A microgel based on acryloyl gelatin, preparation method and application thereof

Abstract

The present invention discloses a microgel based on acryloyl gelatin, a preparation method and an application thereof. A microfluidic method is used in combination with an online photopolymerization reaction to prepare an acryloyl gelatin microgel in high throughput, and the application thereof in the encapsulation of bioactive substances. First, acryloyl gelatin microdroplets are prepared in high throughput using droplet microfluidics technology, and then a visible light source matched with a photoinitiator is placed above the microfluidics pipeline to achieve online continuous production of GelA microdroplets and solidify to form microgel particles. For online encapsulation of substances, it is only necessary to pre-mix the active substances with the aqueous phase. The present invention combines the advantages of droplet microfluidics technology for high throughput production of droplets of uniform size, and also has the advantages of GelA's low viscosity, high photopolymerization efficiency and good biocompatibility. It can be used for high-efficiency and high-quality production of biocompatible microgels. The preparation process is also biocompatible and can meet the needs of online encapsulation of biomolecules and cells.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and relates to a biocompatible microgel and a preparation method thereof, in particular to the preparation of a high-throughput microgel by online photopolymerization using microfluidic technology, and the application of the microgel to online encapsulation of bioactive substances. Background Art

[0002] Biocompatible microgels have a larger specific surface area than macro gels. The substances in the gel spheres allow efficient material exchange with the environment, and they have a certain degree of flowability and injectability. Therefore, they have important application value in fields including biomedical testing, protection and loading of drugs and key biological molecules, and tissue repair. Among the technologies for preparing biocompatible microgels, droplet microfluidics technology has unique advantages in the precision control of size and structure because it can mass-produce monodisperse microgel particles and precisely control their size, composition, and structure. It has become a key technology for microgel production and has received widespread attention. Droplet microfluidics technology has been widely used in the preparation of a variety of biocompatible polymer droplets and their microgels, mainly including polysaccharides (alginate and agarose), PEG derivatives, gelatin, hyaluronic acid, and collagen.

[0003] Among these materials, gelatin has many advantages:

[0004] It has abundant sources, ideal biocompatibility, rich RGD areas for cell attachment, and no immunogenicity, but the physical gel formed near or above body temperature is unstable. Therefore, people have developed many modified gelatins that can be chemically cross-linked. Among them, methacrylamide gelatin (GelMA) is the most widely used modified gelatin in biomanufacturing. It inherits all the advantages of gelatin as a biocompatible gel and can form stable covalently cross-linked gels by chemical or photopolymerization. Therefore, it is very valuable to produce GelMA microgel particles that encapsulate biological substances. However, there are two limitations in the existing droplet microfluidics production of GelMA microgel particles.

[0005] First, the viscosity of GelMA varies greatly between room temperature and body temperature, and the droplet size is very sensitive to temperature, so precise temperature control is required to prepare droplets of uniform size. This preparation requirement that requires precise temperature control increases manufacturing costs and possible manufacturing errors.

[0006] Secondly, the 365nm ultraviolet light source and the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) are both biotoxic. For example, biological molecules such as proteins will denature under ultraviolet light, and even if cells can survive the polymerization process, they may change their phenotype and have toxic side effects. Therefore, only low concentrations of Irgacure 2959 and small doses of ultraviolet light can be used for photopolymerization of microgels. At the same time, GelMA has slow polymerization kinetics, which requires higher concentrations of photoinitiators and higher light doses to form gel microspheres with certain mechanical strength. Such a manufacturing process also makes it difficult to encapsulate drugs, bioactive molecules and even cells online, and it is necessary to strike a balance between high biocompatibility and appropriate gel mechanical strength. Therefore, it is very necessary to use a gelatin derivative photopolymerization system with biocompatibility and faster reaction kinetics in conjunction with a microfluidic online photopolymerization chip. This will effectively protect the biological activity of the encapsulated material and improve the production quality of the entire microgel particles. Summary of the invention

