High-hydrophilicity macroporous microsphere and preparation method thereof

Abstract

The invention discloses a high-hydrophilicity macroporous microsphere and a preparation method thereof, and the preparation method comprises the following steps: firstly, preparing a polystyrene seed microsphere and swelling; styrene, divinyl benzene and vinyl ester monomers are added for a polymerization reaction, then alcoholysis is carried out, and macroporous microspheres with primary hydrophilic groups on the surfaces are obtained; and finally, modifying a secondary hydrophilic group to obtain the high-hydrophilicity macroporous microsphere. The high-hydrophilicity macroporous microsphere provided by the invention is excellent in comprehensive performance, the surface of the microsphere is completely covered with the high-hydrophilicity macroporous microsphere through primary and secondary hydrophilic groups, and the hydrophilic groups are directly connected with the microsphere through chemical bonds, so that the high-hydrophilicity macroporous microsphere has good stability and can effectively reduce non-specific adsorption of base spheres and improve the recovery rate of protein purification; the preparation method of the high-hydrophilicity macroporous microspheres provided by the invention is simple and efficient, has wide raw material sources, can be used for preparing fillers such as affinity chromatography fillers, ion chromatography fillers, composite mode chromatography fillers and the like, and has wide application prospects and good industrialization conditions.

Description

Technical Field

[0001] The invention relates to a preparation method and application of microspheres, in particular to a preparation method and application of highly hydrophilic macroporous microspheres, belonging to the field of chemistry. Background Art

[0002] Chromatography uses differences in physical and chemical properties to separate different components in a mixture. Modifying specific functional groups on the surface of the chromatography filler can achieve specific adsorption of specific substances. When using a chromatography filler with specific adsorption, nonspecific adsorption needs to be avoided to ensure high efficiency of chromatography. In other chromatography techniques except hydrophobic interaction chromatography, hydrophobic interactions may cause nonspecific adsorption. When biological macromolecules such as proteins with hydrophobic groups on the surface pass through a hydrophobic chromatography filler, they will be adsorbed on the filler due to hydrophobic interactions.

[0003] Polystyrene-divinylbenzene microspheres are a commonly used chromatography filler with a large number of benzene rings on their surface, which are very hydrophobic. Before the polystyrene-divinylbenzene microspheres are modified with various specific functional groups, a hydrophilic coating needs to be modified on the surface of the microspheres to shield the hydrophobic interaction brought by the base ball. The current method for modifying the hydrophilic coating on the surface of polystyrene-divinylbenzene microspheres is to disperse and stir the microspheres and hydrophilic polymers, usually polyhydroxy compounds, so that a layer of hydroxyl-containing polymer is coated on the surface of the microspheres. Since there is no chemical bond between the polymer and the microspheres, it is also necessary to cross-link the hydroxyl groups of the polymer itself to fix the hydrophilic polymer on the surface of the microspheres, thereby achieving the purpose of hydrophilicity on the surface of the microspheres. This method has obvious defects. On the one hand, the polymer is unevenly coated on the surface of the microspheres, resulting in areas on the surface of the microspheres that are not covered by the hydrophilic coating; on the other hand, the polymer of the physical property coating has poor stability and is easy to fall off, resulting in the exposure of the hydrophobic groups, and thus the non-specific adsorption caused by the hydrophobic interaction cannot be effectively avoided. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for preparing and applying highly hydrophilic macroporous microspheres in which hydrophilic groups are stably connected to the microspheres and completely cover the surface of the microspheres.

[0005] Technical solution: The present invention provides a method for preparing highly hydrophilic macroporous microspheres, comprising the following steps: (1) preparing polystyrene seed microspheres and swelling them to obtain swollen microspheres; (2) adding styrene, divinylbenzene and vinyl ester monomers to the swollen microspheres for polymerization, and performing alcoholysis on the polymerized microspheres to obtain macroporous microspheres having primary hydrophilic groups on the surface; (3) The surface of the macroporous microspheres with primary hydrophilic groups is modified with secondary hydrophilic groups to obtain highly hydrophilic macroporous microspheres.

[0006] The present invention first uses styrene, divinylbenzene and vinyl ester monomers to polymerize the swollen microspheres to increase the functional groups on their surface for subsequent hydrophilic modification; then alcoholysis is performed to make the surface have primary hydrophilic groups connected by chemical bonds; on this basis, the secondary hydrophilic groups are modified to obtain highly hydrophilic macroporous microspheres. The hydrophilic groups of the highly hydrophilic macroporous microspheres prepared by this method fully cover the surface of the microspheres, and the hydrophilic groups are directly connected to the microspheres by chemical bonds, are not easy to fall off, and have good stability.

