Super-macroporous cross-linked polymer microsphere as well as preparation method and application thereof

Abstract

The invention provides super-macroporous cross-linked polymer microspheres as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing a monomer, a cross-linking agent, a pore-foaming agent and an organic solvent to obtain an oil phase O; preparing an aqueous solution containing a first osmotic pressure regulator as an inner aqueous phase W1; preparing an aqueous solution containing a surfactant as an external water phase W2; (2) mixing the oil phase O with the inner water phase W1 to obtain a water-in-oil W1 / O primary emulsion; (3) mixing the water-in-oil W1 / O primary emulsion with an external water phase W2 to obtain a water-in-oil-in-water multiple emulsion W1 / O / W2; and (4) carrying out polymerization reaction on the water-in-oil-in-water multiple emulsion W1 / O / W2 to obtain the super-macroporous cross-linked polymer microspheres. The super-macroporous cross-linked polymer microsphere has a nano-to-micron through pore structure, the particle size is 1-300 [mu] m, the pore diameter is 0.1-90 [mu] m, and the porosity is 10-80%.

Description

Technical Field

[0001] The present invention belongs to the cross - technical field of polymer materials and biochemistry, and particularly relates to a super - macroporous cross - linked polymer microsphere, a preparation method thereof and an application thereof. Background Art

[0002] People's demand for the purification of biological macromolecules such as polypeptides, nucleic acids, viroid particles, etc. is getting higher and higher. Among many purification methods, liquid chromatography is by far the most effective separation and purification means, and its biocompatible characteristics ensure a relatively high active yield. With the industrialization process of biotechnology, the requirements for chromatography efficiency and chromatography capacity are also getting higher and higher. High - performance chromatography packing materials play an important role in improving product quality and production efficiency. At present, the commonly used chromatography packing materials in scientific research and production can be divided into the following categories:

[0003] 1. Polysaccharide - based microsphere chromatography packing materials are the most commonly used biochemical separation media at home and abroad. Agarose molecules are uncharged and have good hydrophilicity, which can provide a biocompatible interfacial environment; the abundant hydroxyl groups in agarose molecules can be further modified, and after forming spheres, they can be derivatized into various different types of media such as ion - exchange, hydrophobic, and affinity. However, as a kind of soft - gel microspheres, the pore size of agarose media is generally less than 50nm, and the water content of the medium spheres is usually above 90%. During the application process, the flow rate is low and the back - pressure is high, seriously affecting the separation efficiency.

[0004] 2. Porous polymer microspheres: Polymer media can be synthesized from monomers, usually forming a rigid microsphere structure by suspension polymerization, which can withstand higher flow rates and pressures. By regulating the polymerization monomers and polymerization process, the pore structure and interfacial characteristics of polymer microspheres can be effectively controlled. The pore size range is generally 10 - 30nm. When used for chromatography separation, due to the small pore size, the mass transfer process of the mobile phase in the pores mainly relies on molecular diffusion to complete, and the mass transfer speed is slow; it is not conducive to separating biochemical technology products with large molecules.

[0005] 3. Super - macroporous packing materials: In the early 1990s, Afeyan et al. applied for patents (US5019270, US5228989, US5833861) for polymer microsphere media characterized by convective mass transfer, and the trade name is This type of microspheres has two typical pore size distributions: through - pores (600 - 800nm) and diffusion pores (50 - 150nm). The mass transfer inside the particles mainly relies on convective transfer in the through - pores, reducing the residence time of the mobile - phase molecules inside the particles; the existence of diffusion pores also provides a large specific surface area and column capacity. The excellent performance of has attracted wide attention, but the preparation of this type of medium is difficult and the price is expensive.

[0006] Guanghui Ma, Weiqing Zhou et al. were inspired by the two-phase continuous structure of microemulsions and combined the reverse micelle swelling method on the basis of suspension polymerization to prepare supermacroporous polystyrene (PS) microspheres with pore sizes of about 100 - 500 nm. The supermacroporous PS microspheres have the advantages of high rigidity, stable chemical properties, resistance to high pressure and high flow rate, etc., and have good application prospects in the field of chromatographic media (W.Q. Zhou, T.Y. Gu, Z.G. Su, G.H. Ma, Synthesis of macroporous poly(styrene-divinylbenzene) microspheres by surfactant reverse micelles swelling method, Polymer, 2007, 48, 1981).

[0007] In summary, the research on chromatography packing materials has experienced decades of development history, and the separation efficiency has also been significantly improved. However, with the development of biotechnology, chromatography media also face many new challenges. When facing the purification of larger-sized macromolecules, such as influenza virus, adenovirus, rabies virus, phage, etc., the target particle size is usually dozens to hundreds of nanometers. Therefore, a medium with a larger pore size is needed to support it. The pore sizes of existing polymer microspheres are generally small. Therefore, preparing a microsphere with a large pore size to meet the demand for the purification of larger-sized macromolecules is a key issue. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a supermacroporous cross-linked polymer microsphere and its preparation method and application. The method further expands the pore size of the supermacroporous microsphere and can be used in the separation and purification process of macromolecules, such as vaccines, viroid particles, etc. Moreover, the supermacroporous cross-linked polymer microsphere has stronger rigidity and better solvent resistance.