[0007] In order to solve the problems of large viscosity variation range and high toxicity of ultraviolet light source and Irgacure 2959 photoinitiator in the droplet microfluidic production of GelMA microgel in the prior art, the purpose of the present invention is to overcome the shortcomings of the existing technology and provide a microgel based on acrylated gelatin, a preparation method and application thereof, which can continuously, reliably and high-throughput online production of modified gelatin microgel particles in a droplet microfluidic device. The present invention combines the advantages of high throughput and high size uniformity of droplet microfluidics, and has the advantages of low material viscosity, high photopolymerization efficiency and high biocompatibility. It can be used for high-quality production of biocompatible microgel particles, and the preparation process is also biocompatible, which can meet the needs of online encapsulation of active substances, biomolecules and cells.

[0008] In order to achieve the above invention purpose, the present invention adopts the following technical solution:

[0009] A method for preparing a microgel based on acryloyl gelatin comprises the following steps:

[0010] (1) combining temperature-sensitive gelatin with acrylic anhydride having double bonds that can be photocured to form acrylated gelatin (GelA);

[0011] (2) dissolving acrylated gelatin and a visible light photoinitiator in a solvent to obtain a dispersed phase solution; taking the dispersed phase solution as 100%, the amount of acrylated gelatin added is 3-20 wt.% by weight; building a constant temperature device, and using a droplet microfluidics method to make the dispersed phase solution form uniform acrylated gelatin droplets;

[0012] (3) The acrylated gelatin microdroplets were cured online with visible light on the collection channel to prepare acrylated gelatin gel microspheres as microgels for coating bioactive substances.

[0013] Preferably, in step (1), the proportion of acrylic anhydride replacing amino groups on gelatin is 10-99%.

[0014] Preferably, in step (2), the structural formula of the prepared acryloyl gelatin-based microgel is:

[0015]

[0016] Preferably, in the step (2), when preparing the dispersed phase solution, the acrylated gelatin and the visible light initiator are dissolved in at least one of pure water, PBS, TBE, and cell culture medium to obtain a dispersed phase solution; and then droplet microfluidics is used to prepare acrylated gelatin droplets of uniform and controllable size.

[0017] Preferably, in step (2), when using the droplet microfluidics method, the temperature of the aqueous solution entering the chip and the chip forming the droplets are controlled to ensure that the gel precursor is in a flowable state.

[0018] Preferably, in step (2), when using the droplet microfluidics method, microgels of different sizes can be obtained by adjusting the water phase flow rate and the oil phase flow rate, as well as the chip structure and the chip size, and the microgel size range is 10-1000 μm;

[0019] Preferably, in step (2), the mass percentage concentration of the photoinitiator used is 0.01-0.5%.

[0020] Preferably, in step (2), when using the droplet microfluidics method, the constant temperature device used controls the temperature to 35-40°C, and the temperature region covers the range from the water phase inlet to the chip position where the droplets are formed.

[0021] Preferably, in the step (3), gel microspheres with different mechanical strengths are formed by adjusting the visible light illumination distance, light source intensity, GelA concentration and photoinitiator concentration.

[0022] Preferably, in the step (3), the light source used for online curing of the acrylic anhydride gelatin microdroplets with visible light is blue light.

[0023] Preferably, in step (3), a blue light source is placed above the microfluidic channel, 0.5-3 cm away from the GelA microdroplets, and the GelA microdroplets are cured by continuous light irradiation.

[0024] The visible light source of the present invention can initiate the corresponding photoinitiator, and can solidify the microdroplets online in real time under a certain light intensity, and the temperature of the light source is maintained within the range that the biologically active substance can allow.

