A core-shell structured polyvinyl alcohol microsphere and a preparation method and application thereof

Abstract

The application discloses a kind of polyvinyl alcohol microspheres of core-shell structure and its preparation method and application.The core of polyvinyl alcohol microspheres is cellulose nanospheres, and the shell is polyvinyl alcohol.The polyvinyl alcohol microspheres of core-shell structure of some examples of the application have high mechanical strength, have comparable biocompatibility with agarose resin microspheres, can be directly applied to the environment in contact with human body, and do not need to be hydrophilic modified compared with synthetic resin microspheres;The inside and outside of it both contain rich hydroxyl groups, the pore size of the formed microspheres is large, which is beneficial to the entry of ligand into coupling with hydroxyl groups, increases the coupling amount, improves the adsorption effect, and meets the needs of different occasions;Large pore size can improve the contact area and contact time of the adsorbed material and the adsorbent, and further enhance the adsorption performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polyvinyl alcohol microsphere with a core-shell structure and a preparation method and application thereof. BACKGROUND

[0002] Blood purification therapy refers to reducing the concentration of harmful substances in blood through extracorporeal removal, so as to achieve the purpose of treating or slowing down certain diseases. The core of blood purification therapy is an adsorbent material, and obtaining an adsorbent material with good blood compatibility, high mechanical strength and good adsorption performance is the key to the development of blood purification technology.

[0003] Currently, there are three types of commonly used adsorbents. Activated carbon has high porosity and specific surface area, and is a commonly used broad-spectrum adsorbent. However, its mechanical strength is not high, and it needs to be coated to improve the biocompatibility, so its application is limited. Polysaccharide adsorbent has good biocompatibility and no cytotoxicity, and the surface has a large number of active groups for chemical modification. However, it is expensive and has low mechanical strength, and it is easy to be damaged under high flow rate or long-term use. Synthetic resin has high adsorption capacity and specific surface area, and has high mechanical strength. However, it has poor blood compatibility, and needs to be modified to be hydrophilic in advance.

[0004] Polyvinyl alcohol (PVA) is an artificially synthesized water-soluble polymer, which has good formability, non-toxicity and biocompatibility. At the same time, PVA material contains a large number of hydroxyl groups, which can couple different ligands to achieve specific adsorption, so it can be selected as a blood purification adsorbent. However, compared with synthetic resin adsorbent, it still has poor mechanical properties, and cannot be applied to whole blood perfusion or antibody chromatography purification under high flow rate and high pressure. Therefore, it is very important to develop a polyvinyl alcohol microsphere carrier with high mechanical strength, high adsorption performance and high selectivity. SUMMARY

[0005] The present application aims to overcome at least one deficiency of the prior art, and provides a polyvinyl alcohol microsphere with a core-shell structure and a preparation method and application thereof.

[0006] The technical solution adopted by the present application is as follows:

[0007] In a first aspect, the present application provides:

[0008] A polyvinyl alcohol microsphere with a core-shell structure, wherein the core is a cellulose nanomicrosphere, and the shell is polyvinyl alcohol.

[0009] In some examples of the polyvinyl alcohol microsphere, the particle size of the cellulose nanomicrosphere is 100-400 nm.

[0010] In some examples of the polyvinyl alcohol microspheres, the polyvinyl alcohol microspheres have a particle size of 30-200 μm and a surface pore size of 0.10-2.5 μm.

[0011] In some examples of the polyvinyl alcohol microspheres, the cellulose nanomicrospheres have a particle size of 100-400 nm, the polyvinyl alcohol microspheres have a particle size of 30-200 μm and a surface pore size of 0.10-2.5 μm.

[0012] In some examples of the polyvinyl alcohol microspheres, the cellulose is at least one selected from cyanoethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose.

[0013] In a second aspect of the present application, there is provided:

[0014] In a first aspect of the present application, there is provided a preparation method of polyvinyl alcohol microspheres, comprising the following steps:

[0015] S1) mixing cellulose nanomicrospheres, polyvinyl alcohol and water uniformly to obtain an aqueous phase;

[0016] S2) dissolving an emulsifier in a water-insoluble solvent to obtain an oil phase;

[0017] S3) adding the aqueous phase into the oil phase, and stirring and emulsifying and dispersing to obtain a suspension;

[0018] S4) adding a crosslinking agent and a catalyst into the suspension, stirring and crosslinking, and reacting to obtain a crosslinked liquid;

[0019] S5) cooling and forming the crosslinked liquid, filtering and washing to obtain polyvinyl alcohol microspheres with a core-shell structure.

