. Green indicates that the cell activity is good, and it can be seen from the cell morphology that the cells have spread on the surface of the microspheres. After inoculating cells on the surface of the hydrogel microspheres obtained in Example 1, the cell activity state diagram is shown in Figure 4 , after inoculating cells on the surface of the hydrogel microspheres obtained in Comparative Example 2, the cell activity state diagram is shown in Figure 5 , it can be seen that the cell activity of the hydrogel microspheres obtained in Example 1 is much higher than that of the hydrogel microspheres obtained in Comparative Example 2.

[0160] Test on the pore size stability of hydrogel microspheres: Immerse the hydrogel microspheres obtained in the examples and comparative examples in water, and place the water-saturated hydrogel microspheres under a microscope to observe the pore size state. Figure 6 It can be seen that the pore structure of the hydrogel microspheres obtained in Example 1 did not change significantly after water absorption, did not disappear, and the pore structure was stable. As shown in the figure, the red circled part is the micropores that stably exist after the microspheres absorb water (this is an example, and each microsphere has multiple similar bubbles, which are not circled one by one). Figure 7 This is the morphology of the hydrogel microspheres obtained in Comparative Example 1 after water absorption. It can be clearly seen that Figure 7 there are no pores on the micro surface spheres in , indicating that most of the micropores of the hydrogel microspheres obtained in Comparative Example 1 are re-closed after water absorption, and the pore structure stability is poor.

[0161] As described above, it is only the implementation mode of the present invention, and the patent protection scope is not limited thereby. Those skilled in the art make non-substantive changes or substitutions based on the present invention, and still fall within the patent protection scope.

Claims

1. A method for preparing hydrogel microspheres, comprising S1. Adding a pore-forming agent to a hydrogel precursor to obtain a dispersed phase; S2. Obtaining hydrogel microspheres by using a microfluidic method, an electrospray method or an emulsion polymerization method for the continuous phase and the dispersed phase; It is characterized in that In S1, the pore-forming agent is a fluorinated liquid; In S2, the continuous phase contains a fluorinated liquid.

2. The preparation method of the hydrogel microspheres according to claim 1, wherein, The grafting amount of methacrylate groups in the hydrogel microspheres obtained in S2 is 20% to 80%.

3. The preparation method of the hydrogel microspheres according to claim 2, characterized in that, Before the step of S1, there is also S0. Preparing a hydrogel precursor, and S0 includes the following steps: dissolving a water-soluble hydrogel, adding an initiator to obtain a hydrogel precursor; the mass ratio of the water-soluble hydrogel to the initiator is (1 to 30):(0.1 to 1.5).

4. The method for preparing the hydrogel microspheres according to claim 3, wherein A surfactant is also added in the method for preparing the hydrogel precursor in S0; the mass ratio of the water-soluble hydrogel, the photoinitiator to the surfactant is (1 to 30):(0.1 to 1.5):(0.5 to 5).

5. The preparation method of the hydrogel microspheres according to claim 1, characterized in that, The addition amount of the pore-forming agent in S1 is 10% to 50% of the volume of the hydrogel precursor.

6. The preparation method of the hydrogel microspheres according to claim 1, wherein, For the method of preparing hydrogel microspheres by the microfluidic method in S2, specifically, it includes the following steps: S21. Pumping the continuous phase and the dispersed phase into a microfluidic device together to form droplets; S22. Sequentially performing photocrosslinking curing, washing, and freeze-drying on the droplets obtained in S21 to obtain hydrogel microspheres.

7. The method for preparing the hydrogel microspheres according to claim 7, wherein, In S21, the pumping flow rate of the dispersed phase in the pumped liquid phase is (1 to 1000) μL / min; the pumping flow rate of the continuous phase is (10 to 5000) μL / min.

8. The preparation method of the hydrogel microspheres according to claim 1, wherein, The continuous phase in S2 also contains a surfactant, and the addition amount of the surfactant is 0.5% to 5% of the volume of the fluorinated liquid.

9. A hydrogel microsphere prepared by the method for preparing hydrogel microspheres according to any one of claims 1 to 8.

10. Use of a hydrogel microsphere prepared by the method for preparing hydrogel microspheres according to any one of claims 1 to 8 in the preparation of biomedical materials.

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