[0009] To achieve this purpose, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention provides a preparation method of a supermacroporous cross-linked polymer microsphere, and the preparation method includes the following steps:

[0011] (1) Mix the monomer, cross-linking agent, porogen and organic solvent to obtain an oil phase O; prepare an aqueous solution including a first osmotic pressure regulator as an inner aqueous phase W 1 ; prepare an aqueous solution including a surfactant as an outer aqueous phase W 2 ;

[0012] (2) Mix the oil phase O and the inner aqueous phase W 1 to obtain a water-in-oil W 1 / O primary emulsion;

[0013] (3) Mix the water-in-oil W 1 / O primary emulsion with the external aqueous phase W 2 to obtain a water-in-oil-in-water multiple emulsion W 1 / O / W 2 ;

[0014] (4) Subject the water-in-oil-in-water multiple emulsion W 1 / O / W 2 to a polymerization reaction to obtain the supermacroporous crosslinked polymer microspheres.

[0015] The preparation method provided by the present invention adopts a two-step multiple emulsion method to prepare a water-in-oil-in-water multiple emulsion as a template for supermacroporous microspheres; by controlling parameters such as interfacial stability and osmotic pressure gradient, the template morphology is regulated; curing is carried out by means of crosslinking polymerization of monomers in the oil phase. During the polymerization process, the internal aqueous phase droplets escape outward, forming internally and externally interconnected pores. Based on the multiple emulsion method, the pore size regulation of the present invention is more flexible and applicable to various polymer materials. By adjusting the multiple emulsion formulation and emulsification parameters, the particle size, pore size, and porosity of the microspheres can be effectively controlled, which is beneficial to realizing the regulation of the microsphere structure and surface functional groups. During the development process of the present invention, the requirements for production scale-up are considered. Emulsification and polymerization are both conventional unit operations and are suitable for large-scale production.

[0016] The following are preferred technical solutions of the present invention, but do not limit the technical solutions provided by the present invention. Through the following preferred technical solutions, the objectives and beneficial effects of the present invention can be better achieved and realized.

[0017] As a preferred technical solution, the monomer includes any one or a combination of at least two of acrylate esters or styrene;

[0018] Preferably, the acrylate esters include any one or a combination of at least two of glycidyl methacrylate, methyl methacrylate, butyl methacrylate, octadecyl methacrylate, or 2-hydroxyethyl methacrylate.

[0019] Preferably, the crosslinking agent includes ethylene glycol dimethacrylate and / or divinylbenzene.

[0020] Preferably, the mass ratio of the monomer to the crosslinking agent is 1:(0.2 - 5), for example, it can be 1:0.2, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5, etc.

[0021] Preferably, the pore-forming agent includes an oil-soluble surfactant and / or an amphiphilic block copolymer.

[0022] Preferably, the oil-soluble surfactant includes sorbitan monostearate and / or polyoxyethylene sorbitan trioleate.

[0023] Preferably, the hydrophilic segment in the amphiphilic block copolymer includes at least one of a polyoxypropylene ether segment, a polyethylene glycol segment, a polyacrylic acid segment, or a hydroxyethyl acrylate segment.

[0024] Preferably, the hydrophobic segment in the amphiphilic block copolymer includes at least one of a polyoxyethylene segment, a polylactic acid segment, a polystyrene segment, or a polymethyl methacrylate segment.

[0025] Preferably, the amphiphilic block copolymer includes a diblock copolymer and / or a triblock copolymer.

[0026] Preferably, the triblock copolymer includes a linear triblock copolymer and / or a star-shaped triblock copolymer.

[0027] Preferably, the diblock copolymer includes any one or a combination of at least two of a polyoxyethylene polyoxypropylene ether copolymer, a polyethylene glycol polylactic acid copolymer, a polystyrene polyethylene glycol copolymer, a polystyrene polyacrylic acid copolymer, a polystyrene hydroxyethyl acrylate copolymer, a polymethyl methacrylate polyacrylic acid copolymer, and a polymethyl methacrylate hydroxyethyl acrylate copolymer.

[0028] Preferably, the triblock copolymer is obtained by combining at least one of the hydrophobic segments and at least one of the hydrophilic segments.

[0029] The triblock copolymer in the amphiphilic block copolymer can be one kind or a combination of at least two kinds.

[0030] The block copolymer, also known as a graft copolymer, is a special polymer prepared by connecting two or more polymer segments with different properties together. Those skilled in the art can select the amphiphilic block copolymer by themselves to have the dual effects of the thickening property of high polymers and the surface activity of low molecules, and have an affinity for both the aqueous phase and the oil phase.

