A method for preparing carboxylated porous microspheres

Abstract

The application relates to the technical field of microspheres, and particularly provides a preparation method of carboxylated porous microspheres, which comprises the following steps: adding a biocompatible polymer, an acrylic copolymer and an oil-soluble emulsifier into an organic solvent to be dissolved and prepared into a dispersed phase; adding a water-soluble emulsifier and / or a protective colloid into water to be prepared into a continuous phase; mixing the dispersed phase and the continuous phase to obtain an emulsion, continuously stirring until precipitation appears, collecting the precipitation, cleaning, drying and obtaining the carboxylated porous microspheres. In the continuous stirring stage, air blowing or a crosslinking agent can be added. The carboxylated porous microspheres have good fluidity and good nucleic acid extraction and purification effects.

Description

Technical Field

[0001] This application relates to the field of microsphere technology, and more specifically, to a method for preparing carboxylated porous microspheres. Background Technology

[0002] Porous microspheres are a commonly used raw material in biological reagent detection, especially for the separation and purification of nucleic acids. Therefore, the porous surface is required to have active functional groups, such as hydroxyl, carboxyl, and amino groups, which can improve the hydrophilicity of porous microspheres and their binding to nucleic acids, thereby improving the separation and purification effect of nucleic acids.

[0003] Carboxyl-functionalized porous microspheres are usually treated with carboxylating agents, such as long-chain alkyl carboxylic acids like oleic acid and undecanoic acid. However, since the combination is physical and the molecular weight of long-chain alkyl carboxylic acids is relatively small, the stability after carboxylation is not high enough, or the carboxyl groups cannot be effectively present on the surface of the porous microspheres. As a result, the performance of carboxyl-functionalized porous microspheres cannot be effectively demonstrated, and the application effect for nucleic acid extraction and purification is not good.

[0004] Therefore, the technology for carboxylated porous microspheres still needs to be improved. Summary of the Invention

[0005] To address the aforementioned technical problems and improve the application effect of carboxyl-functionalized porous microspheres in nucleic acid extraction and purification, this application provides a method for preparing carboxyl-functionalized porous microspheres.

[0006] The technical solution adopted in this application is as follows:

[0007] A method for preparing carboxylated porous microspheres, comprising the following steps:

[0008] S1. Dissolve the biocompatible polymer, acrylic copolymer, and oil-soluble emulsifier in an organic solvent to prepare a dispersed phase;

[0009] S2. Add water-soluble emulsifier and / or protective colloid to water to prepare a continuous phase;

[0010] S3. Mix the dispersed phase described in step S1 and the continuous phase described in step S2 to obtain an emulsion. Stir continuously until a precipitate appears. Collect the precipitate, wash it, and dry it to obtain the carboxylated porous microspheres.

[0011] Preferably, the weight ratio of the biocompatible polymer and the acrylic copolymer in step S1 is 1:0.01-0.2.

[0012] Preferably, the acrylic acid in the acrylic copolymer in step S1 has a weight ratio of 1-50%.

[0013] Preferably, the organic solvent in step S1 has a boiling point of no more than 120°C at 1 atmosphere.

[0014] Preferably, the concentration of the dispersed phase in step S1 is 1-60 wt%.

[0015] Preferably, the weight of the oil-soluble emulsifier in step S1 is 1-10% of the weight of the dispersed phase.

[0016] Preferably, the water-soluble emulsifier and / or protective colloid in step S2 has a weight content of 0.5-10 wt% in the continuous phase.

[0017] Preferably, the weight ratio of the dispersed phase to the continuous phase in step S3 is 0.1-1:1.

[0018] Preferably, in step S3, the continuous stirring until precipitation occurs involves continuously introducing gas from the bottom at the start of the continuous stirring.

[0019] Preferably, in step S3, the carboxyl crosslinking agent is added at the beginning of continuous stirring until precipitation occurs.

