Preparation method and application of uricase-loaded blood purification adsorbent

Abstract

The invention relates to the technical field of adsorbents, in particular to a preparation method and application of a uricase-loaded blood purification adsorbent. The invention provides a preparation method of a uricase-loaded blood purification adsorbent. The preparation method comprises the following steps: S1, copolymerizing divinylbenzene and methyl acrylate to obtain PDVB-MA microspheres; s2, the PDVB-MA microspheres are hydrolyzed with a sodium hydroxide solution, and PDVB-AA microspheres are obtained after washing and drying; s3, adding the PDVB-AA microspheres into a mixed aqueous solution containing uricase and zinc nitrate hexahydrate, and shaking for chelating; centrifugally collecting and washing to obtain Zn < 2 + >-loaded microspheres; and S4, adding the Zn < 2 + >-loaded microspheres into a 2-methylimidazole aqueous solution, carrying out oscillation reaction, centrifuging, washing with water, and freeze-drying to obtain the Zn2 < + >-loaded microspheres. The uricase-loaded blood purification adsorbent prepared by the preparation method disclosed by the invention is good in thermal stability and excellent in purification and adsorption capacity.

Description

Technical Field

[0001] The present invention relates to the technical field of adsorbents, and particularly to a preparation method and application of a blood purification adsorbent loaded with uricase. Background Art

[0002] Uric acid is the end product of purine metabolism in the human body. Under normal circumstances, the production and excretion of uric acid in the body are in a dynamic balance state. However, when the production of uric acid in the body is excessive or the excretion is reduced, it will lead to an increase in the concentration of uric acid in the blood, thereby causing hyperuricemia. Hyperuricemia is an important risk factor for diseases such as gout and uric acid nephropathy, seriously affecting the quality of life of patients.

[0003] Uricase is an enzyme that can catalyze the rapid oxidation of uric acid. It can convert uric acid into allantoin, which is no longer reabsorbed by the renal tubules, thereby increasing the excretion of uric acid and reducing the content of uric acid in the plasma. Uricase has significant curative effects in the treatment of hyperuricemia, especially suitable for patients who cannot orally take uric acid production inhibitors. However, as an exogenous protein, uricase has high antigenicity and is prone to cause allergic reactions, which to a certain extent limits its clinical application.

[0004] Blood purification technology is a treatment method that removes pathogenic substances in the blood through extracorporeal circulation, and is widely used in fields such as uremia, drug poisoning, and autoimmune diseases. In recent years, with the development of materials science and bioengineering technology, blood purification adsorbents, as a new type of treatment means, have gradually attracted attention. Blood purification adsorbents can selectively remove specific substances in the blood, such as toxins and metabolites, through their unique adsorption properties, so as to achieve the treatment purpose.

[0005] The blood purification adsorbent loaded with uricase is a new type of treatment material that combines the catalytic action of uricase and the adsorption performance of blood purification adsorbents. By loading uricase on a carrier with specific adsorption properties, the catalytic activity of uricase can be maintained while improving its clearance efficiency of uric acid in the blood. This adsorbent not only has the direct catalytic action of uricase, but also can further increase the clearance amount of uric acid through the adsorption performance of the carrier, thus showing great potential in the treatment of hyperuricemia. However, the current loading efficiency of uricase on the carrier is generally low. In addition to the catalytic action of uricase, the adsorption performance of the carrier is also an important factor affecting the uric acid clearance efficiency of the adsorbent. However, the existing blood purification adsorbents loaded with uricase still need to be improved in terms of adsorption performance to meet the needs of clinical treatment. Based on this, the present invention provides a preparation method and application of a blood purification adsorbent loaded with uricase. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method and application of a blood purification adsorbent loaded with uricase, which has good thermal stability and excellent purification and adsorption capacity.

