Coenzyme regeneration material and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a coenzyme regeneration material and a preparation method thereof. The coenzyme regeneration material comprises a ZIF metal organic framework; the glucose dehydrogenase is connected with metal ions in the ZIF metal organic framework through electrostatic adsorption, and the glucose dehydrogenase is wrapped by the ZIF metal organic framework. The coenzyme regeneration material can efficiently realize regeneration of NADH (Nicotinamide Adenine Dinucleotide) in peritoneal dialysis liquid in the portable artificial kidney device through a biotransformation method, so that the cost is reduced, the dosage of an adsorbent and the volume weight of the device are reduced, and the treatment efficiency of urea is improved; meanwhile, the ZIF metal organic framework is used as a carrier, so that the stability of the coenzyme regeneration material is also improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a coenzyme regeneration material and a preparation method thereof. Background Art

[0002] Currently, most systems that meet the requirements of wearable artificial kidney (WAK) are REcirculating DialYsis sorbent system (REDY) filters, which mainly remove urea-based toxins. Currently, REDY filters use glutamate dehydrogenase to convert NH4 + Technology for glutamate to reduce adsorbent consumption and Na+ and H + Leakage, but the disadvantage is that it needs to consume NADH for the reaction.

[0003] NADH is a commonly used coenzyme in the human body. There is a series of coenzyme regeneration systems in the body that can convert NAD + Reduction to NADH realizes the recycling of NADH and promotes the forward progress of the reversible reaction. At present, a variety of coenzyme regeneration routes have been constructed by imitating the reaction system in the body, and are used in chemical synthesis, materials and other fields. Common coenzyme regeneration methods include enzyme catalysis and photoregeneration. However, the regeneration efficiency of the coenzyme NADH (nicotinamide adenine dinucleotide) is currently low. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the present invention provides a coenzyme regeneration material, which can efficiently realize the regeneration of NADH in the peritoneal dialysis fluid in the portable artificial kidney device through a bioconversion method, reduce the cost, reduce the amount of adsorbent and the volume and weight of the device, and improve the treatment efficiency of urea; at the same time, the ZIF metal organic framework as a carrier also improves the stability of the coenzyme regeneration material.

[0005] In one aspect of the present invention, the present invention provides a coenzyme regeneration material, the coenzyme regeneration material comprising: ZIF metal-organic framework; Glucose dehydrogenase, wherein the glucose dehydrogenase is connected to the metal ions in the ZIF metal organic framework through electrostatic adsorption, and the ZIF metal organic framework encapsulates the glucose dehydrogenase.

[0006] Furthermore, the ZIF metal organic framework includes MAF-7 or ZIF-8 or ZIF-90.

[0007] Further, based on the total mass of the coenzyme regeneration material, the size of the coenzyme regeneration material is 1-2 μm.

[0008] In another aspect of the present invention, the present invention provides a method for preparing the coenzyme regeneration material as described above, the preparation method comprising the following steps: The ZIF metal organic framework encapsulates glucose dehydrogenase, and the glucose dehydrogenase is connected to the metal ions in the ZIF metal organic framework through chemical bonds.

[0009] Furthermore, the preparation method comprises the following steps: S1. Adding glucose dehydrogenase and aqueous ammonia to Hmtz solution, 2-methylimidazole solution or 2-formylimidazole solution to obtain a mixed solution; S2, adding the mixed solution dropwise into the zinc salt solution, and reacting under stirring; S3, performing solid-liquid separation on the product obtained after the reaction in step S2; S4. freeze-drying the solid obtained after the solid-liquid separation to obtain the coenzyme regeneration material.

[0010] Furthermore, in the mixed solution, the concentration of glucose dehydrogenase is 0.17-0.67 g / L.

[0011] Furthermore, in the mixed solution, the concentration of glucose dehydrogenase is 0.3-0.5 g / L.

[0012] Furthermore, in step S3, the solid-liquid separation includes a first centrifugal treatment, and the rotation speed of the first centrifugal treatment is 9000-11000 rpm and the time is 5-10 min.

[0013] Furthermore, in step S4, before freeze drying, the solid obtained after the solid-liquid separation is cleaned, and the cleaning process includes: S41, dispersing the solid obtained after the solid-liquid separation in water and subjecting it to ultrasonic treatment for 5 to 10 min to obtain a suspension solution; S42, subjecting the suspension solution to a second centrifugation treatment; The solid obtained by the second centrifugation treatment is dispersed in water and steps S41 and S42 are repeated.