[0031] Preferably, the mass ratio of the total mass of the monomer and the crosslinking agent to the mass of the pore-forming agent is (3 - 300):1, for example, it can be 3:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, 260:1, 270:1, 280:1, 290:1, or 300:1, etc.

[0032] Those skilled in the art can select a good solvent for the polymer solution according to the solubility of the polymer. Preferably, the organic solvent includes any one or a combination of at least two of chloroform, acetone, tetrahydrofuran, toluene, dichloromethane, trichloromethane, dichloroethane, or ethyl acetate.

[0033] Preferably, the oil phase O further includes an initiator.

[0034] Preferably, the initiator includes any one or a combination of at least two of azo initiators or peroxide initiators.

[0035] Preferably, the azo initiator includes any one or a combination of at least two of azodiisobutyronitrile, azodiisoheptonitrile, or dimethyl azodiisobutyrate.

[0036] Preferably, the peroxide initiator includes any one or a combination of at least two of benzoyl peroxide, lauroyl peroxide, dodecanoyl peroxide, diisopropyl peroxydicarbonate, or dicyclohexyl peroxydicarbonate.

[0037] Preferably, based on the total mass of the monomer, crosslinking agent, pore-forming agent, and initiator being 100%, the mass fraction of the initiator is 0.3 - 6%, for example, it can be 0.3%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6%, as well as the specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the range.

[0038] Preferably, the external aqueous phase W 2 further includes a second osmotic pressure regulator.

[0039] Preferably, the first osmotic pressure regulator and the second osmotic pressure regulator each independently include metal ion salts or sugars.

[0040] The addition of metal ion salts or sugars can adjust the surface tension of the inner aqueous phase droplets and control the osmotic pressure. Any metal ion salt known to those skilled in the art can be used to implement the present invention. Preferably, the metal ion salts include any one or a combination of at least two of sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, or magnesium sulfate.

[0041] Preferably, the sugars include any one or a mixture of at least two of sucrose, glucose, or trehalose.

[0042] Preferably, the inner aqueous phase W 1The mass fraction of the first osmotic pressure regulator is 0.01-10%, for example, it can be 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, and specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the said range.

[0043] Preferably, the external aqueous phase W 2 The mass fraction of the second osmotic pressure regulator in it is ≤5%, for example, it can be 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, and specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the said range.

[0044] Preferably, the surfactant includes an ionic surfactant or a non-ionic surfactant, and the surfactant can reduce the aggregation of microspheres during the coalescence and polymerization of the emulsion.

[0045] Preferably, the ionic surfactant includes any one or a mixture of at least two of sodium octadecyl sulfate, sodium dodecyl sulfate or sodium dodecylbenzenesulfonate.

[0046] Preferably, the non-ionic surfactant includes any one or a mixture of at least two of polyvinyl alcohol, fatty alcohol polyoxyethylene ether or polysorbate-20.

[0047] Preferably, the external aqueous phase W 2 The mass fraction of the surfactant in it is 0.01-10%, for example, it can be 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, and specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the said range.

[0048] Preferably, the method of mixing in step (2) includes any one of ultrasonic emulsification method, conventional membrane emulsification method, rapid membrane emulsification method, homogenization emulsification method or mechanical stirring method; more preferably the conventional membrane emulsification method or the rapid membrane emulsification method. By the method of mixing, the uniformity of the W 1 / O primary emulsion is ensured, thereby ensuring the uniformity of the pores.

[0049] Preferably, the method of mixing in step (3) includes any one of ultrasonic emulsification method, conventional membrane emulsification method, premixed membrane emulsification method, homogenization emulsification method or mechanical stirring method; more preferably the conventional membrane emulsification method or the premixed membrane emulsification method. By the method of mixing, the uniformity of the water-in-oil-in-water multiple emulsion is ensured, thereby ensuring the uniformity of the particle size of the supermacroporous crosslinked polymer microspheres.

[0050] The conventional membrane emulsification method refers to the process of pressing the dispersed phase through the small holes on the solid membrane under a certain pressure, so as to form dispersed phase droplets on the other side of the membrane pores, and separating them from the membrane pores by the shear force of the flowing continuous phase. The premixed membrane emulsification method (rapid membrane emulsification method) refers to first preparing the primary emulsion by the traditional emulsification method, and then pressing the primary emulsion through the membrane tube to obtain droplets. During the rapid membrane emulsification process, when the operating pressure is greater than the critical pressure, the primary emulsion of large droplets is broken when passing through the membrane pores, thus forming smaller droplets.

[0051] Preferably, the internal aqueous phase W 1 and the oil phase O have a volume ratio of (0.1 - 3):1. For example, it can be 0.1:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1 or 3:1, as well as the specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the above range.

[0052] Preferably, the internal aqueous phase W 1 and the total volume of the oil phase O and the external aqueous phase W 2 have a volume ratio of 1:(1 - 50). For example, it can be 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50, etc.