[0007] Commonly used methods for preparing and swelling polystyrene (PS) seed microspheres are applicable to the present invention. For example, polyvinyl alcohol, styrene monomer and benzoyl peroxide are used as raw materials to prepare PS seed microspheres through polymerization reaction; PS seed microspheres and porogens are added to the SDS aqueous solution system to react to obtain PS seed swollen microspheres. Commonly used porogens include toluene, n-hexane, n-hexanol, n-heptane, isooctane, dibutyl phthalate, etc.

[0008] Preferably, in step (2), the vinyl ester monomer is at least one of vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, trimethylacetate, vinyl valerate, and vinyl hexanoate.

[0009] Preferably, in step (2), the polymerization reaction step is: (21) Add styrene, divinylbenzene, vinyl ester monomer, initiator and dispersant to a 0.1-0.5% by mass SDS aqueous solution, and mix by ultrasonication to obtain an emulsion; the volume ratio of styrene: divinylbenzene: vinyl ester monomer: SDS aqueous solution is 0.1-5: 0.5-15: 0.1-5: 20-120; the mass ratio of initiator: dispersant is 1: 1; the solid component: liquid ratio is 0.2-2: 20.7-145, and the unit of the solid-liquid ratio is g / mL; (22) Add the emulsion to the swollen microspheres, stir for 6-48 h, then heat to 50-120° C., keep the temperature for 6-48 h, and then wash with anhydrous ethanol and reflux to obtain polymerized microspheres.

[0010] Preferably, in step (21), the initiator is at least one of benzoyl peroxide, azobisisobutyronitrile, lauroyl peroxide, and cumene hydroperoxide.

[0011] Preferably, in step (21), the dispersant is at least one of gelatin, hydroxyethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, and sodium polyacrylate.

[0012] Preferably, in step (2), the fermentation step is: (23) adding methanol to the polymerized microspheres, stirring at 100-300 r / min, and heating under reflux at 40-80° C. for 1-6 h; the material-liquid ratio of the polymerized microspheres to methanol is 1:1-10, and the unit of the material-liquid ratio is g / mL; (24) Add a sodium hydroxide-methanol solution having a mass concentration of 0.2-1.2% to the product obtained in step (23), and keep the reaction warm for 1-24 hours. Then, wash the microspheres with anhydrous ethanol and purified water to obtain macroporous microspheres having primary hydrophilic groups on the surface.

[0013] Furthermore, the fermentation is carried out in a three-necked flask, which is convenient for controlling the addition of materials and the reaction conditions.

[0014] Furthermore, the sodium hydroxide-methanol solution is prepared and used immediately, and after cooling, is added into the reaction system through a dropping funnel.

[0015] Preferably, in step (3), the step of modifying the secondary hydrophilic group is: (31) mixing macroporous microspheres having primary hydrophilic groups on the surface with epoxy compounds; the mass ratio of the macroporous microspheres to the epoxy compounds is 10:3-20; (32) Adding a 10-50% by mass dextran aqueous solution to the product obtained in step (31) and mixing; then adding a 0.2-2 mol / L NaOH solution; reacting at 25-90° C. for 2-24 h to obtain a highly hydrophilic macroporous microsphere; the volume ratio of the dextran aqueous solution to the NaOH solution is 2-10: 1-6; the solid component to liquid component ratio is 13-30: 15-80, and the unit of the solid-liquid ratio is g / mL.

[0016] Furthermore, the dextran aqueous solution is prepared and used immediately.

[0017] Furthermore, in step (32), after the reaction is completed, the reaction product is washed with pure water and the water is drained.

[0018] Preferably, in step (31), the epoxy compound is at least one of ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, di(2-epoxypropyl) ether, dipropylene glycol diglycidyl ether, glycerol triglycidyl ether, and pentaerythritol tetraglycidyl ether.

[0019] Preferably, in step (32), the molecular weight of the dextran is 2000-100000.

[0020] Furthermore, the molecular weight of the dextran is 2000, 4000, 10000, 20000, 40000 or 100000.