[0014] Furthermore, the freeze-drying is carried out under vacuum conditions at a temperature of -60 to -40°C for 12 to 24 hours.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The coenzyme regeneration material of the present invention utilizes glucose dehydrogenase to convert NAD +By converting it into NADH, NADH in the peritoneal dialysis fluid of a portable artificial kidney device can be efficiently regenerated through the method of bioconversion. It has high regeneration efficiency and good biological and blood compatibility, which is conducive to reducing costs, reducing the amount of adsorbent and the volume and weight of the device, and improving the treatment efficiency of urea. At the same time, the ZIF metal organic framework as a carrier also improves the stability of the coenzyme regeneration material, so that the coenzyme regeneration material has good thermal stability and pH stability, can be stored for a long time, has good biocompatibility, and can further optimize the WAK device.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is the result of measuring the coenzyme regeneration efficiency in Experimental Example 1 of the present invention.

[0019] Figure 2 This is the result of measuring the coenzyme regeneration efficiency of GL@MAF-7-2 in the patient's peritoneal dialysis fluid in Experimental Example 2 of the present invention.

[0020] Figure 3 This is the biocompatibility evaluation result in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0022] The terms “first”, “second” and “third” are used for descriptive purposes only and should not be understood as indicating or implying relative importance.

[0023] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0024] In one aspect of the present invention, the present invention provides a coenzyme regeneration material, which includes: a ZIF metal-organic framework; glucose dehydrogenase, wherein the glucose dehydrogenase is connected to the metal ions in the ZIF metal-organic framework through electrostatic adsorption, and the ZIF metal-organic framework encapsulates the glucose dehydrogenase.

[0025] It should be noted that glucose dehydrogenase is connected to the metal ions in the ZIF metal-organic framework through electrostatic adsorption, and there is also a coordination effect between glucose dehydrogenase and the metal ions in the ZIF metal-organic framework.

[0026] It should be noted that the metal ions in the ZIF metal organic framework include zinc ions and / or zinc ion clusters, and the organic ligands include imidazole ligands.

[0027] The coenzyme regeneration material of the present invention utilizes glucose dehydrogenase to convert NAD + By converting it into NADH, NADH in the peritoneal dialysis fluid of a portable artificial kidney device can be efficiently regenerated through the method of bioconversion. It has high regeneration efficiency and good biological and blood compatibility, which is conducive to reducing costs, reducing the amount of adsorbent and the volume and weight of the device, and improving the treatment efficiency of urea. At the same time, the ZIF metal organic framework as a carrier also improves the stability of the coenzyme regeneration material, so that the coenzyme regeneration material has good thermal stability and pH stability, can be stored for a long time, has good biocompatibility, and can further optimize the WAK device.

[0028] It is understood that in the portable artificial kidney device, urea in the peritoneal dialysis fluid is decomposed into NH4 + After that, NADH is consumed and converted into glutamate to remove urea, which further improves the efficiency of urea removal and reduces costs. The coenzyme regeneration material of the present invention contains glucose dehydrogenase, which can convert the generated NAD + Reducing to NADH, placing the coenzyme regeneration material of the present invention in a portable artificial kidney device can achieve the cyclic regeneration of NADH.

[0029] In some embodiments of the present invention, the ZIF metal organic framework includes MAF-7 or ZIF-8 or ZIF-90. MAF-7 is a hydrophilic MOF (metal organic framework) material with a dodecahedral structure and a diameter of about 2 μm.

[0030] In some embodiments of the present invention, the size of the coenzyme regeneration material is 1-2 μm based on the total mass of the coenzyme regeneration material.

[0031] In another aspect of the present invention, the present invention provides a method for preparing the coenzyme regeneration material as described above, the preparation method comprising the following steps: encapsulating glucose dehydrogenase in a ZIF metal-organic framework, and connecting the glucose dehydrogenase to the metal ions in the ZIF metal-organic framework through chemical bonds.

[0032] In some embodiments of the present invention, the preparation method comprises the following steps: S1, adding glucose dehydrogenase and ammonia water to Hmtz (3-methyl-1,2,4-triazole) solution or 2-methylimidazole solution or 2-formylimidazole solution to obtain a mixed solution; S2, adding the mixed solution dropwise to a zinc salt solution, and reacting under stirring; S3, performing solid-liquid separation on the product obtained after the reaction in step S2; S4, freeze-drying the solid obtained after the solid-liquid separation to obtain the coenzyme regeneration material. Thus, the preparation method is simple, convenient, and easy to implement. Glucose dehydrogenase is added during the synthesis process so that it is loaded into the ZIF metal organic framework during in-situ synthesis to achieve biomimetic mineralization of glucose dehydrogenase.