[0020] In some examples of the preparation method, the cellulose nanomicrospheres, the polyvinyl alcohol and the water are mixed uniformly respectively to obtain a cellulose nanomicrosphere suspension and a polyvinyl alcohol aqueous solution.

[0021] In some examples of the preparation method, the cellulose nanomicrosphere suspension has a mass concentration of 0.5-20 mg / ml.

[0022] In some examples of the preparation method, the cellulose nanomicrospheres have a particle size of 100-400 nm.

[0023] In some examples of the preparation method, the cellulose nanomicrosphere suspension has a mass concentration of 0.5-20 mg / ml, and the cellulose nanomicrospheres have a particle size of 100-400 nm.

[0024] In some examples of the preparation method, the polyvinyl alcohol aqueous solution has a mass concentration of 2-8%.

[0025] In some examples of the preparation method, the cellulose nanospheres suspension has a mass concentration of 0.5-20 mg / ml, and the polyvinyl alcohol aqueous solution has a mass concentration of 2-8%.

[0026] In some examples of the preparation method, the cellulose nanospheres suspension and the polyvinyl alcohol aqueous solution are mixed at a volume ratio of 1: (1-5).

[0027] In some examples of the preparation method, the cellulose nanospheres suspension has a mass concentration of 0.5-20 mg / ml, and the polyvinyl alcohol aqueous solution has a mass concentration of 2-8%, and the cellulose nanospheres suspension and the polyvinyl alcohol aqueous solution are mixed at a volume ratio of 1: (1-5).

[0028] In some examples of the preparation method, the water-insoluble solvent is selected from at least one of n-hexane, liquid paraffin, petroleum ether, n-heptane or n-octane.

[0029] In some examples of the preparation method, the emulsification and dispersion is carried out at a temperature of 30-70°C and a stirring speed of 500-1200 rpm.

[0030] In some examples of the preparation method, the emulsification and dispersion is carried out for a stirring time of 30 min-150 min.

[0031] In some examples of the preparation method, the emulsification and dispersion is carried out at a temperature of 30-70°C and a stirring speed of 500-1200 rpm for a stirring time of 30 min-150 min.

[0032] In some examples of the preparation method, the crosslinking agent is selected from at least one of formaldehyde, glutaraldehyde and genipin.

[0033] In some examples of the preparation method, the catalyst is an acid, preferably hydrochloric acid, and further, the concentration of the hydrochloric acid is 0.1-1 M.

[0034] In some examples of the preparation method, the cooling and molding is carried out at a temperature of 0-20°C.

[0035] In a third aspect of the present application, there is provided:

[0036] An adsorbent, which is obtained by covalently coupling a ligand to the polyvinyl alcohol microspheres according to the first aspect of the present application or prepared by the preparation method according to the second aspect of the present application.

[0037] In a fourth aspect of the present application, there is provided:

[0038] The polyvinyl alcohol microspheres according to the first aspect of the present application or prepared by the preparation method according to the second aspect of the present application are used for, but not limited to:

[0039] Preparation of blood purification material;

[0040] Preparation of protein purification material;

[0041] Preparation of drug carrier.

[0042] The beneficial effects of the present application are:

[0043] The polyvinyl alcohol microspheres with core-shell structure of some examples of the present application have high mechanical strength, comparable biocompatibility with agarose resin microspheres, and can be directly applied to environments in contact with the human body, without the need for hydrophilic modification compared with synthetic resin microspheres.

[0044] The polyvinyl alcohol microspheres with core-shell structure of some examples of the present application have abundant hydroxyl groups inside and outside, forming microspheres with a large pore size range, which facilitates the entry of ligands into coupling with hydroxyl groups, increases the coupling amount, improves the adsorption effect, and meets the needs of different occasions.

[0045] The polyvinyl alcohol microspheres with core-shell structure of some examples of the present application have a large pore size, which can increase the contact area and contact time of the adsorbate and the adsorbent, and further enhance the adsorption performance.

[0046] The preparation method of some examples of the present application forms resin microspheres by forming a core-shell structure of nanoparticles and polyvinyl alcohol, and then cross-linking and curing, while auxiliary process conditions are designed, to obtain polyvinyl alcohol microspheres with large pore size and high mechanical strength, expanding the application scenarios of the microspheres.