[0021] The present invention also provides a highly hydrophilic macroporous microsphere, which is prepared by the above-mentioned preparation method of the highly hydrophilic macroporous microsphere.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The highly hydrophilic macroporous microspheres provided by the present invention have superior comprehensive performance. The hydrophilic groups are fully covered on the surface of the microspheres through the primary hydrophilic groups and the secondary hydrophilic groups, and the hydrophilic groups are directly connected to the microspheres through chemical bonds, which have good stability, can effectively reduce the nonspecific adsorption of the base balls, and improve the recovery rate of protein purification; 2. The preparation method of the highly hydrophilic macroporous microspheres provided by the present invention is simple and efficient, and the raw materials are widely available. They can be used for the preparation of affinity chromatography fillers, ion chromatography fillers, composite mode chromatography fillers and other fillers, and have broad application prospects and good industrialization conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A synthetic route map of the highly hydrophilic macroporous microspheres provided by the present invention; Figure 2 A chromatogram of separation of strongly hydrophobic proteins by the highly hydrophilic macroporous microspheres provided by the present invention; Figure 3 The chromatogram of separation of strongly hydrophobic proteins by highly hydrophilic macroporous microspheres provided as comparative examples. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0025] Unless otherwise specified, the raw materials used in the examples of the present invention are commercially available.

[0026] Example 1 A method for preparing highly hydrophilic macroporous microspheres Weigh 5 g of polyvinyl alcohol and dissolve it in ethanol / water. Mix them evenly by ultrasonic treatment. Set the reaction temperature to 80°C and introduce nitrogen. Then add 15 g of styrene monomer and 0.1 g of benzoyl peroxide and keep the polymerization for 18 h. After the polymerization reaction, centrifuge and wash with ethanol and water three times each. Freeze-dry to obtain PS seed microspheres.

[0027] Take 1 g of seed microspheres and mix them in 30 mL of deionized water, mix them evenly with ultrasound; prepare 40 mL of SDS aqueous solution with a mass fraction of 0.15%, add 5 mL of porogen toluene and n-hexanol in a volume ratio of 1:1, and add them to the 40 mL of prepared SDS aqueous solution, mix them evenly with ultrasound, and add them to the seed microsphere dispersion solution and stir at a stirring rate of 600 r / min, and react for 24 h at a certain temperature. The swollen PS microspheres are obtained.

[0028] Prepare 35 mL of SDS aqueous solution with a mass fraction of 0.2% in advance, weigh 3 mL of styrene, 6.5 mL of divinylbenzene, 4 mL of vinyl acetate monomer, 0.35 g of initiator azobisisobutyronitrile and 0.2 g of dispersant polyvinyl alcohol, add them to the SDS aqueous solution and mix them evenly by ultrasonication, then add the ultrasonicated emulsion to the swollen microsphere system and continue stirring for 12 hours. After stirring is complete, increase the system temperature to 85°C and keep the reaction for 24 hours. After the reaction is completed, wash and reflux with anhydrous ethanol to obtain polymerized microspheres.

[0029] Then put 10 g of the obtained polymerized microspheres in a three-necked flask, add 50 ml of methanol to the three-necked flask, stir at 200 r / min, and heat to reflux at 50°C for 5 h. Prepare sodium hydroxide-methanol solution in advance, weigh 0.4 g of NaOH, dissolve it to 100 ml with methanol solution, and pour it into the dropping funnel after cooling. Then add the sodium hydroxide-methanol solution dropwise into the reaction bottle and keep it warm for 6 h. After the reaction, wash the microspheres with anhydrous ethanol and purified water to obtain macroporous microspheres with primary hydrophilic groups on the surface.

[0030] Take 10 g of microspheres with primary hydrophilic groups and add them to the reaction bottle, then add 6 g of ethylene glycol diglycidyl ether and mix well. Prepare 15 mL of 20,000 molecular weight dextran aqueous solution with a mass fraction of 25% in advance, add it to the reaction bottle and stir evenly. Then add 20 mL of 0.8 mol / L NaOH solution to the material. The temperature in the reactor is set to 70°C and the reaction time is 12 h. After the reaction is completed, wash it with pure water and drain the water to obtain highly hydrophilic macroporous microspheres.

[0031] Example 2 A method for preparing highly hydrophilic macroporous microspheres This embodiment is basically the same as the embodiment 1, except that: the porogen is replaced by toluene and isooctane; the vinyl ester monomer is replaced by vinyl n-butyrate; and the dispersant is replaced by hydroxyethyl cellulose.

[0032] Example 3 A method for preparing highly hydrophilic macroporous microspheres This embodiment is basically the same as embodiment 2, except that: the porogen is replaced by n-hexanol and isooctane; the vinyl ester monomer is replaced by vinyl acetate and vinyl propionate, which are added in a ratio of 1:1; and the secondary hydrophilic group is replaced by dextran with a molecular weight of 40,000.

