Preparation method of porous microspheres with adjustable aperture

Abstract

The invention relates to the technical field of hydrogel material preparation, and discloses a preparation method of porous microspheres with adjustable pore diameters. According to the preparation method provided by the invention, liquid drop microfluidics, ice crystal pore-forming and photo-crosslinking technologies are integrated, continuous and seamless production from liquid drops to porous microspheres is realized, the size of an ice crystal template can be flexibly controlled by accurately regulating and controlling the freezing temperature, the freezing time and the concentration of a photo-curable material in a water phase, and the size of the porous microspheres can be accurately controlled. The whole process does not need to introduce a toxic pore-foaming agent and is green and safe, the steps are simplified, the operation is simple and convenient, and the cost is low; according to the adopted device, a micro-fluidic chip, a controllable freezing system and a light source are connected in series through an outlet hose, an integrated platform with a compact structure is constructed, traditional complex freezing hole-forming equipment is greatly simplified, and the manufacturing cost and the operation threshold are reduced.

Description

Technical Field

[0001] This invention relates to the field of hydrogel material preparation technology, and in particular to a method for preparing porous microspheres with adjustable pore size. Background Technology

[0002] Porous microspheres are tiny spheres with a three-dimensional network structure and pores, formed by physical or chemical cross-linking of polymer materials. Due to their excellent biocompatibility, high specific surface area, tunable pore structure, and good cell adhesion and material exchange capabilities, they have broad application prospects in the biomedical field. For example, they can be used as microcarriers for cell culture, drug controlled-release systems, tissue engineering scaffolds, cell therapy carriers, diagnostic imaging tools, wound repair materials, and ink components for 3D bioprinting. The performance of porous microspheres largely depends on their particle size uniformity, pore size, and pore connectivity; therefore, precise control of the preparation method is crucial.

[0003] Traditional methods for preparing porous microspheres mainly involve two steps: forming hydrogel droplets and solidifying the microspheres through pore formation and cross-linking. Early droplet formation methods often employed batch emulsification, but this method suffers from problems such as high droplet size dispersibility and significant batch-to-batch variations, resulting in non-uniform microsphere particle size and hindering its application in biomedicine. To overcome these shortcomings, microfluidic technology has been gradually introduced. Microfluidic technology, by precisely controlling fluids in micrometer-scale channels, can generate highly monodisperse microdroplets, offering advantages such as adjustable microsphere size, high monodispersity, high preparation efficiency, and controllable structure.

[0004] Combining droplet microfluidics with pore-forming processes can produce porous microspheres with uniform particle size, tunable pore size, and uniform porosity. Currently, pore-forming methods combined with microfluidics mainly include photopolymerization with pore-forming agents, two-emulsion methods, and ice crystal pore-forming methods. Among these, the photopolymerization with pore-forming agents method requires adding a pore-forming agent to the aqueous phase, followed by cross-linking and elution, involving multiple steps and potentially introducing biocompatible chemicals. The two-emulsion method prepares hollow microspheres by forming a W / O / W structure, but the process is complex and requires sophisticated equipment. The ice crystal pore-forming method utilizes water in the aqueous phase to form ice crystals at low temperatures; after cross-linking, the ice crystals melt, leaving pores. It has advantages such as not introducing exogenous pore-forming agents, low biotoxicity, and simplified steps, and is considered a green and efficient pore-forming method.

[0005] However, existing ice crystal pore-forming methods still have significant limitations in practical applications. First, the temperature control methods are relatively simple and rigid, usually relying on fixed low temperatures (such as -80°C or -20°C) set by commercial refrigerators, lacking a flexible and adjustable low-temperature environment, making it difficult to achieve precise control over the pore structure. Second, existing ice crystal pore-forming devices are usually quite complex, requiring multiple droplet transfers and independent freezing and cross-linking steps, which is not only cumbersome and time-consuming, but also results in high equipment costs and poor repeatability, limiting their widespread application in laboratories and industrial scales. In addition, the separation of droplet collection and freezing processes in traditional methods can easily lead to droplet aggregation, deformation, or uneven ice crystal growth, affecting the pore uniformity and structural integrity of the final microspheres.

[0006] Therefore, there is an urgent need to develop an integrated, easy-to-operate, low-cost porous microsphere preparation platform with flexible controllable freezing temperature to achieve efficient preparation of high-quality microspheres with uniform particle size, adjustable pore size, and interconnected pores, so as to meet the higher requirements for material performance in tissue engineering, drug delivery and other biomedical applications. Summary of the Invention

[0007] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a method for preparing porous microspheres with adjustable pore size.

[0008] A second objective of this invention is to provide an apparatus for the preparation method of the aforementioned porous microspheres with adjustable pore size.

[0009] The third objective of this invention is to provide a porous microsphere with adjustable pore size.