[0020] In summary, this application has the following beneficial effects:

[0021] 1. This application employs an emulsification method to prepare microspheres. During the emulsion stirring process, as the organic solvent in the dispersed phase (oil phase) evaporates, porous structures are formed on the surface of the microspheres, and the microspheres precipitate and precipitate. An acrylic copolymer is used to provide carboxyl groups for the porous microspheres. Furthermore, the acrylic copolymer has a relatively large molecular weight, resulting in a high binding affinity with biocompatible polymers. Unlike small molecule fatty acids such as oleic acid, it does not plasticize biocompatible polymers, thus providing a more stable carboxyl group content and distribution, which is more conducive to nucleic acid extraction and purification.

[0022] 2. In this application, during the emulsion stirring and organic solvent evaporation process, air is continuously blown from the bottom. This serves two purposes: first, it accelerates the evaporation of the organic solvent, which is beneficial for forming more and larger micropores and speeds up the precipitation of microspheres; second, the air blowing prevents premature sedimentation to the bottom of the container during microsphere formation, which could cause deformation due to gravity before the microspheres are fully solidified and set, resulting in porous microspheres with high sphericity. Porous microspheres with high sphericity have a more uniform contact surface with nucleic acids during nucleic acid extraction, which is more conducive to nucleic acid extraction and purification.

[0023] 3. In this application, during the emulsion stirring and organic solvent evaporation process, an appropriate amount of carboxyl crosslinking agent is added simultaneously. This allows for the gradual crosslinking of some carboxyl groups in the acrylic copolymer during stirring. The partially crosslinked acrylic copolymer forms a semi-interpenetrating polymer network structure with the biocompatible polymer, which can improve the stability of the microspheres. At the same time, it allows the remaining carboxyl groups in the acrylic copolymer to be more stably distributed on the surface of the porous microspheres, which is more conducive to the extraction and purification of nucleic acids. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] This application proposes a method for preparing carboxylated porous microspheres. An O / W emulsion is prepared using an emulsification method. During stirring, as the organic solvent in the oil phase (dispersed phase) of the emulsion gradually evaporates, porous microspheres gradually precipitate from the emulsion, thereby obtaining porous microspheres. Specific steps include:

[0027] S1. Dissolve the biocompatible polymer, acrylic copolymer, and oil-soluble emulsifier in an organic solvent to prepare a dispersed phase;

[0028] S2. Add water-soluble emulsifier and / or protective colloid to water to prepare a continuous phase;

[0029] S3. Mix the dispersed phase from step S1 and the continuous phase from step S2 to obtain an emulsion. Stir continuously until a precipitate appears. Collect the precipitate, wash it, and dry it to obtain carboxylated porous microspheres.

[0030] The dispersed phase in this application uses a biocompatible polymer and an acrylic copolymer. The main material of the resulting porous microspheres is a combination of a biocompatible polymer and an acrylic copolymer, with the biocompatible polymer being the predominant material. This allows for better compatibility between the porous microspheres and nucleic acids. For example, the biocompatible polymer can be polylactic acid (PLA), polylactic-co-glycolic acid copolymer (PLGA), polyglycolic acid (PGA), polycaprolactone (PCL), polycarbonate-based polyurethane, etc.

[0031] There are no particular restrictions on the mixing operation and the apparatus used in step S3 above. A microfluidic injection pump can be used. First, the dispersed phase and the continuous phase are loaded into the microfluidic injection device. Then, according to the weight ratio of the dispersed phase and the continuous phase, the injection volume of the dispersed phase and the continuous phase is adjusted by controlling the injection pressure, injection speed or injection volume of the injection pump. The mixture is then mixed in the pipeline. The mixed emulsion enters a container, such as a beaker, flask or other container. The container is equipped with a stirring device, such as a stirring rod or a magnetic stirring rod, to further improve the mixing uniformity of the emulsion and accelerate the evaporation of organic solvents in the dispersed phase.

[0032] In a preferred embodiment of this application, the weight ratio of the biocompatible polymer to the acrylic copolymer in step S1 is 1:0.01-0.2. Further, the weight ratio of the biocompatible polymer to the acrylic copolymer is 1:0.05-0.15; for example, the weight ratio can be 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, etc.