[0033] Example 4 A method for preparing highly hydrophilic macroporous microspheres This embodiment is basically the same as embodiment 3, except that: the porogen is replaced by toluene; the vinyl ester monomer is replaced by vinyl propionate; the dispersant is replaced by gelatin; and the secondary hydrophilic group is replaced by dextran with a molecular weight of 10,000.

[0034] Example 5 A method for preparing highly hydrophilic macroporous microspheres This example is basically the same as Example 1, except that: when the swollen PS microspheres are polymerized, 120 mL of a 0.5% SDS aqueous solution, 5 mL of styrene, 15 mL of divinylbenzene, 5 mL of vinyl acetate monomer, 1 g of an initiator azobisisobutyronitrile and 1 g of a dispersant polyvinyl alcohol are used. After stirring for 48 h, the system temperature is raised to 120° C. and the reaction is kept warm for 48 h.

[0035] Then, 100 ml of methanol was added to 10 g of the polymerized microspheres, stirred at 300 r / min, and heated to reflux at 80°C for 6 h. A 1.2% sodium hydroxide-methanol solution was added dropwise, and the reaction was kept warm for 24 h.

[0036] Finally, add 20 g of ethylene glycol diglycidyl ether to 10 g of microspheres with primary hydrophilic groups and mix well; then add 50 mL of 20,000 molecular weight dextran aqueous solution with a mass fraction of 50% and stir well; then add 30 mL of 2 mol / L NaOH solution to the material and react at 90°C for 24 h.

[0037] Comparative Example 1 A hydrophilic macroporous microsphere This comparative example is a hydrophilic macroporous microsphere disclosed in the prior art, which is a self-made product of our company, with the model number PSDVB-50OH and the material number 2200509D1. This comparative example is a polystyrene-divinylbenzene microsphere modified with a hydrophilic coating, and the modification method is to disperse and stir the polystyrene-divinylbenzene sphere and the hydrophilic polymer, so that the surface of the microsphere is coated with a layer of hydroxyl-containing polymer, and then the hydroxyl groups of the hydrophilic polymer itself are cross-linked to fix the hydrophilic polymer on the surface of the microsphere.

[0038] Performance Testing The highly hydrophilic macroporous microspheres prepared in Example 1 were used to separate strongly hydrophobic proteins to determine whether the hydrophobic regions on the microsphere surfaces were modified with hydrophilic groups and the quality of the modification.

[0039] Test conditions: The instrument is HPLC. The chromatographic column is a 4.6*50 mm steel column filled with macroporous microspheres prepared in Example 1, and the column temperature is 25°C. The test sample is chicken ovalbumin OVA + chymosin Chy (0.5 mg / mL). Mobile phase A: 2Mammonium sulfate +100mM sodium phosphate (pH7.0); mobile phase B: 100mM sodium phosphate (pH7.0).

[0040] Test method: Use mobile phase A to balance the system and chromatographic column, and then load the sample after the baseline returns to zero; after the system automatically injects the sample, perform gradient elution, 0-100% mobile phase B, and the time is 15 minutes.

[0041] Test results such as Figure 2 As shown. Under the gradient elution condition, OVA and CHY were eluted from the column quickly, indicating that the prepared macroporous microspheres had good hydrophilicity and the hydrophobicity was shielded. And it showed double peaks, indicating that the two proteins were eluted from the microspheres one after another, that is, the macroporous microspheres prepared in Example 1 can effectively separate different substances.

[0042] The microsphere performance of Examples 2-4 and Comparative Example 1 was tested in the same manner. The results are shown in Table 1. Since chicken ovalbumin OVA and chymosin Chy are proteins with strong hydrophobicity, under the condition of gradually increasing polarity of the mobile phase, the earlier the elution, the weaker the binding force between the protein and the microsphere, that is, the better the hydrophilicity of the microsphere. And since the hydrophobicity of CHY is stronger than that of OVA, the elution peak is a double peak, indicating good resolution, that is, less interference from nonspecific adsorption, which also shows that the hydrophilicity of the microsphere is good.

[0043] Table 1 Test results of microsphere performance of Examples 1-5 and Comparative Example 1 The test spectrum of Comparative Example 1 is as follows Figure 3 As shown. Under the gradient elution condition, OVA and CHY could not be eluted from the column quickly, indicating that the prepared macroporous microspheres still had strong hydrophobicity and poor hydrophilicity. And the elution peak was a single peak, indicating that the macroporous microspheres provided in Comparative Example 1 could not effectively separate the two proteins.