[0010] The fourth objective of this invention is to provide applications for such porous microspheres with adjustable pore size.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A first aspect of the present invention provides a method for preparing porous microspheres with adjustable pore size, comprising the following steps:

[0013] S1. Mix the photocurable material, photoinitiator, and solvent to obtain the aqueous phase; mix the emulsifier and oil phase material to obtain the oil phase;

[0014] S2. Inject the aqueous phase and oil phase into the microfluidic chip respectively to obtain hydrogel droplets;

[0015] S3. The hydrogel droplets are flowed into the freezing system, where the water inside the droplets condenses into ice crystals, which are then photocrosslinked and solidified to obtain porous microspheres.

[0016] The temperature of the refrigeration system is regulated by a refrigeration mixture, and the temperature range includes below 0°C and above -40°C, as well as -70°C to -80°C, with a freezing time of 10-60 min; the refrigeration mixture is selected from ice-sodium chloride mixture, ice-calcium chloride mixture, or dry ice-ethanol mixture.

[0017] In some embodiments of the present invention, the concentration of the photocurable material in the aqueous phase is 3wt%-12wt%.

[0018] In some preferred embodiments of the present invention, the concentration of the photocurable material in the aqueous phase is 3wt%-6wt%.

[0019] In some embodiments of the present invention, the photocurable material is selected from at least one of methacrylamide-modified biopolymers and synthetic photocrosslinking polymers.

[0020] In some embodiments of the present invention, the methacrylamide-modified biopolymer is selected from at least one of methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid (HAMA), methacrylated decellularized matrix (dECMMA), methacrylamide chitosan (CSMA), methacrylamide carboxymethyl chitosan (CMCSMA), methacrylamide sodium alginate (AlgMA), methacrylamide silk fibroin (SilMA), methacrylamide dextran (DexMA), methacrylamide chondroitin sulfate (ChSMA), methacrylamide polylysine (PLMA), and acrylamide RGD peptide (Pep-RGDfKAC).

[0021] In some embodiments of the present invention, the synthetic photocrosslinking polymer is selected from at least one of polyethylene glycol diacrylate (PEGDA) and polyether F127 diacrylate (F127DA).

[0022] In some embodiments of the present invention, the concentration of the photoinitiator in the aqueous phase is 0.2wt%-0.5wt%.

[0023] In some preferred embodiments of the present invention, the concentration of the photoinitiator in the aqueous phase is 0.2wt%-0.3wt%.

[0024] In some embodiments of the present invention, the photoinitiator is selected from at least one of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (I2959), and ethyl 2,4,6-trimethylbenzoylphenylphosphonate (TPO-L).

[0025] In some embodiments of the present invention, the solvent is selected from water, phosphate buffer (PBS), Tris-HCl buffer, HEPES buffer, cell culture medium, and sodium bicarbonate aqueous solution.

[0026] In some embodiments of the present invention, the aqueous phase is prepared by a method comprising the following steps: dissolving a photoinitiator in a solvent, adding a photocurable material, and stirring at 40-60°C to dissolve the aqueous phase.

[0027] In some embodiments of the present invention, the volume ratio of the emulsifier to the oil phase material is 1:(16-24).

[0028] In some preferred embodiments of the present invention, the volume ratio of the emulsifier to the oil phase material is 1:(18-22).

[0029] In some embodiments of the present invention, the oil phase material is selected from mineral oil or fluorinated oil.

[0030] In some embodiments of the present invention, the mineral oil comprises liquid paraffin; the fluorinated oil comprises at least one of HFE7500, HFE7100, FC-40, and Fluo-Oil 135.

[0031] In some embodiments of the present invention, when the oil phase material is mineral oil, the emulsifier is selected from at least one of Span 80 and Tween 60; when the oil phase material is fluorinated oil, the emulsifier is selected from at least one of Fluo-Surf and PFPE-PEG-PFPE.

[0032] In some embodiments of the present invention, the aqueous phase is prepared by a method comprising the following steps: stirring an emulsifier and an oil phase material at 30-50°C for 8-12 minutes until they are mixed evenly to obtain the oil phase.

[0033] In some embodiments of the present invention, the internal channel size of the microfluidic chip is 50-1000 μm.

[0034] In some embodiments of the present invention, the flow rate ratio of the aqueous phase to the oil phase injected into the microfluidic chip is 1:(5-10).

[0035] In some preferred embodiments of the present invention, the flow rate ratio of the aqueous phase to the oil phase injected into the microfluidic chip is 1:(6-8). In some embodiments of the present invention, adjusting the temperature of the refrigeration system by means of a freezing mixture specifically includes:

[0036] An ice-sodium chloride mixture with a mass ratio of ice to sodium chloride of (2-10):1 is selected, and the temperature of the refrigeration system is adjusted to be below 0°C and greater than or equal to -21°C;

[0037] An ice-calcium chloride mixture with a mass ratio of ice to calcium chloride of (2-5):1 is selected, and the temperature of the refrigeration system is adjusted to -20°C to -40°C.