[0053] Preferably, the temperature of the polymerization reaction is 10 - 90 °C. For example, it can be 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C or 90 °C, as well as the specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the above range.

[0054] Preferably, the time of the polymerization reaction is 2 - 24 h. For example, it can be 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h or 24 h, as well as the specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the above range.

[0055] Preferably, the preparation method specifically includes the following steps:

[0056] (1) Mix the monomer, crosslinking agent, pore-forming agent, initiator and organic solvent to obtain the oil phase O; prepare an aqueous solution containing the first osmotic pressure regulator as the internal aqueous phase W 1 ; prepare an aqueous solution containing a surfactant and optionally a second osmotic pressure regulator as the external aqueous phase W 2 .

[0057] The mass ratio of the monomer to the crosslinking agent is 1:(0.2 - 5); the mass ratio of the total mass of the monomer and the crosslinking agent to the mass of the porogen is (3 - 300):1.

[0058] Based on the total mass of the monomer, crosslinking agent, porogen and initiator being 100%, the mass fraction of the initiator is 0.3 - 6%.

[0059] The first osmotic pressure regulator in the inner aqueous phase W 1 has a mass fraction of 0.01 - 10%.

[0060] The second osmotic pressure regulator in the outer aqueous phase W 2 has a mass fraction ≤ 5%.

[0061] The surfactant in the outer aqueous phase W 2 has a mass fraction of 0.01 - 10%.

[0062] (2) Mix the oil phase O and the inner aqueous phase W 1 to obtain a water-in-oil W 1 / O primary emulsion.

[0063] The volume ratio of the inner aqueous phase W 1 to the oil phase O is (0.1 - 3):1.

[0064] (3) Mix the water-in-oil W 1 / O primary emulsion and the outer aqueous phase W 2 to obtain a water-in-oil-in-water multiple emulsion W 1 / O / W 2 .

[0065] The total volume of the inner aqueous phase W 1 and the oil phase O to the volume of the outer aqueous phase W 2 is 1:(1 - 50).

[0066] (4) The water-in-oil-in-water multiple emulsion W 1 / O / W 2 undergoes a polymerization reaction to obtain the supermacroporous crosslinked polymer microspheres.

[0067] The temperature of the polymerization reaction is 10 - 90 °C.

[0068] The time of the polymerization reaction is 2 - 24 h.

[0069] Second, the present invention provides a supermacroporous crosslinked polymer microsphere, which is prepared by the preparation method described in the first aspect.

[0070] Preferably, the supermacroporous crosslinked polymer microsphere has a nano- to micron-scale through-channel structure.

[0071] Preferably, the particle size of the macroporous cross-linked polymer microspheres is 1-300 μm, for example, it can be 1 μm, 5 μm, 20 μm, 50 μm, 70 μm, 90 μm, 100 μm, 120 μm, 150 μm, 180 μm, 210 μm, 240 μm, 270 μm or 300 μm, etc., preferably 5-250 μm, and more preferably 10-200 μm.

[0072] Preferably, the pore size of the macroporous cross-linked polymer microspheres is 0.1-90 μm, for example, it can be 0.1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm or 90 μm, preferably 0.2-70 μm, and more preferably 0.5-30 μm.

[0073] Preferably, the porosity of the macroporous cross-linked polymer microspheres is 10-80%, for example, it can be 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, preferably 20-70%, and more preferably 30-70%.

[0074] The macroporous cross-linked polymer microspheres provided by the present invention are conducive to the penetration of biological macromolecules into the interior of the microspheres, can realize the convective mass transfer inside the microspheres, and the rigid structure can withstand higher pressures and flow rates.

[0075] In the third aspect, the present invention provides an application of the macroporous cross-linked polymer microspheres as described in the second aspect as a stationary phase filler for chromatographic separation, an immobilization carrier for enzymes, a microcarrier for cell culture, a micro scaffold material for tissue engineering or an adsorption material.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] (1) The macroporous cross-linked polymer microspheres provided by the present invention have a pore structure that penetrates inside and outside, the porosity can be as high as 80%, the pore size can reach the micron scale at most, up to 90 μm at most, and the particle size can reach 300 μm at most, which is particularly suitable for the separation and purification of biological macromolecules such as viroid particles; as a chromatography medium, it has the characteristics of convective mass transfer and is suitable for the efficient preparation of biochemical products; at the same time, it is suitable for aspects such as an immobilization carrier for enzymes, a microcarrier for cell culture, a micro scaffold material for tissue engineering and an adsorption material;

[0078] (2) Compared with the existing polymerization methods of supermacroporous microspheres, the method of the present invention based on double emulsion enables more flexible regulation of the pore size of supermacroporous crosslinked polymers. The method proposed by the present invention is applicable to various polymer materials. By adjusting the double emulsion formulation and emulsification parameters, the particle size, pore size, and porosity of supermacroporous crosslinked polymer microspheres can be effectively controlled, which is conducive to realizing the regulation of the structure of supermacroporous crosslinked polymer microspheres. Moreover, the emulsification and polymerization in the preparation method provided by the present invention are both conventional unit operations, suitable for large-scale production. Description of the Drawings