[0025] The present invention adopts two strategies to improve the production quality and controllability of droplet microfluidics based on acrylated gelatin microgel particles. First, from the perspective of materials, acrylated gelatin (GelA) is used to replace traditional GelMA to improve its rheological properties and improve the photopolymerization efficiency. By changing the photopolymerization conditions or the properties of the prepolymer, the mechanical properties of GelA microgel particles can be adjusted in a wide range. Secondly, visible light photopolymerization replaces traditional ultraviolet light polymerization, making it safer and more biocompatible. An aqueous solution containing acrylated gelatin and a photoinitiator is used as the aqueous phase, and droplet microfluidics technology is used to form uniform droplets. GelA microdroplets are cured online with visible light on a collection pipeline to prepare GelA microgel particles with good biocompatibility. Bioactive substances such as biomolecules or cells are added to an aqueous solution containing acrylated gelatin and a photoinitiator as the aqueous phase, and the same steps are used to prepare GelA microgels encapsulated with active substances.

[0026] Preferably, a method for online curing of GelA gel microspheres comprises the following specific steps:

[0027] a. Dissolve GelA and a visible light photoinitiator in at least one solution of pure water, PBS, TBE, or cell culture medium as the dispersed phase of droplet microfluidics, and use an incompatible oil as the continuous phase; the visible light photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP); the incompatible oil is preferably at least one of HFE 7500 and hexadecane;

[0028] b. To form uniform and stable GelA microdroplets, the GelA temperature is controlled at 35-40°C to maintain it in a low viscosity state to stably form uniform microdroplets;

[0029] c. Place a visible light source matched with a photoinitiator above the microfluidic channel to continuously solidify the GelA microdroplets.

[0030] A microgel based on acryloyl gelatin is prepared by using the preparation method of the microgel based on acryloyl gelatin of the present invention.

[0031] An application of the microgel based on acryloyl gelatin described in the present invention, the microgel is used for coating biologically active substances to form an outer layer or protective layer of the biologically active substances, and the biologically active substances include cells and at least one biologically active substance among nucleic acids, proteins, and enzymes.

[0032] Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

[0033] 1. The present invention adopts two strategies to improve the ability of droplet microfluidics to produce high-quality gel microspheres, which can then be used to encapsulate bioactive substances: first, the strategy adopted from the perspective of materials is to use acrylic anhydride to modify gelatin to provide a more efficient modified gelatin photocuring efficiency and low viscosity characteristics with better fluidity; second, the strategy adopted from the perspective of photoinitiator is to use visible light and visible light initiator to replace ultraviolet light and Irgacure 2959 initiator to achieve a preparation process with higher safety, better biocompatibility and lower cost;

[0034] 2. The results show that compared with methacrylic anhydride gelatin (GelMA), GelA has lower viscosity and less dependence on temperature. From 30°C to 40°C, the viscosity change rate of GelMA is 73.97%, while that of GelA is 15.77%, which supports the generation of smaller microdroplets and improves the stability of droplet size.

[0035] 3. In the present invention, under the action of a blue light source and a LAP photoinitiator, the photocuring efficiency of GelA is higher than that of GelMA; by changing the photopolymerization conditions or the properties of the prepolymer, the mechanical properties of the gel can be effectively adjusted;

[0036] 4. The present invention adopts a droplet microfluidic device to achieve continuous, reliable and high-throughput online production of gel microspheres, and the production process also has good biocompatibility, and can encapsulate bioactive molecules or cells online to retain their biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The graphs show the changes of viscosity of GelA and GelMA with temperature. Inset a shows the changes of GelA with shear rate at different temperatures; inset b shows the changes of GelMA with shear rate at different temperatures; inset c shows the comparison of viscosity of GelA and GelMA at different temperatures.

[0038] Figure 2 Comparison of the photocuring kinetics of GelA and GelMA. Inset a. GelA and inset b. H NMR spectra of GelMA under different blue light irradiation times. Inset c. Comparison of the photocuring kinetics of GelA and GelMA

[0039] Figure 3 Comparison of the size of GelA and GelMA microspheres controlled by microfluidics. GelA and GelMA droplets of different sizes were prepared at different flow rates using droplet microfluidics. Inset a is a microscopic image and inset b is a statistical data chart. (Scale bar is 100μm)

[0040] Figure 4 This is a comparison of the blue light curing effects of GelA and GelMA microspheres at 3 cm (scale is 200 μm).

[0041] Figure 5 The encapsulation of MCF7 and PC12 cells by GelA microgels (green represents live cells, and red represents dead cells).