[0038] A dry ice-ethanol mixture is used, and the temperature of the refrigeration system is adjusted to -70°C to -80°C.

[0039] In some preferred embodiments of the invention, the temperature of the refrigeration system is -4°C to -40°C and -72°C to -79°C.

[0040] In some preferred embodiments of the present invention, adjusting the temperature of the refrigeration system by means of a freezing mixture specifically includes:

[0041] An ice-sodium chloride mixture with ice and sodium chloride mass ratios of 10:1, 5:1, 3:1, and 2:1 is selected, and the temperature of the refrigeration system is adjusted to -4°C to -6°C, -8°C to -10°C, -15°C to -18°C, and approximately -21°C.

[0042] An ice-calcium chloride mixture with ice and calcium chloride in mass ratios of 5:1, 3:1, and 2:1 was selected, and the temperature of the refrigeration system was adjusted to approximately -20°C, approximately -30°C, and approximately -40°C, respectively.

[0043] A dry ice-ethanol mixture is selected, and the dry ice is immersed in ethanol. The temperature of the refrigeration system is adjusted to -72°C to -79°C.

[0044] In some embodiments of the present invention, the conditions for photocrosslinking curing include: using a 400-410 nm blue light source and an illumination power density of 30-85 mW / cm². 2 The irradiation time is 1-5 minutes.

[0045] In some preferred embodiments of the present invention, the conditions for photocrosslinking curing include: using a 400-410 nm blue light source and an illumination power density of 36-44 mW / cm². 2 The irradiation time is 1-5 minutes.

[0046] In some embodiments of the present invention, after the photocrosslinking and curing, the process further includes the step of collecting and cleaning the microspheres.

[0047] A second aspect of the present invention provides an apparatus for the preparation method of porous microspheres with adjustable pore size as described in the first aspect of the present invention, comprising:

[0048] Microfluidic chips are used to generate hydrogel droplets;

[0049] The outlet hose has its inlet end connected to the droplet outlet of the microfluidic chip for conveying hydrogel droplets;

[0050] A refrigeration system includes an insulated container filled with a refrigeration mixture and a clamp fixed inside the insulated container; the clamp includes a base and a top cover, and the outlet hose is coiled around the base of the clamp and confined between the base and the top cover;

[0051] A light source, located at the outlet end of the outlet hose, is used to perform photocrosslinking and curing of the frozen hydrogel droplets.

[0052] In some embodiments of the present invention, the clamp is made of a thermally conductive material.

[0053] In some preferred embodiments of the present invention, the clamping plate is a stainless steel plate.

[0054] In some embodiments of the present invention, the heat-insulating container is a foam box.

[0055] In some embodiments of the present invention, the length of the outlet hose is 0.8-1.5 m, the outer diameter is 2-3 mm, and the inner diameter is 0.8-1.5 mm.

[0056] In some embodiments of the present invention, the device further includes an injection pump for injecting the aqueous phase and oil phase into the microfluidic chip.

[0057] A third aspect of the present invention provides a porous microsphere with adjustable pore size, comprising a porous microsphere prepared by the method for preparing porous microspheres with adjustable pore size described in the first aspect of the present invention.

[0058] In some embodiments of the present invention, the diameter of the porous microspheres with adjustable pore size is 50-1000 μm.

[0059] In some embodiments of the present invention, the pore size of the adjustable porous microsphere is 5-50 μm.

[0060] The fourth aspect of the present invention provides the application of the tunable porous microspheres described in the third aspect of the present invention in the preparation of cell culture microcarriers and / or interventional embolization microsphere materials.

[0061] In some embodiments of the present invention, when the tunable porous microspheres are used to prepare cell culture microcarriers, the cells include rat bone marrow mesenchymal stem cells (BMSCs).

[0062] The basic principle of the method and apparatus for preparing porous microspheres with adjustable pore size of the present invention is explained as follows:

[0063] 1) In this invention, an aqueous phase containing photocurable materials and photoinitiators, and an oil phase containing emulsifiers are injected into a microfluidic chip via injection pumps. Within the chip's microchannels, the oil phase acts as a continuous phase, shearing the aqueous phase to generate uniformly sized hydrogel droplets. The droplet diameter can be precisely controlled by adjusting parameters such as the flow rates of the two phases and the chip channel size.

[0064] 2) The generated droplets flow into the freezing system through the outlet hose, where the internal water condenses into ice crystals at low temperatures (below 0°C and above -80°C). The freezing system contains a freezing mixture, specifically an ice-sodium chloride mixture, an ice-calcium chloride mixture, or a dry ice-ethanol mixture. By adjusting the ratio of ice to sodium chloride, ice to calcium chloride, or dry ice to ethanol, precise temperature control can be achieved within the range of below 0°C and above -40°C, or between -70°C and -80°C. The lower the temperature, the more ice crystals nucleate and the smaller their size, resulting in smaller pores. The higher the temperature, the larger the ice crystals grow, resulting in larger pores. Controlling the freezing time also helps; the longer the freezing time, the more fully the ice crystals grow, resulting in larger pores. Furthermore, the lower the concentration of the photocurable material in the aqueous phase and the higher the water content, the larger the ice crystal growth space, resulting in larger pores and better permeability. By adjusting the concentration of the photocurable material in the aqueous phase, the freezing temperature, and the freezing time, the pore size of the microspheres can be controlled.