[0033] In a preferred embodiment of this application, the weight percentage of acrylic acid in the acrylic copolymer in step S1 is 1-50%. Further, the weight percentage of acrylic acid in the acrylic copolymer is 5-40%, for example, the weight percentage can be 5%, 7%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 27%, 28%, 30%, 31%, 33%, 35%, 38%, 40%, etc. In this application, by controlling the amount of acrylic copolymer added and the content of acrylic acid in the acrylic copolymer, the carboxyl content of the porous microspheres can be adjusted, thereby affecting the extraction and purification of nucleic acids by the porous microspheres. In this application, the acrylic copolymer is not particularly limited and can be a copolymer of acrylic acid with one or more of the following monomers: acrylate monomers, styrene monomers, acrylamide monomers, etc., such as acrylic acid-acrylate copolymer, acrylic acid-styrene copolymer, acrylic acid-acrylamide copolymer, acrylic acid-acrylate-styrene copolymer, etc. The acrylate monomers can be ethyl acrylate, butyl acrylate, methyl methacrylate, isooctyl acrylate, lauryl acrylate, butyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, etc., or one or more combinations thereof; the styrene monomers can be styrene, α-methylstyrene, 4-methylstyrene, etc., or one or more combinations thereof; the acrylamide monomers can be acrylamide, N,N-dimethylacrylamide, N-methylacrylamide, N,N-hydroxymethylacrylamide, N,N-diethylacrylamide, etc. The acrylic copolymers of this application can be random copolymers or block copolymers, and the preparation methods are well known to those skilled in the art, such as free radical polymerization, which can use water or an organic solvent as the reaction medium.

[0034] In a preferred embodiment of this application, the boiling point of the organic solvent in step S1 at 1 atmosphere does not exceed 120°C. Organic solvents with lower boiling points evaporate faster, making it easier for the microspheres to form porous structures due to the action of the organic solvent, thus accelerating the formation of porous microspheres. For example, in this application, the organic solvent can be dichloromethane, trichloromethane, dichloroethane, 1,1,2-trichloroethane, 1,1,1-trichloroethane, tetrahydrofuran, acetone, methyl ethyl ketone, ethyl acetate, etc., without particular limitation.

[0035] In a preferred embodiment of this application, the concentration of the dispersed phase in step S1 is 1-60 wt%. If the concentration of the dispersed phase is too high, the viscosity is too high, which is not conducive to emulsification; if the concentration is too low, there is too little biocompatible polymer and acrylic copolymer, which is not conducive to the formation of porous microspheres. Further, the concentration of the dispersed phase is 10-40 wt%, for example, it can be 10 wt%, 12 wt%, 15 wt%, 18 wt%, 20 wt%, 22 wt%, 24 wt%, 25 wt%, 27 wt%, 28 wt%, 30 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 37 wt%, 38 wt%, 40 wt%, etc.

[0036] In a preferred embodiment of this application, the weight of the oil-soluble emulsifier in step S1 is 1-10% of the weight of the dispersed phase. Further, the weight of the oil-soluble emulsifier is 1-8% of the weight of the dispersed phase, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc. In this application, the oil-soluble emulsifier refers to an emulsifier with an HLB value not higher than 7, or further, an HLB value not higher than 6, and can be selected from monoglycerides, stearyl alcohol polyether-2, glyceryl stearate, glyceryl oleate, glyceryl lauryl ester, polyglycerol-3 diisostearate, AEO-3, Span 85, Span 80, Span 65, glyceryl monostearate, Span 60, diethylene glycol fatty acid esters, etc., or a combination thereof.