[0007] On the one hand, the present invention provides a blood purification adsorbent loaded with uricase, and the steps include:

[0008] S1. Copolymerize divinylbenzene and methyl acrylate to obtain PDVB-MA microspheres;

[0009] S2. Hydrolyze the PDVB-MA microspheres with a sodium hydroxide solution, wash and dry them to obtain PDVB-AA microspheres;

[0010] S3. Add the PDVB-AA microspheres to a mixed aqueous solution containing uricase and zinc nitrate hexahydrate, shake and chelate; after centrifugation and collection, wash to obtain microspheres loaded with Zn 2+ ;

[0011] S4. Add the microspheres loaded with Zn 2+ to an aqueous solution of 2-methylimidazole, shake and react, centrifuge, wash with water, and then freeze-dry to obtain the product.

[0012] Further, the preparation method of the PDVB-MA microspheres includes: uniformly mixing divinylbenzene, methyl acrylate, toluene, and azobisisobutyronitrile, adding an aqueous solution of polyvinyl alcohol and reacting, then filtering, washing successively with ethanol and water, and vacuum drying to constant weight to obtain the product.

[0013] Further, the reaction rotation speed is 200-300 rpm, the temperature is 65-75 °C, and the reaction time is 5-6 h.

[0014] Further, in step S2, the sodium hydroxide solution is an aqueous sodium hydroxide solution with a mass concentration of 5-10%, the hydrolysis temperature is 60-70 °C, and the hydrolysis time is 10-12 h.

[0015] Further, in step S3, the dosage ratio of the PDVB-AA microspheres, uricase, and zinc nitrate hexahydrate is (1-1.2) g: (0.5-0.6) g: (1.4-1.6) g.

[0016] Further, in step S3, the shaking temperature is 20-30 °C, the time is 120-140 min, and the stirring speed during shaking is 150-200 rpm.

[0017] Further, the weight ratio of the PDVB-AA microspheres to the 2-methylimidazole is (1-1.2): (3-4).

[0018] Further, in step S4, the shaking temperature is 20-30 °C, the time is 10-12 h, and the stirring speed during shaking is 100-140 rpm.

[0019] On the other hand, the present invention also provides a blood purification adsorbent loaded with uricase, which is prepared by the aforementioned method.

[0020] On the other hand, the blood purification adsorbent loaded with uricase and its preparation method in the present invention are applied as core reaction materials in the preparation of hemoperfusion cartridges, hemoadsorbents or plasma adsorbents.

[0021] The beneficial effects of the present invention are as follows:

[0022] In the preparation method of the present invention, PDVB-MA microspheres are obtained by copolymerizing divinylbenzene and methyl acrylate, and then converted into PDVB-AA microspheres containing carboxylic acid groups (-COOH) by hydrolysis with sodium hydroxide. This modification step not only increases the hydrophilicity of the microsphere surface, but the carboxylic acid groups also serve as chelating sites for metal ions. By forming stable metal-carboxylic acid complexes, a firm binding between uricase and the microsphere is achieved. This binding method not only improves the enzyme loading amount, but also enhances the stability of the enzyme on the microsphere surface, preventing the shedding and inactivation of the enzyme, and also enhancing the interaction between the microsphere and uricase, preventing the shedding and inactivation of the enzyme, thereby improving the enzyme loading efficiency and stability.

[0023] Based on the PDVB-AA microspheres, the present invention utilizes the chelating effect of carboxylic acid groups with Zn 2+ to achieve effective immobilization of uricase. Compared with other metal ions (such as Co 2+ and Fe 3+ ), it has significant advantages; on the one hand, Zn 2+ has a strong chelating ability with carboxylic acid groups and can form stable complexes; on the other hand, Zn 2+ has a promoting effect on the catalytic activity of uricase and will not inhibit the enzyme activity or cause enzyme inactivation; therefore, selecting Zn 2+ as the chelating metal ion is the key to achieving efficient enzyme immobilization, that is, the present invention realizes efficient enzyme immobilization by precisely controlling the type of metal ion and chelating conditions, while maintaining the catalytic activity of the enzyme.