[0065] 3) After freezing, the droplets are irradiated by a light source, and the photoinitiator is activated, which triggers a cross-linking reaction of the photocurable material, solidifying and fixing the droplet structure containing ice crystals to form the final three-dimensional network of microspheres. The microspheres are collected and the temperature is raised to room temperature. The ice crystals inside naturally melt into water, leaving the space occupied by the ice crystals, thus forming a through-porous structure. After cleaning, the final porous microspheres can be obtained.

[0066] The apparatus for preparing porous microspheres with adjustable pore size according to this invention is a highly integrated and continuous process. Its core lies in integrating three core processes—droplet generation, ice crystal pore formation, and photocrosslinking curing—into a seamlessly connected flow system, specifically as follows:

[0067] The microfluidic chip receives the aqueous and oil phases from the syringe pump. Within its internal microchannels, shear flow generates highly monodisperse hydrogel droplets. These droplets immediately enter an outlet hose fluidly connected to the chip's outlet. This hose is pre-coiled and fixed between the base and top cover of a clamp (made of thermally conductive material). The entire clamp is placed within an insulated container filled with a freezing mixture, forming a freezing system. The clamp effectively protects the microspheres from damage. As the droplets flow through this low-temperature hose, their internal water gradually condenses into ice crystals. By adjusting the ratio of the freezing mixture (controlling the temperature below 0°C and above -40°C, or -70°C to -80°C) and controlling the hose length / flow rate (controlling the freezing time between 10-60 seconds), the freezing system can be controlled. The concentration of photocurable material in the aqueous phase also affects the growth space of ice crystals. The combined effect of these three factors determines the final size and distribution of ice crystals, which serves as a template for subsequent pores. The outlet hose containing the frozen droplets is led out from the insulated container, and a light source is placed near its outlet end. When the droplets flow to this point, they are irradiated with light of a specific wavelength and power. The photoinitiator in the light is activated, which quickly triggers the cross-linking polymerization reaction of the photocurable material. This process instantly fixes the microstructure of the droplets containing ice crystals, forming a solidified microsphere network. The cross-linked microspheres flow into the collection container with the fluid. At room temperature, the ice crystals inside the microspheres, which serve as templates, gradually melt into water, forming an interconnected porous structure, thus obtaining porous microspheres.

[0068] Compared with the prior art, the beneficial effects of the present invention are:

[0069] 1) The method for preparing porous microspheres with adjustable pore size provided by the present invention integrates droplet microfluidics, ice crystal pore formation and photocrosslinking technology, realizing continuous and seamless production from droplets to porous microspheres. By precisely controlling the freezing temperature, time and the concentration of photocurable material in the aqueous phase, the size of the ice crystal template can be flexibly controlled, thereby achieving precise and programmable control of the final microsphere pore size and porosity. The whole process does not require the introduction of toxic pore-forming agents, is green and safe, and has simplified steps, is easy to operate and has low cost.

[0070] 2) The apparatus for preparing porous microspheres with adjustable pore size provided by the present invention connects the microfluidic chip, the controllable freezing system and the light source in series through the outlet hose, thus constructing a compact integrated platform. The heat-conducting clamp ensures the uniformity of freezing. Using ice-sodium chloride mixture, ice-calcium chloride mixture or dry ice-ethanol mixture as the cold source, it achieves stable low temperature in a wide range (below 0℃ and greater than or equal to -40℃, and -70℃ to -80℃) in a simple insulated container, which greatly simplifies the traditional complex cryogenic pore-forming equipment and reduces manufacturing costs and operating threshold.

[0071] 3) The porous microspheres provided by this invention have a wide range of material systems and are suitable for various photocurable biopolymers. The microspheres have high monodispersity, uniform and controllable particle size, adjustable pore size and three-dimensional interconnection, which is conducive to cell ingrowth and material transport. They have good biocompatibility, are non-toxic and degradable. CCK-8 and live / dead staining have confirmed that they can support good cell adhesion and proliferation, and have great potential as cell culture microcarriers and interventional embolization microsphere materials. Attached Figure Description

[0072] Figure 1 This is a schematic diagram of the apparatus for preparing porous microspheres with adjustable pore size in Example 1;

[0073] Figure 2 This is a schematic diagram of the freezing system clamp in the apparatus for preparing porous microspheres with adjustable pore size in Example 1.