[0042] Figure 6 This is a schematic diagram of a method for preparing gel microspheres based on acryloyl gelatin according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0043] The above scheme is further described below in conjunction with specific implementation examples. The preferred embodiments of the present invention are described in detail as follows:

[0044] Embodiment 1

[0045] In this embodiment, if Figure 6 As shown, a method for preparing microgel based on acryloyl gelatin comprises the following steps:

[0046] A gel microsphere based on acryloyl gelatin is prepared by a droplet microfluidics method through blue light online curing, which specifically includes the following steps:

[0047] Firstly, a dispersed phase solution was prepared with ultrapure water, acryloyl gelatin and visible light photoinitiator LAP. In the dispersed phase solution, the mass percentage concentration of acryloyl gelatin was 10%, and the mass percentage concentration of LAP was 0.1% according to the mass percentage measurement; and insoluble fluorinated oil was used as the oil phase, and a constant temperature device was built to maintain the temperature at 40°C, and the water phase flow rate was controlled to be 50μL / h and the oil phase flow rate was 100μL / h, and uniform GelA microdroplets were generated by droplet microfluidics technology;

[0048] A blue light source was placed above the microfluidic channel, 1 cm away from the GelA microdroplets, and the GelA microdroplets were continuously illuminated to achieve solidification to obtain microgels. The structural formula of the prepared microgel based on acryloyl gelatin is:

[0049] The proportion of amino groups substituted by acrylic anhydride on gelatin is 10-99%. The size of microgels ranges from 10 to 1000 μm.

[0050] Experimental test analysis:

[0051] Figure 1The following is a graph showing the viscosity of GelA and GelMA as a function of temperature. Inset a shows the viscosity of GelA at different temperatures and shear rate; inset b shows the viscosity of GelMA at different temperatures and shear rate; inset c shows the viscosity comparison of GelA and GelMA at different temperatures. Figure 1 It can be seen that Figure 1 (a) GelA has a high viscosity at 10℃-20℃, and its viscosity drops sharply at 30℃-40℃. At 30℃-40℃, its viscosity does not change with the shear rate and is stable at about 30 mPa·s. Figure 1 (b) GelMA has a higher viscosity at 10℃-20℃, and the viscosity decreases significantly at 30℃-40℃. At the same time, the viscosity still decreases with the increase of shear rate. Figure 1 (c) Comparison of the viscosity of GelA and GelMA at different temperatures. It was found that the viscosity of the two was not much different from 5℃ to 25℃. The viscosity of GelA dropped sharply at 25℃ and dropped to 30 mPa·s at 30℃, while the viscosity of GelMA dropped to 900 mPa·s at 30℃, which is 30 times that of GelA. This means that GelA has better fluidity at the phase transition temperature and has a more sensitive temperature response.

[0052] Figure 2 The following is a comparison of the photocuring kinetics of GelA and GelMA in this example. Inset a. GelA and inset b. H NMR spectra of GelMA under different blue light irradiation times. Inset c. Comparison of the photocuring kinetics curves of GelA and GelMA. Figure 2 It can be seen that the blue light polymerization kinetics of GelA and GelMA were analyzed within 1 minute. Figure 2 (a) is the blue light polymerization kinetics NMR analysis of GelA. The hydrogen on the double bond of acrylate has a pair of characteristic peaks at 5.75 ppm and 6.0 ppm. Figure 2 (b) Blue light polymerization kinetics NMR analysis of GelMA, the hydrogen on the double bond of methacrylate is at 6.1ppm and 6.5ppm. Figure 2 (c) Based on the peak integrated area, the kinetic curves of GelA and GelMA were derived. In the first 15 seconds, the kinetic constants of the two were close, but after 15 seconds, the kinetic constant of GelA was more than twice that of GelMA.