[0037] In a preferred embodiment of this application, the water-soluble emulsifier and / or protective colloid in step S2 has a weight content of 0.5-10 wt% in the continuous phase. Further, the weight content of the water-soluble emulsifier and / or protective colloid in the continuous phase is 1-10 wt%, for example, it can be 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, etc. In this application, the water-soluble emulsifier and / or protective colloid refers to either a single water-soluble emulsifier or a single protective colloid, or a combination of water-soluble emulsifier and protective colloid. Water-soluble emulsifiers refer to emulsifiers with an HLB value of not less than 7, or further, an HLB value of not less than 8, and can be selected from tetraethylene glycol monostearate, tetraethylene glycol monooleate, Span 20, AEO-9, AEO-12, Tween 80, Tween 65, Tween 60, triethanolamine oleate, etc.; there are no particular restrictions on the protective colloid, and it can be selected from PVA, sodium polyacrylate, carboxymethyl cellulose, PVP, etc.

[0038] In a preferred embodiment of this application, the weight ratio of the dispersed phase to the continuous phase in step S3 is 0.1-1:1. More preferably, the weight ratio of the dispersed phase to the continuous phase is 0.4-1:1, and for example, it can be 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, etc.

[0039] In a preferred embodiment of this application, in step S3, when stirring continuously until precipitation occurs, gas is continuously introduced from the bottom at the start of continuous stirring. Continuously introducing gas from the bottom utilizes the upward force of the gas to move the gradually precipitating microspheres upwards, preventing the microspheres from settling to the bottom of the container before complete solidification and deforming due to gravity, and also accelerating the evaporation of the organic solvent. Continuously introducing gas from the bottom of the container can be done by opening the bottom and introducing gas, or by inserting a gas inlet pipe into the bottom of the container and introducing gas. There can be one or more gas inlet locations, which can be set as needed. The gas flow rate can be set according to the volume of the emulsion in the container. If the emulsion volume is X, the gas flow rate can be set to (0.1X-10X) / min. For example, if the emulsion volume is 100ml, the gas flow rate can be set to 10-1000ml / min.

[0040] In a preferred embodiment of this application, in step S3, stirring is continued until precipitation occurs, and a carboxyl crosslinking agent is added at the start of continuous stirring. The added carboxyl crosslinking agent can dissolve in the continuous phase (aqueous phase) and can partially crosslink with the carboxyl groups in the acrylic polymer in the dispersed phase. This allows the acrylic polymer to form a semi-interpenetrating polymer network structure with the biocompatible polymer on the surface of the porous microspheres, improving the structural stability of the porous microspheres. Furthermore, the acrylic polymer contains the remaining carboxyl groups, providing a relatively abundant range of carboxyl groups for the surface of the porous microspheres. Further, the carboxyl crosslinking agent can be an aziridine crosslinking agent, and the dosage is 0.1-0.3 equivalents of aziridine crosslinking agent per carboxyl group in the acrylic polymer.

[0041] The technical solution of this application will be described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the parts in the following embodiments and comparative examples are all parts by weight.

[0042] The acrylic copolymers used in the various embodiments and comparative examples are as follows:

[0043] Acrylic copolymer 1: A random copolymer of acrylic acid and styrene, with an acrylic acid content of 8% by weight;

[0044] Acrylic copolymer 2: Random copolymer of acrylate and butyl acrylate, with an acrylic acid content of 23%;

[0045] Acrylic copolymer 3: Random copolymer of acrylic acid-ethyl acrylate-butyl acrylate, with an acrylic acid content of 36% and an ethyl acrylate content of 17%;

[0046] Example 1

[0047] PLA and acrylic copolymer 1 were added to dichloromethane at a weight ratio of 1:0.06, and then oil-soluble emulsifier AEO-3 was added to prepare a dispersed phase with a concentration of 15wt%, wherein AEO-3 accounted for 2% of the weight of the dispersed phase.

[0048] Tetraethylene glycol monooleate was added to water to prepare a continuous phase with a concentration of 2 wt%.

[0049] The dispersed phase and continuous phase were respectively loaded into a microfluidic injection pump device and mixed at a weight ratio of 0.5:1. The mixture was then transferred to a 200ml beaker containing 100ml of emulsion and stirred continuously for 2 hours under magnetic stirring. After stirring was stopped, the mixture was filtered, washed twice with deionized water, and dried at -20℃ in a freeze-drying oven to obtain carboxylated porous microspheres.