[0047] The preparation method of some examples of the present application produces polyvinyl alcohol microspheres with core-shell structure with large pore size and high mechanical strength.

[0048] The preparation method of some examples of the present application can well control the pore size of the microspheres by controlling the temperature of the cooling molding, meeting the needs of different applications. DETAILED DESCRIPTION

[0049] In a first aspect of the present application, there is provided:

[0050] A polyvinyl alcohol microsphere with core-shell structure, wherein the core is a cellulose nanomicrosphere, and the shell is polyvinyl alcohol.

[0051] In some examples of polyvinyl alcohol microspheres, the particle size of the cellulose nanomicrosphere is 100-400 nm. Such cellulose nanomicrospheres with a particle size of 100-400 nm have good reinforcing effect, can effectively improve the mechanical strength of the polyvinyl alcohol microspheres, and meet the application of adsorption at the same time.

[0052] In some examples of the polyvinyl alcohol microspheres, the polyvinyl alcohol microspheres have a particle size of 30-200 μm and a surface pore size of 0.10-2.5 μm. This is conducive to obtaining a proper specific surface area, facilitating the coupling of ligands, and increasing the adsorption area and contact time during adsorption.

[0053] In some examples of the polyvinyl alcohol microspheres, the cellulose nanospheres have a particle size of 100-400 nm, and the polyvinyl alcohol microspheres have a particle size of 30-200 μm and a surface pore size of 0.10-2.5 μm.

[0054] The type of cellulose is not particularly limited, and in some examples of the polyvinyl alcohol microspheres, the cellulose is at least one selected from the group consisting of cyanoethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose. These celluloses are mature celluloses, and their safety has been fully verified, and their cost is more controllable.

[0055] The cellulose nanospheres can be directly purchased from commercial products or prepared according to known methods.

[0056] In a second aspect of the present application, there is provided:

[0057] The method for preparing the polyvinyl alcohol microspheres according to the first aspect of the present application comprises the following steps:

[0058] S1) mixing cellulose nanospheres, polyvinyl alcohol, and water uniformly to obtain an aqueous phase;

[0059] S2) dissolving an emulsifier in a water-insoluble solvent to obtain an oil phase;

[0060] S3) adding the aqueous phase into the oil phase, and stirring and emulsifying and dispersing to obtain a suspension;

[0061] S4) adding a crosslinking agent and a catalyst into the suspension, stirring and crosslinking, and reacting to obtain a crosslinked liquid;

[0062] S5) cooling and forming the crosslinked liquid, filtering, and washing to obtain polyvinyl alcohol microspheres with a core-shell structure.

[0063] In some examples of the method, the cellulose nanospheres, the polyvinyl alcohol, and the water are mixed uniformly to obtain a cellulose nanosphere suspension and a polyvinyl alcohol aqueous solution, respectively. This can facilitate more uniform dispersion and mixing.

[0064] In some examples of the method, the cellulose nanosphere suspension has a mass concentration of 0.5-20 mg / ml. The specific concentration can be adjusted according to actual conditions.

[0065] In some examples of the method, the cellulose nanospheres have a particle size of 100-400 nm.

[0066] In some examples of the preparation method, the mass concentration of the cellulose nanospheres suspension is 0.5-20 mg / ml, and the particle size of the cellulose nanospheres is 100-400 nm.

[0067] In some examples of the preparation method, the mass concentration of the polyvinyl alcohol aqueous solution is 2-8%. This concentration range can better coat the cellulose nanospheres to form a core-shell structure.

[0068] In some examples of the preparation method, the mass concentration of the cellulose nanospheres suspension is 0.5-20 mg / ml, and the mass concentration of the polyvinyl alcohol aqueous solution is 2-8%.

[0069] In some examples of the preparation method, the volume ratio of the mixture of the cellulose nanospheres suspension and the polyvinyl alcohol aqueous solution is 1: (1-5).

[0070] In some examples of the preparation method, the mass concentration of the cellulose nanospheres suspension is 0.5-20 mg / ml, the mass concentration of the polyvinyl alcohol aqueous solution is 2-8%, and the volume ratio of the mixture of the cellulose nanospheres suspension and the polyvinyl alcohol aqueous solution is 1: (1-5).