[0053] Figure 3 This is a comparison of the size of GelA and GelMA microspheres controlled by microfluidics in this example. GelA and GelMA droplets of different sizes were prepared at different flow rates using droplet microfluidics. Inset a is a microscopic image and inset b is a statistical data chart. (Scale bar is 100 μm), Figure 3 It can be seen that Figure 3(a) GelA and GelMA droplets were prepared at the same temperature, and the oil phase flow rate ranged from 100 μL / h to 400 μL / h. Figure 3 (b) Statistics of the droplet sizes of the two materials at different oil phase flow rates. At 100 μL / h, the droplet size of GelA is 28 μm, and the size of GelMA is 38 μm, with a difference of 10 μm. As the oil phase flow rate increases, the droplet size gradually decreases. At 400 μL / h, the droplet size of GelA reaches 25 μm, and that of GelMA is 34 μm, which indicates that the droplet size generated by low-viscosity GelA in droplet microfluidics is smaller than that of GelMA.

[0054] Figure 4 This is a comparison of the blue light curing effects of GelA and GelMA microspheres at 3 cm in this example (the scale is 200 μm). Figure 4 It can be seen that under the same lighting conditions, Figure 4 The upper part shows GelA and GelMA microgels suspended in the oil phase, and the lower part shows the removal of the oil phase and the transfer to the aqueous phase. The morphology of the gel spheres formed by GelA solidification under 3cm illumination is better than that of GelMA, which once again proves that the curing efficiency of GelA is better than that of GelMA.

[0055] Figure 5 The encapsulation of MCF7 and PC12 cells by GelA microgel in this example (green represents live cells, red represents dead cells). Figure 5 It can be seen that GelA has good biocompatibility and the survival rate is above 95%.

[0056] The results show that compared with methacrylic anhydride gelatin (GelMA), the viscosity of GelA prepared in this embodiment is lower and less dependent on temperature. From 30°C to 40°C, the viscosity change rate of GelMA is 73.97%, and the change rate of GelA is 15.77%, which supports the generation of smaller microdroplets and improves the stability of droplet size; under the action of blue light source and LAP photoinitiator, the photocuring efficiency of GelA is higher than that of GelMA; by changing the photopolymerization conditions or the properties of the prepolymer, the mechanical properties of the gel can be effectively adjusted. The method of this embodiment can continuously, reliably and high-throughput online production of modified gelatin microgel particles in a droplet microfluidic device. The present invention combines the advantages of high throughput and high size uniformity of droplet microfluidics, and has the advantages of low material viscosity, high photopolymerization efficiency and high biocompatibility. It can be used for high-quality production of biocompatible microgel particles, and the preparation process is also biocompatible, which can meet the needs of online encapsulation of active substances, biomolecules and cells.

[0057] Embodiment 2

[0058] This embodiment is basically the same as the first embodiment, except that:

[0059] In this embodiment, a method for online blue light curing of gel microspheres based on acrylated gelatin using a droplet microfluidics method and encapsulating bioactive substances therewith specifically includes the following steps:

[0060] First, the MCF-7 cells in the exponential growth phase were digested and resuspended in PBS to adjust the cell concentration to 5×10 6 , pieces / ml, a dispersed phase solution was prepared with PBS solution, acryloyl gelatin and visible light photoinitiator LAP, in which the mass percentage concentration of acryloyl gelatin was 10% and the mass percentage concentration of LAP was 0.1% according to the mass percentage measurement; and insoluble fluorinated oil was used as the oil phase, a constant temperature device was built to maintain the temperature at 40°C, the water phase flow rate was controlled to be 50μL / h, the oil phase flow rate was controlled to be 100μL / h, and uniform GelA microdroplets were formed by droplet microfluidics;

[0061] A blue light source was placed above the microfluidic channel, 2 cm away from the GelA microdroplets, and the GelA microdroplets were solidified by continuous light irradiation; the solidified GelA gel containing MCF-7 cells was washed into the cell culture medium and cultured in a 37°C incubator for 7 days, during which the culture medium was changed every other day to allow the GelA microgel to encapsulate the bioactive substances.