[0074] Figure 3 This is an optical microscope image of the porous microspheres in Example 1;

[0075] Figure 4 This is a statistical diagram of the particle size distribution of the porous microspheres in Example 1;

[0076] Figure 5 The image shows a SEM image of the porous microspheres in Example 1.

[0077] Figure 6 The infrared spectrum of the porous microspheres in Example 1;

[0078] Figure 7 The degradation curve of the porous microspheres in Example 1 is shown.

[0079] Figure 8 This is a schematic diagram illustrating the preparation principle of GelMA-FITC in Example 2;

[0080] Figure 9 The hydrogen nuclear magnetic resonance spectrum of GelMA-FITC in Example 2;

[0081] Figure 10 The infrared spectrum of GelMA-FITC in Example 2;

[0082] Figure 11 The image shows a fluorescence confocal microscope image of the fluorescent microspheres in Example 2.

[0083] Figure 12 The results of the CCK-8 cytotoxicity assessment of porous microspheres in the application examples;

[0084] Figure 13 The results of live and dead cell staining of cell-loaded porous microspheres in the application example;

[0085] Figure 14The results of cytoskeleton staining of cell-loaded porous microspheres in the application example;

[0086] Figure 15 This is a SEM image of cell-loaded porous microspheres in an application example. Detailed Implementation

[0087] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0088] Figure 1 This is a schematic diagram of the apparatus for preparing porous microspheres with adjustable pore size in Example 1, wherein, Figure 1 (A) in the diagram is a schematic diagram of the overall structure of the preparation device. Figure 1 (B) in the diagram is a partially enlarged schematic of the microfluidic chip.

[0089] Figure 2 This is a schematic diagram of the freezing system clamp in the apparatus for preparing porous microspheres with adjustable pore size in Example 1, wherein... Figure 2 (A) in the figure is an isometric view of the upper and lower sections of the top cover of the clamping plate. Figure 2 (B) in the figure shows the three views of the top cover of the clamp plate. Figure 2 (C) in the figure is the upper and lower isometric view of the clamp base. Figure 2 (D) in the figure represents the three views of the base of the clamping plate.

[0090] The following will combine Figure 1 and Figure 2 The preparation process of the porous microspheres in Example 1 is described below:

[0091] Example 1

[0092] This embodiment prepares a porous microsphere using the following steps:

[0093] S11. Methacrylamide gelatin and methacrylamide hyaluronic acid are dissolved in a phosphate buffer containing 0.25 wt% phenyl (2,4,6-trimethylbenzoyl)lithium phosphate at a mass ratio of 5:1. The solution is stirred at 50°C to obtain an aqueous phase, wherein the concentration of methacrylamide gelatin is 4 wt% and the concentration of methacrylamide hyaluronic acid is 0.8 wt%.

[0094] S12. Take an appropriate amount of liquid paraffin and add 5% (v / v) Span 80 to it. Stir at 40°C for 10 minutes to ensure uniform mixing and obtain the oil phase.

[0095] S21. Using a commercial microfluidic chip (YX-1001, internal channel size 100μm), an aqueous phase and an oil phase are injected into the microfluidic chip using an injection pump. The flow rate of the aqueous phase is 10μL / min, and the flow rate of the oil phase is 70μL / min. The aqueous phase is subjected to shearing action by the oil phase to form uniform hydrogel droplets.

[0096] S31. The freezing system consists of a foam box filled with an ice-salt mixture (2:1, w / w) (internal temperature controlled at approximately -20°C) and a stainless steel clamp fixed within it. The stainless steel clamp consists of a top cover and a base. The inlet end of a silicone tube (length: 1.5 m, outer diameter: 2.5 mm, inner diameter: 1.5 mm) is connected to the droplet outlet of the microfluidic chip, extending to the insulation system, coiled around the base of the stainless steel clamp, and confined between the base and the top cover. The outlet end of the silicone tube extends to the outside of the freezing system, with a blue light source positioned above it. Hydrogel droplets flowing from the microfluidic chip flow into the freezing system through the silicone tube and are frozen at -20°C. The water inside the droplets condenses into ice crystals and continues to grow for 20 minutes. After the ice crystals have grown to a certain size, the droplets are discharged from the freezing system through the silicone tube and exit through a 405 nm blue light source (light power density of 40 mW / cm²) at the outlet end. 2 Cross-linking and curing were performed by irradiation for 2 minutes;

[0097] S32. Collect and clean the cross-linked and cured microspheres to obtain porous microspheres. Observe the morphology of the microspheres with an optical microscope and count their particle size distribution. Then freeze-dry the microspheres and store them at room temperature.

[0098] Figure 3 The image shown is an optical microscope image of the porous microspheres in Example 1, wherein... Figure 3 Images (A) and (B) in the figure are optical microscope images at different magnifications, derived from... Figure 3 As can be seen, when observed under an optical microscope, the porous microspheres appear as dark brown spheres with slightly rough edges, and their network porous structure is clearly visible with good pore connectivity. This indicates that the method and apparatus provided by the present invention have successfully prepared spherical microspheres with regular shape, completeness, and porous structure, without any cracking or collapse.