[0071] The water-insoluble solvent is not particularly limited, and in some examples of the preparation method, the water-insoluble solvent is at least one selected from the group consisting of n-hexane, liquid paraffin, petroleum ether, n-heptane, and n-octane. These solvents are relatively safe, easy to remove, and have controllable costs.

[0072] In some examples of the preparation method, the emulsification and dispersion temperature is 30-70°C, and the stirring speed is 500-1200 rpm.

[0073] In some examples of the preparation method, the stirring time for emulsification and dispersion is 30 min-150 min.

[0074] In some examples of the preparation method, the emulsification and dispersion temperature is 30-70°C, the stirring speed is 500-1200 rpm, and the stirring time is 30 min-150 min.

[0075] In some examples of the preparation method, the crosslinking agent is at least one selected from the group consisting of formaldehyde, glutaraldehyde, and genipin. These crosslinking agents can crosslink the polyvinyl alcohol to form a polyvinyl alcohol shell.

[0076] In some examples of the preparation method, the catalyst is an acid, preferably hydrochloric acid, and further, the concentration of the hydrochloric acid is 0.1-1 M.

[0077] In some examples of the preparation method, the temperature of the cooling molding is 0-20℃. In this way, the structure of the core-shell structure polyvinyl alcohol microspheres obtained by the cross-linking reaction can be better maintained and stabilized.

[0078] The technical solutions of the present application are further described below with reference to examples. For the sake of comparison, the solvent that is not soluble in water is n-hexane.

[0079] Example 1, preparation of cyanoethyl cellulose-polyvinyl alcohol microspheres

[0080] (1) Take 5 mg / ml cyanoethyl cellulose nanomicrospheres suspension (prepared by emulsification-solvent evaporation method, particle size range 200-400 nm) and a certain amount of PVA124 solid, dissolve in water at 85℃ under magnetic stirring to prepare a 6% (w / v) solution, mix the cyanoethyl cellulose nanomicrospheres water suspension and the polyvinyl alcohol aqueous solution in a volume ratio of 1:1 at 37℃ to obtain an aqueous phase;

[0081] (2) Prepare 100 ml of 2% span80 and tween20 n-hexane solution as oil phase, add 20 ml of the aqueous phase obtained in step (1) to the oil phase, mechanically stir and emulsify and disperse at 50℃ and 850 rpm for 1 h to form a suspension;

[0082] (3) Add 3 ml of 50% glutaraldehyde aqueous solution, stir for 30 min; add 1M hydrochloric acid solution, continue to stir for 1 h to cross-link the microspheres;

[0083] (4) Cool the obtained suspension to 10℃, filter to obtain core-shell structure cyanoethyl cellulose-polyvinyl alcohol microspheres, and wash with petroleum ether, anhydrous ethanol and pure water respectively.

[0084] Example 2, preparation of hydroxyethyl cellulose-polyvinyl alcohol microspheres

[0085] (1) Take 20 mg / ml hydroxyethyl cellulose nanomicrospheres suspension (prepared by membrane emulsification method, particle size range 100-400 nm), take a certain amount of PVA124 solid, dissolve in water at 85℃ under magnetic stirring to prepare a 2% (w / v) solution, mix the cyanoethyl cellulose nanomicrospheres water suspension and the polyvinyl alcohol aqueous solution in a volume ratio of 1:5 at 37℃ to obtain an aqueous phase;

[0086] (2) Prepare 100 ml of 2% span80 n-hexane solution as oil phase, add 20 ml of the aqueous phase obtained in step (1) to the oil phase, mechanically stir and emulsify and disperse at 70℃ and 500 rpm for 30 min to form a suspension;

[0087] (3) 3 ml of 50% aqueous formaldehyde solution was added and stirred for 30 min; 1 M hydrochloric acid solution was added and stirred for 1 h to crosslink the microspheres;

[0088] (4) The obtained suspension was rapidly cooled to 20°C by pre-cooled organic solvent, and the cellulose-polyvinyl alcohol microspheres with core-shell structure were obtained by filtration, and were washed with petroleum ether, anhydrous ethanol and pure water respectively to obtain the cyanethyl cellulose-polyvinyl alcohol microspheres with core-shell structure.