[0024] To further improve the stability of the enzyme, the present invention constructs a layer of ZIF-8 (zeolitic imidazolate framework material) protective layer on the surface of the microspheres loaded with uricase. Among them, ZIF-8, as a porous material, has excellent chemical stability and mechanical strength. Constructing a ZIF-8 protective layer on the microsphere surface can effectively prevent enzyme leakage and interference from the external environment, thereby maintaining the catalytic activity and stability of the enzyme and extending the service life of the adsorbent. At the same time, the porous structure of ZIF-8 is also beneficial to the diffusion and adsorption of uric acid molecules, improving the uric acid clearance rate.

[0025] In summary, the blood purification adsorbent loaded with uricase and its preparation method proposed by the present invention achieve efficient loading and stable immobilization of uricase through an innovative synthesis strategy and precise control conditions. This adsorbent not only has excellent uric acid clearance rate and reusability, but also maintains good biocompatibility and thermal stability. Detailed Embodiments

[0026] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention.

[0027] Example 1

[0028] This example provides a blood purification adsorbent loaded with uricase, and the preparation steps include:

[0029] S1. Mix 80 mL of divinylbenzene, 20 mL of methyl acrylate, 100 mL of toluene, and 1 g of azobisisobutyronitrile evenly, add 200 mL of an aqueous solution of polyvinyl alcohol with a mass concentration of 4.5%, react at a rotation speed of 250 rpm and a temperature of 70 °C for 5.5 h, then filter, wash successively with ethanol and water, and vacuum dry at 60 °C until constant weight to obtain;

[0030] S2. Hydrolyze the PDVB-MA microspheres with an aqueous solution of sodium hydroxide with a mass concentration of 7% at a temperature of 65 °C for 11 h, wash with water until neutral, filter, and dry at 60 °C until constant weight to obtain PDVB-AA microspheres;

[0031] S3. Add 1.1 g of PDVB-AA microspheres to 55 mL of a mixed aqueous solution containing 0.55 g of uricase and 1.5 g of zinc nitrate hexahydrate, and shake and chelate at a temperature of 25 °C and a stirring speed of 170 rpm for 130 min; after centrifugal collection, wash with deionized water to obtain microspheres loaded with Zn 2+ ;

[0032] S4. Add the microspheres loaded with Zn 2+ to an aqueous solution of 2-methylimidazole with a concentration of 3.5 g / 100 mL, and shake and react at a temperature of 25 °C and a stirring speed of 120 rpm for 11 h. After centrifugation and washing with water, freeze-dry at -40 °C for 26 h to obtain the blood purification adsorbent loaded with uricase.

[0033] Example 2

[0034] This example provides a blood purification adsorbent loaded with uricase, and the preparation steps include:

[0035] S1. Mix 75 mL of divinylbenzene, 15 mL of methyl acrylate, 90 mL of toluene, and 0.8 g of azobisisobutyronitrile evenly. Add 200 mL of a 4% (by mass) aqueous solution of polyvinyl alcohol and react at a rotation speed of 200 rpm and a temperature of 65 °C for 5 h. Then filter, wash successively with ethanol and water, and vacuum dry at 60 °C until a constant weight is obtained.

[0036] S2. Hydrolyze the PDVB-MA microspheres with a 5% (by mass) aqueous solution of sodium hydroxide at 60 °C for 10 h, wash with water until neutral, filter, and dry at 60 °C until a constant weight is obtained to obtain PDVB-AA microspheres.

[0037] S3. Add 1 g of PDVB-AA microspheres to 50 mL of a mixed aqueous solution containing 0.5 g of uricase and 1.4 g of zinc nitrate hexahydrate, and oscillate and chelate at 20 °C and a stirring speed of 150 rpm for 120 min. After centrifugally collecting, wash with deionized water to obtain the microspheres loaded with Zn 2+ ;

[0038] S4. Add the microspheres loaded with Zn 2+ to an aqueous solution of 2-methylimidazole with a concentration of 3 g / 100 mL, and oscillate and react at 20 °C and a stirring speed of 100 rpm for 10 h. After centrifugation and washing with water, freeze-dry at -40 °C for 24 h to obtain the blood purification adsorbent loaded with uricase.