[0079] Figure 1 is the scanning electron microscope photograph of the supermacroporous crosslinked polymer microspheres provided in Example 1;

[0080] Figure 2 is the scanning electron microscope photograph of the supermacroporous crosslinked polymer microspheres provided in Example 2;

[0081] Figure 3 is the scanning electron microscope photograph of the supermacroporous crosslinked polymer microspheres provided in Example 3;

[0082] Figure 4 is the scanning electron microscope photograph of the supermacroporous crosslinked polymer microspheres provided in Example 4;

[0083] Figure 5 is the scanning electron microscope photograph of the supermacroporous crosslinked polymer microspheres provided in Example 5. Detailed Embodiments

[0084] The technical solutions of the present invention will be further described below in conjunction with the drawings and through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0085] The sources of some components in the following examples and comparative examples are as described below:

[0086] (1) Polyvinyl alcohol: PVA-217, purchased from Kuraray, Japan.

[0087] Example 1

[0088] A supermacroporous crosslinked polymer microsphere and a preparation method thereof, the preparation method specifically includes the following steps:

[0089] (1) Prepare 100 mL of an aqueous solution of 0.01% sodium chloride as the inner aqueous phase W 1 ; Mix 70 g of glycidyl methacrylate, 60 g of ethylene glycol dimethacrylate, 0.75 g of azobisisobutyronitrile, 1 g of polyoxyethylene polyoxypropylene ether copolymer, and 100 g of dichloromethane to obtain the oil phase O; Prepare 1 L of an aqueous solution of 2% sodium octadecyl sulfate as the outer aqueous phase W2 ;

[0090] (2) Homogenize and emulsify the inner aqueous phase W 1 and the oil phase O at 18,000 rpm for 5 min to obtain a W 1 / O primary emulsion;

[0091] (3) Mix the W 1 / O primary emulsion with the outer aqueous phase W 2 and stir mechanically at 200 rpm for pre-emulsification for 30 min. Pour the pre-emulsified emulsion into a membrane emulsifier and pass it through a 50-μm membrane tube 6 times under a pressure of 0.5 Mpa to obtain a water-in-oil-in-water double emulsion W 1 / O / W 2 ;

[0092] (4) Pour the water-in-oil-in-water double emulsion W 1 / O / W 2 into a 5-L reaction kettle, stir at 70 rpm, and carry out a polymerization reaction at 55 °C for 4 h. The resulting macroporous cross-linked polymer microspheres are first washed repeatedly 5 times with deionized water and ethanol, and finally washed with deionized water to remove the residual ethanol to obtain the macroporous cross-linked polymer microspheres. The volume average particle size of the macroporous cross-linked polymer microspheres is 42 μm, the porosity is 60%, and the average pore diameter is 1.2 μm.

[0093] Use a cold field emission scanning electron microscope (model: JEM-6700F) to test the surface morphology of the obtained macroporous cross-linked polymer microspheres, as shown in Figure 1 the scanning electron microscope photograph of the macroporous cross-linked polymer microspheres provided in Example 1.

[0094] Example 2

[0095] A macroporous cross-linked polymer microsphere and a preparation method thereof. The preparation method specifically includes the following steps:

[0096] (1) Prepare 120 mL of an aqueous solution of 0.2% potassium chloride as the inner aqueous phase W 1 ; Mix 70 g of octadecyl methacrylate, 60 g of ethylene glycol dimethacrylate, 0.75 g of azobisisobutyronitrile, 0.5 g of polyoxyethylene sorbitan trioleate, and 100 g of chloroform to obtain the oil phase O; Prepare 1 L of an aqueous solution of 3% sodium dodecyl sulfate as the outer aqueous phase W 2 ;

[0097] (2) Mix the W 1 / O primary emulsion with the outer aqueous phase W 2 and stir mechanically at 500 rpm for pre-emulsification for 3 min. Pour the pre-emulsified emulsion into a membrane emulsifier and pass it through a 2.8-μm membrane tube 3 times under a pressure of 0.5 Mpa to obtain a W1 / O primary emulsion;

[0098] (3) Mix the W 1 / O primary emulsion and the external aqueous phase W 2 Homogenize and emulsify at 3000 rpm for 5 min to obtain a water-in-oil-in-water double emulsion W 1 / O / W 2 ;

[0099] (4) Pour the water-in-oil-in-water double emulsion W 1 / O / W 2 into a 5 L reactor, stir at 70 rpm, and react at 55 °C for 4 h. The resulting macroporous cross-linked polymer microspheres are first washed repeatedly 5 times with deionized water and ethanol. Finally, wash away the residual ethanol with deionized water to obtain the macroporous cross-linked polymer microspheres. The volume average particle size of the macroporous cross-linked polymer microspheres is 51 μm, the porosity is 80%, and the average pore size is 2.2 μm.