[0089] Example 3, preparation of hydroxypropyl methyl cellulose-polyvinyl alcohol microspheres

[0090] (1) A certain amount of PVA124 solid was weighed and dissolved in water at 85°C to prepare a 3% (w / v) solution to obtain an aqueous phase;

[0091] (2) 100 ml of 2% span80 n-hexane solution was prepared as an oil phase, 20 ml of the aqueous phase obtained in step (1) was added to the oil phase, and mechanical stirring emulsification and dispersion was carried out at 30°C and 1200 rpm for 150 min to form a suspension;

[0092] (3) 3 ml of 50% aqueous formaldehyde solution was added and stirred for 30 min; 1 M hydrochloric acid solution was added and stirred for 1 h to crosslink the microspheres;

[0093] (4) The obtained suspension was rapidly cooled to 0°C by pre-cooled organic solvent, and the cellulose-polyvinyl alcohol microspheres with core-shell structure were obtained by filtration, and were washed with petroleum ether, anhydrous ethanol and pure water respectively to obtain the hydroxypropyl methyl cellulose-polyvinyl alcohol microspheres with core-shell structure.

[0094] Comparative Example 1: Preparation of polyvinyl alcohol microspheres

[0095] (1) A certain amount of PVA124 solid was weighed and dissolved in water at 85°C to prepare a 3% (w / v) solution to obtain an aqueous phase;

[0096] (2) 100 ml of 2% span80 and Tween20 n-hexane solution was prepared as an oil phase, 20 ml of the aqueous phase obtained in step (1) was added to the oil phase, and mechanical stirring emulsification and dispersion was carried out at 50°C and 850 rpm for 1 h to form a suspension;

[0097] (3) 3 ml of 50% aqueous formaldehyde solution was added and stirred for 30 min; 1 M hydrochloric acid solution was added and stirred for 1 h to crosslink the microspheres;

[0098] (4) The obtained suspension liquid is rapidly cooled to 10 DEG C by pre-cooled organic solvent, and polyvinyl alcohol microspheres are obtained by filtration, and are washed by petroleum ether, anhydrous ethanol and pure water respectively to obtain polyvinyl alcohol microspheres.

[0099] Performance test:

[0100] 1. Microsphere morphology test

[0101] The microspheres are soaked in water for 24 hours, and a certain amount of microspheres are placed on a glass slide, and the morphology of the microspheres is observed under a microscope, and the appropriate magnification is selected, and the particle size of the microspheres is counted to obtain the average particle size of the microspheres. The pore size of the microspheres is tested by a pore size tester. The results are shown in Table 1.

[0102] Table 1, test results of microspheres and pore sizes of different examples

[0103] Sample No. Particle size range (um) Pore size range (nm) Example 1 30-100 500-2000 Example 2 50-150 1000-2500 Example 3 50-200 100-1000 Comparative Example 1 30-150 800-1500 Agarose microspheres 45-160 20-35 Polystyrene synthetic resin microspheres 180-300 2-30

[0104] The microstructure test results of the microspheres show that the polyvinyl alcohol resin microspheres of Examples 1-3 have an average particle size in the range of 30-200 um, which can meet the use requirements in the field of blood purification compared with the commercially available agarose microspheres and polystyrene synthetic resin microspheres. The pore size of the polyvinyl alcohol resin microspheres reaches microns, which is significantly larger than the existing agarose microspheres and polystyrene synthetic resin microspheres, thereby being more conducive to the coupling of ligands of subsequent adsorbents and the improvement of adsorption performance.

[0105] The pore size is affected by multiple factors:

[0106] (1) Compared with agarose resin, the pore size of polyvinyl alcohol microspheres is more easily controlled in a stable range by process conditions.

[0107] (2) The cellulose-polyvinyl alcohol microspheres of the present application first form a core-shell structure, and are directly crosslinked without separation. In addition to the crosslinking and solidification of the hydroxyl groups inside the microspheres, the initially formed microspheres with smaller diameters also undergo different degrees of crosslinking between them, thereby resulting in the formation of microspheres with larger particle size and pore size.

[0108] (3) The microsphere suspension liquid formed by the scheme of the present application is rapidly reduced to a lower temperature for cooling and separation. The micron-level pore size formed during the emulsification and crosslinking process is more easily fixed and thus better retained.