[0050] Example 2

[0051] The difference between Example 2 and Example 1 is that air at a flow rate of 200 ml / min is introduced from the bottom of the beaker while the magnetic stirring is performed. The remaining steps remain the same.

[0052] Example 3

[0053] The difference between Example 3 and Example 1 is that, while magnetically stirring, 0.2 equivalents of aziridine crosslinking agent CX-100 are added to each carboxyl group in acrylic polymer 1. The remaining steps remain the same.

[0054] Example 4

[0055] PLGA and acrylic copolymer 2 were added to dichloroethane at a weight ratio of 1:0.12, and then oil-soluble emulsifier Span 80 was added to prepare a dispersed phase with a concentration of 30 wt%, where Span 80 accounted for 3% of the weight of the dispersed phase.

[0056] PVA1792 was added to 90℃ and stirred to dissolve. AEO-12 was then added to prepare a continuous phase with 2.5 wt% AEO-12 and 1 wt% PVA 1792.

[0057] The dispersed phase and continuous phase were respectively loaded into a microfluidic injection pump device and mixed at a weight ratio of 1:1. The mixture was then transferred to a 200ml beaker. Under magnetic stirring, 100ml of the emulsion was placed in the beaker. 0.12 equivalents of aziridine crosslinking agent CX-100 were added for every carboxyl group in acrylic polymer 2. The mixture was stirred continuously for 3 hours, then the stirring was stopped. The mixture was filtered, washed twice with deionized water, and dried in a freeze-drying vacuum dryer at -20℃ to obtain carboxylated porous microspheres.

[0058] Example 5

[0059] The difference between Example 5 and Example 4 is that in Example 4, after adding the aziridine crosslinking agent CX-100, air was introduced from the bottom of the beaker at a flow rate of 120 ml / min. The remaining steps remained unchanged.

[0060] Example 6

[0061] The difference between Example 6 and Example 4 is that in Example 4, the weight ratio of PLGA and acrylic copolymer 2 was adjusted from 1:0.12 to 1:0.2. The remaining steps remained unchanged.

[0062] Example 7

[0063] The difference between Example 7 and Example 4 is that in Example 4, acrylic copolymer 2 is replaced with an equal weight of acrylic copolymer 3. The remaining steps remain unchanged.

[0064] Example 8

[0065] The difference between Example 8 and Example 4 is that in Example 4, acrylic copolymer 2 is replaced with an equal weight of acrylic copolymer 1. The remaining steps remain unchanged.

[0066] Comparative Example 1

[0067] The difference between Comparative Example 1 and Example 4 is that in Example 4, acrylic copolymer 2 was replaced with an equal weight of oleic acid. The remaining steps remained unchanged.

[0068] Comparative Example 2

[0069] The difference between Comparative Example 2 and Comparative Example 1 is that, in Comparative Example 1, the aziridine crosslinking agent CX-100 was not added. The remaining steps remained unchanged.

[0070] Comparative Example 3

[0071] The difference between Comparative Example 3 and Example 4 is that in Example 4, the amount of aziridine crosslinking agent CX-100 added was 0.6 equivalents. The remaining steps remained unchanged.

[0072] Comparative Example 4

[0073] The difference between Comparative Example 4 and Example 4 is that in Example 4, the amount of aziridine crosslinking agent CX-100 added was 1 equivalent. The remaining steps remained unchanged.

[0074] Comparative Example 5

[0075] The difference between Comparative Example 5 and Example 4 is that in Example 4, acrylic copolymer 2 was replaced with an equal weight of PLGA. The remaining steps remained unchanged.

[0076] Performance testing and flowability results: The flowability of the microspheres was tested using an angle of repose tester. The larger the angle of repose, the lower the flowability of the microspheres, indicating that the sphericity of the microspheres is lower.