[0100] The surface morphology of the macroporous cross-linked polymer microspheres is as Figure 2 shown in the scanning electron microscope photograph of the macroporous cross-linked polymer microspheres provided in Example 2.

[0101] Example 3

[0102] A macroporous cross-linked polymer microsphere and a preparation method thereof. The preparation method specifically includes the following steps:

[0103] (1) Prepare a 60 mL aqueous solution of 0.5% sodium sulfate as the internal aqueous phase W 1 ; Mix 70 g of glycidyl methacrylate, 60 g of ethylene glycol dimethacrylate, 0.75 g of azobisisobutyronitrile, 1.5 g of polystyrene-polyethylene glycol copolymer, and 100 g of ethyl acetate to obtain the oil phase O; Prepare a 1 L aqueous solution of 3% polyvinyl alcohol (PVA) as the external aqueous phase W 2 ;

[0104] (2) Homogenize and emulsify the internal aqueous phase W 1 and the oil phase O at 5000 rpm for 5 min to obtain a W 1 / O primary emulsion;

[0105] (3) Mix the W 1 / O primary emulsion and the external aqueous phase W 2 and homogenize and emulsify at 3000 rpm for 5 min to obtain a water-in-oil-in-water double emulsion W 1 / O / W 2 ;

[0106] (4) The water-in-oil-in-water double emulsion W 1 / O / W 2Pour into a 5 L reactor, stir at 70 rpm, and react at 55 °C for 4 hours. The resulting macroporous crosslinked polymer microspheres were washed repeatedly 5 times with deionized water and ethanol, and finally the residual ethanol was washed away with deionized water to obtain the macroporous crosslinked polymer. The volume average particle size of the macroporous crosslinked polymer microspheres is 51 μm, the porosity is 43%, and the average pore size is 4.3 μm.

[0107] The surface morphology of the macroporous crosslinked polymer microspheres is as Figure 3 shown in the scanning electron microscope photograph of the macroporous crosslinked polymer microspheres provided in Example 3.

[0108] Example 4

[0109] A macroporous crosslinked polymer microsphere and a preparation method thereof, the preparation method specifically includes the following steps:

[0110] (1) Prepare 10 mL of an aqueous solution of 0.2% potassium sulfate as the internal aqueous phase W 1 ; Mix 40 g of styrene, 30 g of divinylbenzene, 0.75 g of benzoyl peroxide, 1 g of polyoxyethylene polyoxypropylene ether copolymer, and 30 g of dichloromethane to obtain the oil phase O; Prepare 300 mL of an aqueous solution of 4% PVA as the external aqueous phase W 2 ;

[0111] (2) Homogenize and emulsify the internal aqueous phase W 1 and the oil phase O at 15000 rpm for 5 min to obtain the W 1 / O primary emulsion;

[0112] (3) Mix the W 1 / O primary emulsion with the external aqueous phase W 2 and homogenize and emulsify at 5000 rpm for 5 min to obtain a water-in-oil-in-water multiple emulsion W 1 / O / W 2 ;

[0113] (4) Pour the water-in-oil-in-water multiple emulsion W 1 / O / W 2 into a 1 L reactor, stir at 70 rpm, and react at 60 °C for 4 hours. The resulting macroporous crosslinked polymer microspheres were washed three times each with deionized water and ethanol, using acetone as the washing solution, extracted 30 times with a Soxhlet extractor, and then the residual acetone was washed away with deionized water to obtain the macroporous crosslinked polymer microspheres. The volume average particle size of the macroporous crosslinked polymer microspheres is 11 μm, the porosity is 10%, and the average pore size is 1.5 μm.

[0114] The surface morphology of the macroporous crosslinked polymer microspheres is as Figure 4 shown in the scanning electron microscope photograph of the macroporous crosslinked polymer microspheres provided in Example 4.

[0115] Example 5

[0116] A supermacroporous crosslinked polymer microsphere and a preparation method thereof. The preparation method specifically comprises the following steps:

[0117] (1) Prepare 50 mL of an aqueous solution of 0.2% magnesium sulfate as the internal aqueous phase W 1 ; Mix 70 g of butyl methacrylate, 60 g of ethylene glycol dimethacrylate, 0.75 g of azobisisobutyronitrile, 1.5 g of polystyrene-polyethylene glycol copolymer, and 100 g of dichloromethane to obtain the oil phase O; Prepare 1 L of an aqueous solution of 3% PVA as the external aqueous phase W 2 ;

[0118] (2) Mix the W 1 / O primary emulsion with the external aqueous phase W 2 ; Mechanically stir the pre-emulsion at 500 rpm for 3 min, and pour the pre-emulsified emulsion into a membrane emulsifier. Pass it through a 2.8 μm membrane tube 3 times under a pressure of 0.5 Mpa to obtain the W 1 / O primary emulsion;