[0109] 2. Mechanical property test of microspheres

[0110] The empty column tube is taken, and the two ends are blocked with a 300-mesh nylon screen. The liquid inlet is connected to a peristaltic pump. 2.5 ml of the above resin microspheres are filled into the column, and the microspheres are allowed to stand until the volume is constant. The height of the microspheres in the tube is measured and recorded as h0;

[0111] Plug the upper end with the lid, adjust the peristaltic pump flow rate to increase slowly to 20 ml / min constant flow rate to flush the adsorption column, when the microsphere volume is constant, record the microsphere height, repeat three times to take the average value, record as h1;

[0112] Calculate the volume ratio L = h1 / h0 before and after compression.

[0113] The test results are shown in Table 2.

[0114] Table 2, mechanical property test results of microspheres of different examples

[0115] Sample No. Compressed volume ratio L Example 1 5.2 Example 2 3.6 Example 3 6.8 Comparative Example 1 12.5 Agarose microspheres Sepharose 6FF 16.3 Polystyrene synthetic resin microspheres 2.5

[0116] As can be seen from Table 2, the mechanical strength of the polyvinyl alcohol microspheres reinforced by cellulose nanomicrospheres (Examples 1-3) is significantly improved, and the compression volume ratio under high flow rate is close to that of polystyrene synthetic resin microspheres, and is significantly higher than that of agarose resin microspheres. The compression resistance of the microspheres without cellulose nanomicrospheres reinforcement is improved compared with that of agarose microspheres, but is still lower than that of the reinforced microspheres.

[0117] 3. Hemolysis rate test:

[0118] 2 g of samples of Examples 1-3 and agarose microspheres were respectively weighed in test tubes, and 10 mL of physiological saline was injected as a test sample. The test sample tube, negative tube (only 10 mL of physiological saline was injected), and positive tube (only 10 mL of distilled water was injected) were placed in a 37℃ water bath for 30 min. 0.2 mL of diluted anticoagulant rabbit blood was added to the test sample tube, negative tube, and positive tube, and mixed gently. Continue to incubate in a 37℃ constant temperature water bath for 60 min. After the water bath is finished, pour out the liquid in the tube, centrifuge at 800 g for 5 min, and take the supernatant to the ultraviolet spectrophotometer, measure the absorbance value at 545 nm, and calculate the hemolysis rate (hemolysis rate should be ≤5%), the hemolysis rate calculation formula is as follows:

[0119]

[0120] In the formula: A is the absorbance of the test sample liquid; B is the absorbance of the negative control liquid; and C is the absorbance of the positive control liquid.

[0121] The test results are shown in Table 3.

[0122] Table 3, hemolysis rate test results of microspheres of different examples

[0123] Sample Name Sample 1 Sample 2 Sample 3 Average Hemolysis rate (%) Positive control 0.718 0.732 0.720 0.7233 / Negative control 0.015 0.020 0.025 0.0183 / Example 1 0.023 0.025 0.028 0.0253 0.99 Example 2 0.019 0.025 0.023 0.0223 0.56 Example 3 0.029 0.027 0.025 0.027 1.23 Comparative Example 1 0.019 0.023 0.019 0.020 0.24 Agarose microspheres 0.023 0.019 0.021 0.021 0.38

[0124] As can be seen from Table 3, the hemolysis rate of the polyvinyl alcohol microspheres synthesized by the method of the present application meets the standard requirement of ≤5%, and has good biocompatibility.

[0125] Preparation of microsphere adsorbent

[0126] The microspheres prepared in Examples 1-3 and Comparative Example 1, and the agarose microspheres were used to prepare the microsphere adsorbent by the method of Example 1 and Example 2 in CN110026166B.

[0127] S1) After rinsing with about 10 times the volume of water for injection, 100 mL of 1M NaOH solution, 30 mL of epoxy bromopropane and 70 mL of 1,4-butanediol diglycidyl ether and 0.2 g of sodium borohydride were added, stirred, and reacted at 30°C for 1.5 h. After the reaction was completed, a large amount of water for injection was used to rinse clean, and epoxy-activated resin microspheres were obtained;

[0128] S2) In a 500 mL reaction vessel, 100 mL of the above-mentioned activated resin microspheres and 150 mL of 0.1 mol / L borate buffer were added, the pH of the system was controlled at 7.5 to 8.5, 14 g of genetically engineered recombinant protein A was added, and the system was reacted at 37°C for 20 h. The reaction was stopped, the packing was rinsed with about 10 times the volume of water for injection, and after rinsing, 200 mL of 0.2M ethanolamine solution was added to block the unreacted epoxy groups, and the reaction was carried out at 20°C for 10 h. After the reaction was completed, a large amount of water for injection was used to rinse, and finally it was stored in a solution containing a preservative;