[0062] Embodiment 3

[0063] This embodiment is basically the same as the above embodiment, with the following features:

[0064] In this embodiment, a method for online blue light curing of gel microspheres based on acrylated gelatin using droplet microfluidics and encapsulating bioactive substances therewith specifically includes the following steps:

[0065] First, the MCF-7 cells in the exponential growth phase were digested and resuspended in PBS to adjust the cell concentration to 8×10 6 / ml, a dispersed phase solution was prepared with PBS solution, acryloyl gelatin and visible light photoinitiator LAP, in which the mass percentage concentration of acryloyl gelatin was 20%, and the mass percentage concentration of LAP was 1% according to mass percentage; insoluble fluorinated oil was used as the oil phase, a constant temperature device maintained at 40°C was built, the water phase flow rate was controlled to be 600 μL / h, and the oil phase flow rate was 1200 μL / h, and droplet microfluidics was used to form uniform GelA microdroplets; a blue light source was placed above the microfluidic channel, 1 cm away from the GelA microdroplets, and the GelA microdroplets were cured by continuous light.

[0066] Embodiment 4

[0067] This embodiment is basically the same as the above embodiment, with the following features:

[0068] In this embodiment, a method for online blue light curing of gel microspheres based on acrylated gelatin using droplet microfluidics and encapsulating bioactive substances therewith specifically includes the following steps:

[0069] The protein was first dissolved in PBS solution, and a dispersed phase solution was prepared with PBS solution, acryloyl gelatin and visible light photoinitiator LAP. In the dispersed phase solution, the mass percentage concentration of acryloyl gelatin was 20%, and the mass percentage concentration of LAP was 1% according to mass percentage. Insoluble fluorinated oil was used as the oil phase, and a constant temperature device maintained at 35°C was built. The water phase flow rate was 600 μL / h, and the oil phase flow rate was 1200 μL / h. Droplet microfluidics was used to form uniform GelA microdroplets. A blue light source was placed above the microfluidic channel, 2 cm away from the GelA microdroplets, and the GelA microdroplets were cured by continuous light.

[0070] Embodiment 5

[0071] This embodiment is basically the same as the above embodiment, with the following features:

[0072] In this embodiment, a method for online blue light curing of gel microspheres based on acrylated gelatin using droplet microfluidics and encapsulating bioactive substances therewith specifically includes the following steps:

[0073] Firstly, DNA was dissolved in PBS solution, and a dispersed phase solution was prepared with PBS solution, acryloyl gelatin and visible light photoinitiator LAP. In the dispersed phase solution, the mass percentage concentration of acryloyl gelatin was 3%, and the mass percentage concentration of LAP was 0.5% according to mass percentage. Insoluble fluorinated oil was used as the oil phase, and a constant temperature device was built to maintain the temperature at 35°C. The water phase flow rate was 100 μL / h, and the oil phase flow rate was 300 μL / h. Droplet microfluidics was used to form uniform GelA microdroplets. A blue light source was placed above the microfluidic channel, 3 cm away from the GelA microdroplets, and the GelA microdroplets were cured by continuous light irradiation.

[0074] Embodiment 6

[0075] This embodiment is basically the same as the above embodiment, with the following features:

[0076] In this embodiment, a method for online blue light curing of gel microspheres based on acrylated gelatin using droplet microfluidics and encapsulating bioactive substances therewith specifically includes the following steps:

[0077] Horseradish peroxidase (HRP) was first dissolved in PBS solution, and a dispersed phase solution was prepared with PBS solution, acryloyl gelatin and visible light photoinitiator LAP. In the dispersed phase solution, the mass percentage concentration of acryloyl gelatin was 3%, and the mass percentage concentration of LAP was 0.5% according to mass percentage. Insoluble fluorinated oil was used as the oil phase, and a constant temperature device maintained at 35°C was built. The water phase flow rate was 100 μL / h, and the oil phase flow rate was 300 μL / h. Droplet microfluidics was used to form uniform GelA microdroplets. A blue light source was placed above the microfluidic channel at a distance of 0.5 cm from the GelA microdroplets, and the GelA microdroplets were cured by continuous light irradiation.