[0039] Example 3

[0040] This example provides a blood purification adsorbent loaded with uricase, and the preparation steps include:

[0041] S1. Mix 85 mL of divinylbenzene, 25 mL of methyl acrylate, 110 mL of toluene, and 1.2 g of azobisisobutyronitrile evenly. Add 200 mL of a 5% (by mass) aqueous solution of polyvinyl alcohol and react at a rotation speed of 300 rpm and a temperature of 75 °C for 6 h. Then filter, wash successively with ethanol and water, and vacuum dry at 60 °C until a constant weight is obtained.

[0042] S2. Hydrolyze the PDVB-MA microspheres with a 10% (by mass) aqueous solution of sodium hydroxide at 70 °C for 12 h, wash with water until neutral, filter, and dry at 60 °C until a constant weight is obtained to obtain PDVB-AA microspheres.

[0043] S3. Add 1.2 g of PDVB-AA microspheres to 60 mL of a mixed aqueous solution containing 0.6 g of uricase and 1.6 g of zinc nitrate hexahydrate, and oscillate and chelate at 30 °C and a stirring speed of 200 rpm for 140 min. After centrifugally collecting, wash with deionized water to obtain the microspheres loaded with Zn2+ microspheres;

[0044] S4. Add the microspheres loaded with Zn 2+ to an aqueous solution of 2-methylimidazole with a concentration of 4 g / 100 mL, and carry out an oscillating reaction at a temperature of 30 °C and a stirring speed of 140 rpm for 12 h. After centrifugation and washing with water, freeze-dry at -40 °C for 28 h to obtain the blood purification adsorbent loaded with uricase.

[0045] Example 4

[0046] This example provides a blood purification adsorbent loaded with uricase, and the preparation steps include:

[0047] S1. Mix 75 mL of divinylbenzene, 25 mL of methyl acrylate, 90 mL of toluene, and 1.2 g of azobisisobutyronitrile evenly, add 200 mL of an aqueous solution of polyvinyl alcohol with a mass concentration of 4%, react at a rotation speed of 300 rpm and a temperature of 65 °C for 6 h, then filter, wash successively with ethanol and water, and vacuum dry at 60 °C to constant weight to obtain;

[0048] S2. Hydrolyze the PDVB-MA microspheres with an aqueous solution of sodium hydroxide with a mass concentration of 5% at a temperature of 70 °C for 12 h, wash with water until neutral, filter, and dry at 60 °C to constant weight to obtain PDVB-AA microspheres;

[0049] S3. Add 1 g of PDVB-AA microspheres to 60 mL of a mixed aqueous solution containing 0.6 g of uricase and 1.4 g of zinc nitrate hexahydrate, and carry out an oscillating chelation at a temperature of 20 °C and a stirring speed of 200 rpm for 120 min; after centrifugation and collection, wash with deionized water to obtain the microspheres loaded with Zn 2+ ;

[0050] S4. Add the microspheres loaded with Zn 2+ to an aqueous solution of 2-methylimidazole with a concentration of 4 g / 100 mL, and carry out an oscillating reaction at a temperature of 20 °C and a stirring speed of 140 rpm for 10 h. After centrifugation and washing with water, freeze-dry at -40 °C for 28 h to obtain the blood purification adsorbent loaded with uricase.

[0051] Comparative Example 1

[0052] On the basis of Example 1, directly perform step S3 on the PDVB-MA microspheres prepared in step S1, and keep other steps and conditions the same as those in Example 1.