[0119] (3) Mix the W 1 / O primary emulsion with the external aqueous phase W 2 ; Homogenize and emulsify at 3000 rpm for 5 min to obtain a water-in-oil-in-water multiple emulsion W 1 / O / W 2 ;

[0120] (4) Pour the water-in-oil-in-water multiple emulsion W 1 / O / W 2 into a 5 L reaction kettle, stir at 70 rpm, and react at 55 °C for 4 hours. The obtained supermacroporous crosslinked polymer microspheres are first washed three times each with deionized water and ethanol, extracted 30 times using a Soxhlet extractor with acetone as the washing solution, and then the residual acetone is washed away with deionized water to obtain the supermacroporous crosslinked polymer microspheres. The volume average particle size of the supermacroporous crosslinked polymer microspheres is 35 μm, the porosity is 43%, and the average pore diameter is 2.5 μm.

[0121] The surface morphology of the supermacroporous crosslinked polymer microspheres is as shown in Figure 5 the scanning electron microscope photograph of the supermacroporous crosslinked polymer microspheres provided in Example 5.

[0122] Product characterization and performance testing:

[0123] (1) Volume average particle size: Measured using a laser particle size analyzer (Mastersizer2000);

[0124] (2) Porosity and average pore diameter: Measured by mercury intrusion method using a mercury intrusion porosimeter (AutoPore IV 9500).

[0125] The applicant declares that the detailed process flow of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed process flow, that is, it does not mean that the present invention must rely on the above detailed process flow to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A preparation method of supermacroporous cross-linked polymer microspheres, characterized in that, the preparation method comprises the following steps: (1) Mix a monomer, a cross-linking agent, a pore-forming agent and an organic solvent to obtain an oil phase O; Prepare an aqueous solution containing a first osmotic pressure regulator as the internal aqueous phase W 1 ; Prepare an aqueous solution containing a surfactant as the outer aqueous phase W 2 ; (2) Mix the oil phase O and the internal water phase W 1 to obtain a water-in-oil W 1 / O primary emulsion; (3) Mix the water-in-oil W 1 / O primary emulsion with the external aqueous phase W 2 to obtain a water-in-oil-in-water multiple emulsion W 1 / O / W 2 ; (4) The water-in-oil-in-water double emulsion W 1 / O / W 2 undergoes a polymerization reaction to obtain the supermacroporous crosslinked polymer microspheres.

2. The preparation method according to claim 1, characterized in that, the monomer comprises any one or a combination of at least two of acrylate esters or styrene; Preferably, the acrylate esters comprise any one or a combination of at least two of glycidyl methacrylate, methyl methacrylate, butyl methacrylate, octadecyl methacrylate or 2-hydroxyethyl methacrylate.

3. The preparation method according to claim 1 or 2, characterized in that, the cross-linking agent comprises ethylene glycol dimethacrylate and / or divinylbenzene; Preferably, the mass ratio of the monomer to the cross-linking agent is 1:(0.2-5); Preferably, the pore-forming agent comprises an oil-soluble surfactant and / or an amphiphilic block copolymer; Preferably, the oil-soluble surfactant comprises sorbitan monostearate and / or polyoxyethylene sorbitan trioleate; Preferably, the hydrophilic segment of the amphiphilic block copolymer comprises at least one of a polyoxypropylene ether segment, a polyethylene glycol segment, a polyacrylic acid segment or a 2-hydroxyethyl polyacrylate segment; Preferably, the hydrophobic segment of the amphiphilic block copolymer comprises at least one of a polyoxyethylene segment, a polylactic acid segment, a polystyrene segment or a polymethyl methacrylate segment; Preferably, the amphiphilic block copolymer comprises a diblock copolymer and / or a triblock copolymer; Preferably, the triblock copolymer comprises a linear triblock copolymer and / or a star-shaped triblock copolymer; Preferably, the mass ratio of the total mass of the monomer and the cross-linking agent to the mass of the pore-forming agent is (3-300):

1.

4. The preparation method according to any one of claims 1 to 3, characterized in that, the organic solvent comprises any one or a combination of at least two of chloroform, acetone, tetrahydrofuran, toluene, dichloromethane, trichloromethane, dichloroethane or ethyl acetate; Preferably, the oil phase O further comprises an initiator; Preferably, the initiator comprises any one or a combination of at least two of azo initiators or peroxide initiators; Preferably, the azo initiators comprise any one or a combination of at least two of azobisisobutyronitrile, azobisisoheptonitrile or dimethyl azobisisobutyrate; Preferably, the peroxide initiators comprise any one or a combination of at least two of benzoyl peroxide, lauroyl peroxide, dodecanoyl peroxide, diisopropyl peroxydicarbonate or dicyclohexyl peroxydicarbonate; Preferably, based on the total mass of the monomer, the cross-linking agent, the pore-forming agent and the initiator being 100%, the mass fraction of the initiator is 0.3-6%.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The external aqueous phase W 2 further includes a second osmotic pressure regulator; Preferably, the first osmotic pressure regulator and the second osmotic pressure regulator each independently comprise metal ion salts or sugars; Preferably, the metal ion salts include any one or a combination of at least two of sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate or magnesium sulfate; Preferably, the saccharides include any one or a mixture of at least two of sucrose, glucose or trehalose; Preferably, the mass fraction of the first osmotic pressure regulator in the inner aqueous phase W 1 is 0.01-10%; Preferably, the external aqueous phase W 2 has a mass fraction of the second osmotic pressure regulator ≤ 5%; Preferably, the surfactant includes an ionic surfactant or a non-ionic surfactant; Preferably, the ionic surfactant includes any one or a mixture of at least two of sodium octadecyl sulfate, sodium dodecyl sulfate or sodium dodecylbenzenesulfonate; Preferably, the non-ionic surfactant includes any one or a mixture of at least two of polyvinyl alcohol, fatty alcohol polyoxyethylene ether or polysorbate-20; Preferably, the external aqueous phase W 2 has a surfactant mass fraction of 0.01-10%.