[0099] Figure 4 This is a statistical diagram of the particle size distribution of porous microspheres in Example 1, where the statistical sample size n=600. Figure 4 It can be seen that the diameter of the porous microspheres in Example 1 is mainly distributed at around 190 μm, exhibiting a normal distribution characteristic, indicating that the method provided by the present invention can achieve precise control of the microsphere particle size.

[0100] Figure 5 Here is a SEM image of the porous microspheres in Example 1, wherein... Figure 5 In the image, (A) and (B) are SEM images at different magnifications, derived from... Figure 5 It can be seen that the porous microspheres in Example 1 have a significant porous structure with a pore size of about 20 μm and interconnected pores.

[0101] Figure 6 The infrared spectrum of the porous microspheres in Example 1 is shown below. Figure 6 It can be seen that the porous microspheres in Example 1 have characteristic peaks of amide bonds, which is consistent with the chemical structure of the photocurable material used, proving that the successful synthesis of the microspheres is consistent with the expected chemical composition.

[0102] Figure 7 The degradation curve of the porous microspheres in Example 1 is shown below. Figure 7 It was found that the porous microspheres in Example 1 exhibited a slow degradation trend in simulated body fluid, and almost completely degraded after two months, indicating that they have good biodegradability. The degradation measurement steps for the porous microspheres were as follows: 10 mg of lyophilized microspheres were placed in a 1.5 mL EP tube, and 1 mL of PBS and simulated body fluid (prepared from PBS to contain a mixed solution of 2 U / mL type II collagenase and 1 U / mL hyaluronidase) were added respectively. The tubes were incubated in a shaker (37℃, 100 rpm), with the degradation solution changed every 2 days. Samples were taken out every two days for the first two weeks, and weekly for weeks 2-8. The supernatant was removed, and the samples were washed once with deionized water and then lyophilized. The residual weight at each time point was measured. Three parallel samples were used for each time point. The residual percentage (RP) of the microspheres was calculated using the following formula:

[0103] 00%

[0104] In the formula, m1 refers to the total mass of each residual lyophilized microsphere and EP tube, and m0 refers to the mass of each empty EP tube. Plotting the residual percentage of microspheres against time yields the degradation curve of the microspheres over time.

[0105] Example 2

[0106] To clearly observe the pore structure of the microspheres and optimize the microsphere preparation parameters in real time, this embodiment prepares a porous microsphere with green fluorescence, as follows:

[0107] (1) Dissolve an appropriate amount of methacrylamide gelatin (GelMA) in a 0.1 mol / L NaHCO3 solution at 40℃, and dissolve an appropriate amount of fluorescein isothiocyanate (FITC) in dimethylformamide. Then, mix the two solutions and react at 40℃ for 6 h. After the reaction is complete, dialyze with pure water for 7 days and freeze dry to obtain GelMA-FITC.

[0108] (2) GelMA-FITC and methacryloyl hyaluronic acid (HAMA) were dissolved in a phosphate buffer containing 0.25 wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate at a mass ratio of 5:1. The mixture was stirred to dissolve and an aqueous phase was obtained, wherein the concentration of GelMA-FITC was 4 wt% and the concentration of HAMA was 0.8 wt%. An appropriate amount of liquid paraffin was taken and 5% (v / v) Span 80 was added to it. The mixture was stirred at 40 °C for 10 min to ensure uniform mixing and an oil phase was obtained.

[0109] (3) Using a commercial microfluidic chip (YX-1001, internal channel size 100 μm), the aqueous phase and oil phase are injected into the microfluidic chip by an injection pump. The flow rate of the aqueous phase is 10 μL / min and the flow rate of the oil phase is 70 μL / min. The aqueous phase is subjected to shearing action by the oil phase to form uniform hydrogel droplets.

[0110] (4) The freezing system consists of a foam box filled with an ice-salt mixture (2:1, w / w) (with an internal temperature controlled at around -20°C) and a stainless steel clamp fixed inside. The stainless steel clamp consists of a top cover and a base. The inlet end of the silicone tube is connected to the droplet outlet of the microfluidic chip, extends to the insulation system, is coiled around the base of the stainless steel clamp, and is confined between the base and the top cover. The outlet end of the silicone tube extends to the outside of the freezing system, and a blue light source is set above it. The hydrogel droplets flowing out of the microfluidic chip flow into the freezing system through the silicone tube and are frozen at -20°C. The water inside the droplets condenses into ice crystals and continues to grow for 20 minutes. After the ice crystals grow to a certain extent, the droplets are discharged from the freezing system through the silicone tube and exit through the 405 nm blue light source at the outlet end (light power density of 40 mW / cm). 2 Cross-linking and curing were performed by irradiation for 2 minutes;

[0111] (5) Collect the cross-linked and cured microspheres and wash them to obtain light green fluorescent microspheres, which are then observed under a fluorescence confocal microscope.