[0053] Comparative Example 2

[0054] Based on Example 1, step S4 was deleted, and no ZIF-8 protective layer was formed. Other steps and conditions were the same as those in Example 1, that is, the microspheres loaded with Zn 2+ obtained in step S3 were the adsorbents in Comparative Example 2.

[0055] Comparative Example 3

[0056] Based on Example 1, zinc nitrate hexahydrate in step S3 was replaced with an equimolar amount of cobalt nitrate hexahydrate, and other steps and conditions were the same as those in Example 1.

[0057] Comparative Example 4

[0058] Based on Example 1, zinc nitrate hexahydrate in step S3 was replaced with an equimolar amount of ferric nitrate nonahydrate, and other steps and conditions were the same as those in Example 1.

[0059] Comparative Example 5

[0060] Based on Example 1, zinc nitrate hexahydrate was not added in step S3, and other steps and conditions were the same as those in Example 1.

[0061] Comparative Example 6

[0062] Based on Example 1, uricase was not added in step S3, and other steps and conditions were the same as those in Example 1.

[0063] Comparative Example 7

[0064] Based on Example 1, 90 mL of divinylbenzene and 10 mL of methyl acrylate were used as raw materials in step S1, and other steps and conditions were the same as those in Example 1.

[0065] Comparative Example 8

[0066] Based on Example 1, the oscillation chelation time in step S3 was shortened to 60 min, and other steps and conditions were the same as those in Example 1.

[0067] Test Example: The blood purification adsorbents loaded with uricase prepared in the foregoing Examples 1-4 and Comparative Examples 1-8 were tested as follows.

[0068] 1. Thermal stability (placed at 37 °C for 7 days): According to the uric acid assay kit (uricase peroxidase coupling method) YYT1207-2013, the enzyme activity was tested, and the thermal stability of the adsorbent was characterized by the enzyme activity. The thermal stability retention rate (%) = (enzyme activity after treatment / initial enzyme activity) × 100%;

[0069] 2. Reusability: Use the unused adsorbent to measure the initial enzyme activity. Reuse the adsorbent in the uric acid determination reaction. Thoroughly wash the resin after each reaction to remove residues. After repeating 20 times, measure the enzyme activity again. Reusability (%) = (enzyme activity after the 20th use / initial enzyme activity) × 100%;

[0070] 3. Uric acid adsorption and clearance rate: Pack the adsorbent into a hemoperfusion cartridge and purify the blood simulant with a uric acid content of 5 mmol / L, and detect the uric acid adsorption and clearance rate;

[0071] The test results are shown in Table 1:

[0072] Table 1: Effect test

[0073]

[0074]

[0075] The above data show that in Comparative Example 1, due to the absence of carboxylic acid groups (-COOH) and the lack of Zn2+ chelating ability, the enzyme could hardly be loaded, resulting in poor thermal stability, low reusability, and a significant decrease in uric acid clearance rate, proving that carboxylic acid groups are the basis for metal chelation and enzyme immobilization. In Comparative Example 2, when the ZIF-8 protective layer was lacking, the enzyme was prone to leakage and inactivation, and the reusability and thermal stability were greatly reduced. The uric acid clearance rate was also lower than that of the example due to the decrease in enzyme activity. Comparative Examples 3 and 4 showed that the chelating ability of Zn2+ was significantly better than that of Co2+ and Fe3+. Co2+ inhibited the enzyme activity, while Fe3+ caused more serious enzyme inactivation, and both reduced biocompatibility. In Comparative Example 8, insufficient chelation led to a decrease in enzyme loading, and the uric acid clearance rate and reusability were also significantly lower than those of Example 1. In Comparative Example 7, too high a proportion of divinylbenzene led to a decrease in the porosity of the microspheres, limited enzyme loading, a uric acid clearance rate of only 32%, and poor reusability and thermal stability. Comparative Examples 5 and 6 showed that the physically adsorbed enzyme was prone to shedding, and without the enzyme, only physical adsorption was relied on, and the uric acid clearance rate was only 11%.