6. The preparation method according to any one of claims 1 to 5, characterized in that the method of mixing in step (2) includes any one of ultrasonic emulsification method, conventional membrane emulsification method, rapid membrane emulsification method, homogenization emulsification method or mechanical stirring method; further preferably the conventional membrane emulsification method or the rapid membrane emulsification method; Preferably, the method of mixing in step (3) includes any one of ultrasonic emulsification method, conventional membrane emulsification method, premixed membrane emulsification method, homogenization emulsification method or mechanical stirring method; further preferably the conventional membrane emulsification method or the premixed membrane emulsification method.

7. The preparation method according to any one of claims 1 to 6, characterized in that The inner aqueous phase W 1 has a volume ratio to the oil phase O of (0.1 - 3):1; Preferably, the total volume of the inner aqueous phase W 1 and the oil phase O to the volume of the outer aqueous phase W 2 is 1:(1 - 50); Preferably, the temperature of the polymerization reaction is 10-90 °C; Preferably, the time of the polymerization reaction is 2-24 h.

8. The preparation method according to any one of claims 1 to 7, characterized in that the preparation method specifically includes the following steps: (1) Mix the monomer, crosslinking agent, pore-forming agent, initiator and organic solvent to obtain an oil phase O; Prepare an aqueous solution containing a first osmotic pressure regulator as the internal aqueous phase W 1 ; Prepare an aqueous solution containing a surfactant and optionally a second osmotic pressure regulator as the external aqueous phase W 2 ; The mass ratio of the monomer to the crosslinking agent is 1:(0.2-5); The mass ratio of the total mass of the monomer and the crosslinking agent to the mass of the pore-forming agent is (3-300):1; Based on the total mass of the monomer, crosslinking agent, pore-forming agent and initiator being 100%, the mass fraction of the initiator is 0.3-6%; The inner aqueous phase W 1 contains a first osmotic pressure regulator with a mass fraction of 0.01-10%; The external aqueous phase W 2 has a mass fraction of the second osmotic pressure regulator ≤ 5%; The external aqueous phase W 2 has a surfactant mass fraction of 0.01-10%; (2) Mix the oil phase O and the internal aqueous phase W 1 to obtain a water-in-oil W 1 / O primary emulsion; The inner aqueous phase W 1 has a volume ratio to the oil phase O of (0.1 - 3):1; (3) Mix the water-in-oil W 1 / O primary emulsion with the external aqueous phase W 2 to obtain a water-in-oil-in-water multiple emulsion W 1 / O / W 2 ; The inner aqueous phase W 1 and the total volume of the oil phase O and the outer aqueous phase W 2 is in a volume ratio of 1:(1 - 50); (4) The water-in-oil-in-water double emulsion W 1 / O / W 2 undergoes a polymerization reaction to obtain the supermacroporous crosslinked polymer microspheres; The temperature of the polymerization reaction is 10-90 °C; The time of the polymerization reaction is 2-24 h.

9. A supermacroporous crosslinked polymer microsphere, characterized in that the supermacroporous crosslinked polymer microsphere is prepared by the preparation method according to any one of claims 1 to 8; Preferably, the supermacroporous crosslinked polymer microsphere has a nano- to micron-scale through-pore structure; Preferably, the particle size of the supermacroporous crosslinked polymer microsphere is 1-300 μm, preferably 5-250 μm, and further preferably 10-200 μm; Preferably, the pore diameter of the supermacroporous crosslinked polymer microsphere is 0.1-90 μm, preferably 0.2-70 μm, and further preferably 0.5-30 μm; Preferably, the porosity of the supermacroporous crosslinked polymer microsphere is 10-80%, preferably 20-70%, and further preferably 30-70%.

10. An application of the supermacroporous crosslinked polymer microsphere according to claim 9 as a stationary phase filler for chromatographic separation, an immobilization carrier for enzymes, a cell culture microcarrier, a tissue engineering micro scaffold material or an adsorption material.

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