[0112] Figure 8 This is a schematic diagram illustrating the fabrication principle of GelMA-FITC in Example 2. Figure 8 It is known that porous microspheres with green fluorescence are obtained by grafting fluorescein isothiocyanate (FITC) onto methacrylamide gelatin (GelMA). Figure 9 The image shows the hydrogen nuclear magnetic resonance spectrum of GelMA-FITC in Example 2. Figure 10 The infrared spectrum of GelMA-FITC in Example 2 is shown below. Figure 9 and Figure 10 This demonstrates the successful synthesis of GelMA-FITC, providing a suitable raw material for the subsequent preparation of fluorescent microspheres.

[0113] Figure 11 The image shown is a fluorescence confocal microscope image of the fluorescent microspheres in Example 2, created by... Figure 11 It can be seen that the three-dimensional image obtained by scanning the fluorescent microspheres from top to bottom at 10 μm layers non-destructively demonstrates the spatial distribution and connectivity of the pores inside the microspheres. The image clearly shows that the fluorescent signal is distributed throughout the entire interior of the microspheres, forming a network, which intuitively proves that the pores are three-dimensionally interconnected.

[0114] Application examples

[0115] The porous microspheres prepared in Example 1 were used as cell culture microcarriers, with rat bone marrow mesenchymal stem cells (BMSCs) as the culture medium.

[0116] (1) Isolation and culture of BMSCs: BMSCs were isolated from the bone marrow cavities of the femur and tibia of rats and cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The second to fifth generation cells were selected for use.

[0117] (2) Cytotoxicity assessment of porous microspheres: Weigh an appropriate amount of porous microspheres and place them in a centrifuge tube. After UV sterilization, add DMEM / F12 complete medium and incubate at 37℃ for 72 h. Seed the digested P2-P5 generation BMSCs at a density of 5000 cells / well in a 96-well plate. After the cells adhere to the plate, replace the medium with microsphere extract and continue culturing for 1-3 days. Then add CCK-8 reagent to measure cytotoxicity.

[0118] (3) Staining of live and dead cells on cell-loaded porous microspheres: First, the microspheres were sterilized by immersing them in 75% ethanol. After half an hour, the ethanol was removed, and the microspheres were washed three times with PBS solution. Then, cell counting was performed, and the microspheres were placed in a 6-well plate without TC treatment according to the predetermined number. Next, the digested P2-P5 generation BMSCs cell suspension was seeded onto the surface of the microspheres at a ratio of 200:1. After standing for 3 hours, when the cells adhered, sufficient culture medium was added, and the microspheres were cultured at 37°C for 1-7 days, with the culture medium being changed every two days. On the 1st, 3rd, 5th, and 7th days of culture, the porous microspheres were removed, the culture medium was removed, and the microspheres were gently washed several times with PBS solution. Then, propidium iodide (PI) staining solution prepared in PBS was added, and the microspheres were incubated at room temperature for 10 minutes. The PI staining solution was removed, and the microspheres were gently washed once with PBS. Then, the prepared AM staining solution was added, and the microspheres were incubated at room temperature for 30 minutes. The AM staining solution was removed, and the microspheres were thoroughly washed with PBS. The microspheres were then observed under a confocal microscope.

[0119] (4) Cytoskeleton staining of cell-loaded porous microspheres: First, the microspheres were sterilized by immersing them in 75% ethanol. After half an hour, the ethanol was removed, and the microspheres were washed three times with PBS solution. Cell counting was then performed, and the microspheres were seeded into 6-well plates without TC treatment according to the predetermined number. Next, the digested P2-P5 generation BMSCs cell suspension was seeded onto the surface of the microspheres at a ratio of 200:1. After standing for 3 hours, sufficient culture medium was added, and the microspheres were cultured at 37°C for 5 days. On the 5th day, the microspheres were removed, the culture medium was removed, and the microspheres were washed twice with PBS. Then, 4% paraformaldehyde solution was added for fixation for 30 min, and the paraformaldehyde was removed. The microspheres were washed 2-3 times with PBS for 5 min each time. Then, 0.5% Triton X-100 solution was used for permeation treatment for 5 min, and the microspheres were washed 2-3 times with PBS for 3 min each time. Finally, a pre-prepared rhodamine-labeled pyrotropin solution was added, and the microspheres were incubated at room temperature in the dark for 30 minutes. min; next, counterstain the cell nuclei with an appropriate amount of DAPI solution for about 10-30 min; after removing the staining solution, wash the sample with PBS for 10 min each time, for a total of 3-6 washes; finally, observe under a confocal microscope;

[0120] (5) SEM observation of cell-loaded porous microspheres: The above cells were seeded onto the surface of the microspheres and cultured for 5 days. The culture medium was removed and the microspheres were washed with PBS 3 times. Then, 4% paraformaldehyde was added for fixation for 2-3 h. The microspheres were then dehydrated with a gradient of 30%, 50%, 70%, 90%, 95%, and 100% ethanol for 10 min each time to remove as much water as possible from the cells. After the samples were air-dried, they were carefully removed with tweezers, sprayed with gold, and attached to conductive tape for observation under a scanning electron microscope.