[0076] As used in the specification and claims, certain terms are used to refer to specific components. Those skilled in the art should understand that hardware manufacturers may use different terms to refer to the same component. The specification and claims do not distinguish components by the difference in names, but by the difference in functions. As used throughout the specification and claims, the terms "comprising" and "including" are open-ended terms and should be interpreted as "comprising / including but not limited to". "Substantially" means within an acceptable error range. Those skilled in the art can solve the technical problems within a certain error range and basically achieve the technical effects. The following description in the specification is a preferred embodiment for implementing the present application, but the description is for the purpose of explaining the general principles of the present application and is not intended to limit the scope of the present application. The protection scope of the present application shall be subject to what is defined by the appended claims.

[0077] It should also be noted that the term "including", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that a commodity or system including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such commodity or system. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of another identical element in the commodity or system including the said element.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and not to limit the technical solutions described in the present invention; those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing a blood purification adsorbent loaded with uricase, characterized in that the steps include: S1, copolymerizing divinylbenzene and methyl acrylate to obtain PDVB-MA microspheres; S2, hydrolyzing the PDVB-MA microspheres with a sodium hydroxide solution, washing and drying to obtain PDVB-AA microspheres; S3, adding PDVB-AA microspheres to a mixed aqueous solution containing uricase and zinc nitrate hexahydrate, shaking for chelation; collecting by centrifugation and washing to obtain loaded Zn 2+ of microspheres; S4, load Zn 2+ The microspheres are added to a 2-methylimidazole aqueous solution, shaken for reaction, centrifuged, washed with water and freeze-dried to obtain the microspheres.

2. The method for preparing a uricase-loaded blood purification adsorbent according to claim 1, characterized in that: The preparation method of the PDVB-MA microspheres comprises: uniformly mixing divinylbenzene, methyl acrylate, toluene and azobisisobutyronitrile, adding polyvinyl alcohol aqueous solution for reaction, filtering, washing with ethanol and water in sequence, and vacuum drying to constant weight to obtain the microspheres.

3. The method for preparing a uricase-loaded blood purification adsorbent according to claim 2, characterized in that: The usage ratio of divinylbenzene, methyl acrylate, toluene and azobisisobutyronitrile is (75-85) mL: (15-25) mL: (90-110) mL: (0.8-1.2) g.

4. The method for preparing a uricase-loaded blood purification adsorbent according to claim 1, characterized in that: The sodium hydroxide solution in step S2 is a sodium hydroxide aqueous solution with a mass concentration of 5-10%, a hydrolysis temperature of 60-70° C., and a hydrolysis time of 10-12 hours.

5. The method for preparing a uricase-loaded blood purification adsorbent according to claim 1, characterized in that: In the step S3, the usage ratio of PDVB-AA microspheres, uricase and zinc nitrate hexahydrate is (1-1.2) g: (0.5-0.6) g: (1.4-1.6) g.

6. The method for preparing a uricase-loaded blood purification adsorbent according to claim 1, characterized in that: In step S3, the shaking temperature is 20-30° C., the time is 120-140 min, and the stirring speed during shaking is 150-200 rpm.

7. The method for preparing a uricase-loaded blood purification adsorbent according to claim 1, characterized in that: The weight ratio of the PDVB-AA microspheres to the 2-methylimidazole is (1-1.2):(3-4).

8. The method for preparing a uricase-loaded blood purification adsorbent according to claim 1, characterized in that: In step S4, the shaking temperature is 20-30° C., the time is 10-12 h, and the stirring speed during shaking is 100-140 rpm.

9. A blood purification adsorbent loaded with uricase, characterized in that: The method is prepared by any one of claims 1 to 8.

10. A method for preparing a uricase-loaded blood purification adsorbent according to any one of claims 1 to 8 or use of the uricase-loaded blood purification adsorbent according to claim 9 as a core reaction material in the preparation of a hemoperfusion device, a hemoadsorber or a plasmaadsorber.

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