[0077] Nucleic acid extraction test: Porous microspheres were used to replace the silicon-based material of existing centrifuge columns in the various examples and comparative examples for the extraction of nucleic acid from the novel coronavirus. Novel coronavirus samples were obtained from throat swabs. The samples were added to commercially available lysis buffer (Universal RNA Extraction Kit (Centrifuge Column Type) from Shanghai Yiyan Biotechnology Co., Ltd.) and mixed thoroughly. The mixture was then adsorbed onto porous microspheres, centrifuged at 8000 rpm for half an hour, washed, and eluted with eluent (Universal RNA Extraction Kit (Centrifuge Column Type) from Shanghai Yiyan Biotechnology Co., Ltd.) to obtain the extracted RNA. The concentration of the extracted RNA and the OD260 / OD280 value were then measured.

[0078] The results are shown in Table 1 below.

[0079] Table 1 Test Results

[0080]

[0081]

[0082] Comparative analysis of the data results in Table 1 shows that in Comparative Examples 1-3, the porous microspheres exhibited better fluidity and RNA purification effect when aeration was initiated from the bottom during stirring. Adding a certain amount of crosslinking agent resulted in even better fluidity and RNA purification compared to porous microspheres without crosslinking agent. This may be related to the roundness of the porous microspheres. Continuous aeration during stirring helps prevent or reduce microsphere deposition at the bottom of the container during formation. Higher roundness of the porous microspheres, coupled with the addition of a certain amount of crosslinking agent, can improve their mechanical strength and resist deformation caused by gravity. Comparative Example 4 and Comparative Examples 1-2 showed that replacing oleic acid with acrylic copolymer improved fluidity and RNA extraction effect. This may be due to two reasons: (1) Acrylic copolymer is a macromolecule, which is more stable and less prone to loss during porous microsphere formation compared to the smaller molecule oleic acid; (2) Oleic acid acts as a plasticizer in PLGA, leading to reduced mechanical strength of the porous microspheres and easier deformation and reduced roundness during formation. Compared with Example 4 and Comparative Examples 3-4, appropriate cross-linking of porous microspheres can retain more carboxyl groups while maintaining good flowability. When there is more cross-linking agent, fewer carboxyl groups are retained, resulting in poor extraction and purification of RNA.

[0083] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing carboxylated porous microspheres, characterized in that the steps include... include: S1. A biocompatible polymer, an acrylic copolymer, and an oil-soluble emulsifier are dissolved in an organic solvent to prepare a dispersed phase; the biocompatible polymer is selected from polylactic acid or polylactic acid-glycolic acid copolymer. S2. Add water-soluble emulsifier and / or protective colloid to water to prepare a continuous phase; S3. Mix the dispersed phase described in step S1 and the continuous phase described in step S2 to obtain an emulsion. Stir continuously until a precipitate appears. Collect the precipitate, wash it, and dry it to obtain the carboxylated porous microspheres.

2. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The weight ratio of the biocompatible polymer and the acrylic copolymer in step S1 is 1:0.01-0.

2.

3. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The acrylic acid in the acrylic copolymer described in step S1 has a weight ratio of 1-50%.

4. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The organic solvent described in step S1 has a boiling point of no more than 120°C at 1 atmosphere.

5. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The concentration of the dispersed phase in step S1 is 1-60 wt%.

6. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The weight of the oil-soluble emulsifier in step S1 is 1-10% of the weight of the dispersed phase.

7. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The water-soluble emulsifier and / or protective colloid mentioned in step S2 has a weight content of 0.5-10 wt% in the continuous phase.

8. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, The weight ratio of the dispersed phase to the continuous phase in step S3 is 0.1-1:

1.

9. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, In step S3, the stirring continues until a precipitate forms, and gas is continuously introduced from the bottom at the start of the stirring.

10. The method for preparing carboxylated porous microspheres according to claim 1, characterized in that, In step S3, the stirring continues until precipitation occurs, and a carboxyl crosslinking agent is added at the beginning of the stirring.

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