[0121] Figure 12 The results of the CCK-8 cytotoxicity assessment of porous microspheres in the application examples were obtained by... Figure 12 It can be seen that the survival rate of BMSCs is close to or even exceeds 1, which indicates that the components of the microsphere extract did not inhibit cell growth, that is, the porous microspheres have no obvious cytotoxicity.

[0122] Figure 13 The results of live and dead cell staining of cell-loaded porous microspheres in the application example were obtained by... Figure 13 It can be seen that the cells maintained good morphology after 7 days of culture on the microspheres, and the number of live cells increased significantly while the number of dead cells (red) was very small. This indicates that the porous microspheres are conducive to cell adhesion and proliferation and maintain their good condition.

[0123] Figure 14 The cytoskeleton staining results of cell-loaded porous microspheres in the application example are obtained from... Figure 14It can be seen that after 5 days of cell culture, the actin filaments (red) almost covered the entire microsphere, indicating that the porous microspheres facilitate cell spreading and migration.

[0124] Figure 15 The image shows a SEM image of cell-loaded porous microspheres in the application example. Figure 15 It can be seen that after 5 days of culture, the cells have covered the surface of the microspheres, and the microspheres have maintained a normal morphology.

Claims

1. A method for preparing porous microspheres with adjustable pore size, characterized in that, Includes the following steps: S1. Mix the photocurable material, photoinitiator, and solvent to obtain the aqueous phase; mix the emulsifier and oil phase material to obtain the oil phase; S2. Inject the aqueous phase and oil phase into the microfluidic chip respectively to obtain hydrogel droplets; S3. The hydrogel droplets are flowed into the freezing system, where the water inside the droplets condenses into ice crystals, which are then photocrosslinked and solidified to obtain porous microspheres. The temperature of the refrigeration system is regulated by a refrigeration mixture, and the temperature range includes below 0°C and above -40°C, as well as -70°C to -80°C, with a freezing time of 10-60 min; the refrigeration mixture is selected from ice-sodium chloride mixture, ice-calcium chloride mixture, or dry ice-ethanol mixture.

2. The preparation method according to claim 1, characterized in that, The concentration of the photocurable material in the aqueous phase is 3wt%-12wt%.

3. The preparation method according to claim 2, characterized in that, The photocurable material is selected from at least one of methacrylamide-modified biopolymers and synthetic photocrosslinking polymers.

4. The preparation method according to claim 1, characterized in that, The flow rate ratio of the aqueous phase to the oil phase injected into the microfluidic chip is 1:(5-10).

5. The preparation method according to claim 1, characterized in that, Adjusting the temperature of the refrigeration system by using a freezing mixture specifically includes: An ice-sodium chloride mixture with a mass ratio of ice to sodium chloride of (2-10):1 is selected, and the temperature of the refrigeration system is adjusted to be below 0°C and greater than or equal to -21°C; An ice-calcium chloride mixture with a mass ratio of ice to calcium chloride of (2-5):1 is selected, and the temperature of the refrigeration system is adjusted to -20°C to -40°C. A dry ice-ethanol mixture is used, and the temperature of the refrigeration system is adjusted to -70°C to -80°C.

6. The preparation method according to claim 1, characterized in that, The conditions for photocrosslinking and curing include: using a 400-410 nm blue light source and a light power density of 30-85 mW / cm². 2 The irradiation time is 1-5 minutes.

7. An apparatus for preparing porous microspheres with adjustable pore size according to any one of claims 1-6, characterized in that, include: Microfluidic chips are used to generate hydrogel droplets; The outlet hose has its inlet end connected to the droplet outlet of the microfluidic chip for conveying hydrogel droplets; A refrigeration system includes an insulated container filled with a refrigeration mixture and a clamp fixed inside the insulated container; the clamp includes a base and a top cover, and the outlet hose is spirally wound around the base of the clamp and confined between the base and the top cover; A light source, located at the outlet end of the outlet hose, is used to perform photocrosslinking and curing of the frozen hydrogel droplets.

8. The apparatus according to claim 7, characterized in that, The outlet hose has a length of 0.8-1.5 m, an outer diameter of 2-3 mm, and an inner diameter of 0.8-1.5 mm.

9. A porous microsphere with adjustable pore size, characterized in that, The porous microspheres with adjustable pore size are prepared using the preparation method described in any one of claims 1-6.

10. The use of the tunable porous microspheres of claim 9 in the preparation of cell culture microcarriers and / or interventional embolization microsphere materials.

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