[0078] The above-mentioned embodiment adopts microfluidic technology combined with online photopolymerization reaction to prepare an acryloyl gelatin microgel in high throughput, and its application in the encapsulation of bioactive substances. First, the acryloyl gelatin microdroplets are prepared in high throughput using droplet microfluidic technology, and then a visible light source matched with the photoinitiator is placed above the microfluidic pipeline to realize the online continuous production of GelA microdroplets and solidify them to form microgel particles. To encapsulate the substance online, it is only necessary to pre-mix the active substance with the aqueous phase. This technology combines the advantages of droplet microfluidic technology for high-throughput production of uniform size droplets, and also has the advantages of GelA's low viscosity, high photopolymerization efficiency and good biocompatibility. It can be used for high-efficiency and high-quality production of biocompatible microgels. The preparation process is also biocompatible and can meet the needs of online encapsulation of biomolecules and cells.

[0079] The above describes the embodiments of the present invention in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made according to the purpose of the invention. Any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical solution of the present invention should be equivalent replacement methods. As long as they meet the purpose of the invention and do not deviate from the technical principles and inventive concepts of the present invention, they belong to the protection scope of the present invention.

Claims

1. A method for preparing microgel based on acryloyl gelatin, characterized in that: The steps include: (1) Combining temperature-sensitive gelatin with acrylic anhydride having double bonds that can be photocured to form acrylic anhydride gelatin GelA; (2) Dissolving acrylated gelatin and visible light photoinitiator in a solvent to obtain a dispersed phase solution; taking the dispersed phase solution as 100%, the amount of acrylated gelatin added is 3-20wt.% by weight; building a constant temperature device, and using a droplet microfluidics method to make the dispersed phase solution form uniform acrylated gelatin droplets; the structure of the prepared microgel based on acrylated gelatin is: ； (3) Acrylic anhydride gelatin microdroplets were cured online with visible light on the collection tube to prepare acrylated gelatin gel microspheres as microgels for coating bioactive substances.

2. The method for preparing microgel based on acryloyl gelatin according to claim 1, characterized in that: In the step (1), the proportion of amino groups substituted by acrylic anhydride on gelatin is 10-99%.

3. The method for preparing microgel based on acryloyl gelatin according to claim 1, characterized in that: In the step (2), when preparing the dispersed phase solution, the acrylated gelatin and the visible light initiator are dissolved in at least one of pure water, PBS, TBE, and cell culture medium to obtain a dispersed phase solution; and then droplet microfluidics is used to prepare acrylated gelatin droplets with uniform and controllable size.

4. The method for preparing microgel based on acryloyl gelatin according to claim 1, characterized in that: In the step (2), when using the droplet microfluidics method, the temperature of the aqueous solution entering the chip and the chip forming the droplets are controlled to ensure that the gel precursor is in a flowable state; Alternatively, microgels of different sizes can be obtained by adjusting the water phase flow rate and the oil phase flow rate, as well as the chip structure and the chip size, and the microgel size range is 10-1000 μm.

5. The method for preparing microgel based on acryloyl gelatin according to claim 1, characterized in that: In the step (2), the mass percentage concentration of the photoinitiator used is 0.01-0.5%.

6. The method for preparing microgel based on acryloyl gelatin according to claim 1, characterized in that: In step (2), when using the droplet microfluidics method, the constant temperature device used controls the temperature to 35-40°C, and the temperature region covers the range from the water phase inlet to the chip position where the droplets are formed.

7. The method for preparing microgel based on acryloyl gelatin according to claim 1, characterized in that: In the step (3), the light source used for online curing of the acrylic anhydride gelatin microdroplets using visible light is blue light.

8. A microgel based on acryloyl gelatin, characterized in that: The microgel is prepared by the method for preparing the acryloyl gelatin-based microgel according to claim 1.

9. An application of the acryloyl gelatin-based microgel according to claim 8, characterized in that: The microgel is used for coating biologically active substances, and the biologically active substances include cells and at least one biologically active substance among nucleic acids, proteins and enzymes.

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