[0044] In summary, the microspheres prepared by the method for preparing highly hydrophilic macroporous microspheres provided by the present invention can well shield the hydrophobic effect and reduce nonspecific adsorption by introducing a primary hydrophilic group in the microsphere polymerization stage and subsequently modifying the secondary hydrophilic group on the microsphere surface, and have obvious advantages in hydrophilicity. The highly hydrophilic macroporous microspheres provided by the present invention can be widely used in the fields of affinity chromatography fillers, ion chromatography fillers, composite mode chromatography fillers, etc.

Claims

1. A method for preparing highly hydrophilic macroporous microspheres, characterized in that: The following steps are involved: (1) preparing polystyrene seed microspheres and swelling them to obtain swollen microspheres; (2) adding styrene, divinylbenzene and vinyl ester monomers to the swollen microspheres for polymerization, and performing alcoholysis on the polymerized microspheres to obtain macroporous microspheres having primary hydrophilic groups on the surface; (3) The surface of the macroporous microspheres with primary hydrophilic groups is modified with secondary hydrophilic groups to obtain highly hydrophilic macroporous microspheres.

2. The preparation method according to claim 1, characterized in that: In step (2), the vinyl ester monomer is at least one of vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, trimethyl vinyl acetate, vinyl valerate, and vinyl hexanoate.

3. The preparation method according to claim 1, characterized in that: In step (2), the polymerization reaction steps are: (21) Add styrene, divinylbenzene, vinyl ester monomer, initiator and dispersant to a 0.1-0.5% by mass SDS aqueous solution, and mix by ultrasonication to obtain an emulsion; the volume ratio of styrene: divinylbenzene: vinyl ester monomer: SDS aqueous solution is 0.1-5: 0.5-15: 0.1-5: 20-120; the mass ratio of initiator: dispersant is 1: 1; the solid component: liquid ratio is 0.2-2: 20.7-145, and the unit of the solid-liquid ratio is g / mL; (22) Add the emulsion to the swollen microspheres, stir for 6-48 h, then heat to 50-120° C., keep the temperature for 6-48 h, and then wash with anhydrous ethanol and reflux to obtain polymerized microspheres.

4. The preparation method according to claim 3, characterized in that: In step (21), the initiator is at least one of benzoyl peroxide, azobisisobutyronitrile, lauroyl peroxide, and cumene hydroperoxide.

5. The preparation method according to claim 3, characterized in that: In step (21), the dispersant is at least one of gelatin, hydroxyethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, and sodium polyacrylate.

6. The preparation method according to claim 1, characterized in that: In step (2), the fermentation step is: (23) adding methanol to the polymerized microspheres, stirring at 100-300 r / min, and heating under reflux at 40-80° C. for 1-6 h; the material-liquid ratio of the polymerized microspheres to methanol is 1:1-10, and the material-liquid ratio is expressed in g / mL; (24) Add a sodium hydroxide-methanol solution having a mass concentration of 0.2-1.2% to the product obtained in step (23), and keep the reaction warm for 1-24 hours. Then, wash the microspheres with anhydrous ethanol and purified water to obtain macroporous microspheres having primary hydrophilic groups on the surface.

7. The preparation method according to claim 1, characterized in that: In step (3), the step of modifying the secondary hydrophilic group is: (31) mixing macroporous microspheres having primary hydrophilic groups on the surface with epoxy compounds, wherein the mass ratio of the macroporous microspheres to the epoxy compounds is 10:3-20; (32) Adding a 10-50% by mass dextran aqueous solution to the product obtained in step (31) and mixing; then adding a 0.2-2 mol / L NaOH solution; reacting at 25-90° C. for 2-24 h to obtain a highly hydrophilic macroporous microsphere; the volume ratio of the dextran aqueous solution to the NaOH solution is 2-10: 1-6; the solid component to liquid component ratio is 13-30: 15-80, and the unit of the solid-liquid ratio is g / mL.

8. The preparation method according to claim 7, characterized in that: In step (31), the epoxy compound is at least one of ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, di(2-epoxypropyl) ether, dipropylene glycol diglycidyl ether, glycerol triglycidyl ether, and pentaerythritol tetraglycidyl ether.

9. The preparation method according to claim 7, characterized in that: In step (32), the molecular weight of the dextran is 2000-100000.

10. A highly hydrophilic macroporous microsphere, characterized in that: The highly hydrophilic macroporous microspheres are prepared by the preparation method of any one of claims 1 to 9.

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