[0129] S3) 0.5 mL of the above-mentioned prepared adsorbent microspheres were taken in a 10 mL EP tube, 5 mL of human plasma was added, and the mixture was slowly shaken for 1 h at room temperature. After the reaction was completed, the reaction solution was poured into a disposable affinity chromatography column, rinsed with about 100 mL of equilibration buffer (PBS), then eluted with 40 mL of elution buffer (citric acid 2.1 g / L, NaCl 8.0 g / L), and the elution peak was collected and detected by ultraviolet detection of OD280.

[0130] The adsorption capacity (mg / mL) = [(OD280 / 1.38) x 40] / 0.5, and the adsorption capacity was determined as shown in Table 4.

[0131] Table 4, adsorption capacity test results of microsphere adsorbents of different examples

[0132] Sample No. Adsorption performance to IgG (mg / mL) Example 1 60 Example 2 58 Example 3 61 Comparative Example 1 56 Agarose microspheres 52

[0133] As can be seen from the results in Table 4, compared with the adsorbent prepared from agarose microspheres, the adsorbent prepared from nanocellulose-reinforced polyvinyl alcohol microspheres has a higher adsorption capacity for IgG. Both the internal cellulose and the external polyvinyl alcohol contain abundant hydroxyl groups, and the large pore size structure is conducive to the entry and coupling of protein A ligands, and increases the contact area and contact time of IgG with the adsorbent during the adsorption process, thereby further enhancing the adsorption capacity.

[0134] The above is a further detailed description of the present application, which cannot be considered as a limitation of the specific implementation of the present application. For those skilled in the art to which the present application belongs, simple deductions or replacements without departing from the concept of the present application are within the protection scope of the present application.

Claims

1. A core-shell structured polyvinyl alcohol microsphere, characterized in that, Its core is cellulose nanospheres and its shell is polyvinyl alcohol. The preparation method includes the following steps: S1) Cellulose nanospheres, polyvinyl alcohol, and water are mixed evenly to obtain a cellulose nanosphere suspension and a polyvinyl alcohol aqueous solution, wherein the mass concentration of the cellulose nanosphere suspension is 0.5-20 mg / ml, the mass concentration of the polyvinyl alcohol aqueous solution is 2-8%, and the mixing volume ratio of the cellulose nanosphere suspension and the polyvinyl alcohol aqueous solution is 1:(1-5), thus obtaining an aqueous phase; S2) Dissolve the emulsifier in a water-insoluble solvent to obtain the oil phase; S3) The aqueous phase is added dropwise to the oil phase, and the mixture is stirred to emulsify and disperse to obtain a suspension. The emulsification and dispersion temperature is 30-70℃. S4) Add a crosslinking agent and a catalyst to the suspension, stir and crosslink, and the reaction yields a crosslinked liquid. The catalyst is an acid. S5) The crosslinking solution is cooled and molded at 0-20℃, filtered, and washed to obtain core-shell structured polyvinyl alcohol microspheres.

2. The polyvinyl alcohol microspheres according to claim 1, characterized in that, The cellulose nanospheres have a particle size of 100–400 nm.

3. The polyvinyl alcohol microspheres according to claim 1, characterized in that, The polyvinyl alcohol microspheres have a particle size of 30–200 μm and a surface pore size of 0.10–2.5 μm.

4. The polyvinyl alcohol microspheres according to claim 1, characterized in that, The cellulose is selected from at least one of cyanoethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose.

5. The polyvinyl alcohol microspheres according to claim 1, characterized in that, The stirring speed is 500-1200 rpm.

6. The polyvinyl alcohol microspheres according to claim 1, characterized in that, The crosslinking agent is selected from at least one of formaldehyde, glutaraldehyde, and genipin; and / or The water-insoluble solvent is selected from at least one of n-hexane, liquid paraffin, petroleum ether, n-heptane, or n-octane.

7. An adsorbent obtained by covalently coupling a ligand onto polyvinyl alcohol microspheres according to any one of claims 1 to 6.

8. Application of polyvinyl alcohol microspheres, wherein the polyvinyl alcohol microspheres are as described in any one of claims 1 to 6, the application comprising: Preparation of blood purification materials; Preparation of protein purification materials; Preparation of drug carriers.