[0033] In some embodiments of the present invention, in the mixed solution, the concentration of the glucose dehydrogenase is 0.17-0.67 g / L. In some preferred embodiments of the present invention, in the mixed solution, the concentration of the glucose dehydrogenase is 0.3-0.5 g / L.

[0034] In some embodiments of the present invention, in step S3, the solid-liquid separation includes a first centrifugal treatment, and the rotation speed of the first centrifugal treatment is 9000-11000 rpm (revolutions / minute) and the time is 5-10 min.

[0035] In some embodiments of the present invention, in step S4, before freeze-drying, the solid obtained after the solid-liquid separation is cleaned, and the cleaning treatment includes: S41, dispersing the solid obtained after the solid-liquid separation in water and performing ultrasonic treatment for 5 to 10 minutes to obtain a suspension solution; S42, performing a second centrifugation treatment on the suspension solution; dispersing the solid obtained by the second centrifugation treatment in water and repeating steps S41 and S42.

[0036] In some embodiments of the present invention, the freeze-drying is carried out under vacuum conditions at a temperature of -60 to -40°C for 12 to 24 hours.

[0037] In some specific embodiments of the present invention, the method for preparing the coenzyme regeneration material comprises the following steps: (1) Weigh a certain amount of Zn(NO3)2·6H2O and dissolve it in water; (2) Weigh a certain amount of Hmtz or 2-methylimidazole or 2-formylimidazole and dissolve it in water; (3) adding a certain amount of glucose dehydrogenase to the solution obtained in step (2), and adding a small amount of ammonia water, and mixing; (4) adding the mixed solution obtained in step (3) dropwise to the solution obtained in step (1), stirring at room temperature for 24 h; (5) subjecting the product obtained in step (4) to high-speed centrifugation and discarding the supernatant; (6) adding a certain amount of water to the solid obtained in step (5) to resuspend the precipitate, ultrasonically clean the mixture, centrifuge it again at high speed, discard the supernatant, and repeat the operation three times; (7) The obtained precipitate is freeze-dried to obtain the coenzyme regeneration material.

[0038] In some specific embodiments of the present invention, glucose dehydrogenase is added during the synthesis of MAF-7, so that glucose dehydrogenase is combined with zinc ions through electrostatic interaction, and a process of combining Hmtz with zinc ions as the center is accompanied, thereby preparing an immobilized enzyme material. It can efficiently achieve coenzyme regeneration through a bioconversion method, and the MAF-7 carrier also improves the stability of the coenzyme regeneration material.

[0039] In some embodiments of the present invention, the present invention is that urease decomposes urea into NH4 + , while glutamate dehydrogenase consumes NADH to convert NH4 + Glucose dehydrogenase is added to further convert it into glutamate to achieve NAD + The coenzyme regeneration material can realize the efficient regeneration of NADH in the portable artificial kidney device, further improve the efficiency of urea removal and reduce the cost. In addition, the coenzyme regeneration material loaded with glucose dehydrogenase has good stability, good thermal stability and pH stability, can be stored for a long time, has good biocompatibility, and can further optimize the WAK device.

[0040] The present invention is further described below in conjunction with specific examples. It should be noted that the following examples are only used to explain the present invention and are not to be construed as limiting the present invention.

[0041] Example Example 1 The preparation method of the coenzyme regeneration material comprises the following steps: Weigh 0.357 g of Zn(NO3)2·6H2O and dissolve it in 15 mL of distilled water for later use; weigh 0.299 g of Hmtz and dissolve it in 10 mL of distilled water, add 5 mg of glucose dehydrogenase, and then add 600 μL of 10% ammonia water, mix well, and make up to 15 mL; add the above mixed solution dropwise to the Zn(NO3)2 solution, stirring while adding and stirring at room temperature for 24 h. Then, the mixture was centrifuged at 9000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 30 mL of distilled water, ultrasonically treated for 10 min, and then centrifuged at 9000 rpm for 10 min again, the supernatant was discarded, and the washing was repeated 3 times. The obtained precipitate was freeze-dried overnight to obtain the coenzyme regeneration material, which was labeled as GL@MAF-7-1.

[0042] Example 2 The preparation method of the coenzyme regeneration material comprises the following steps: Weigh 0.357 g of Zn(NO3)2·6H2O and dissolve it in 15 mL of distilled water for later use; weigh 0.299 g of Hmtz and dissolve it in 10 mL of distilled water, add 10 mg of glucose dehydrogenase, and then add 600 μL of 10% ammonia water, mix well, and make up to 15 mL; add the above mixed solution dropwise to the Zn(NO3)2 solution, stirring while adding and stirring at room temperature for 24 h. Then, the mixed solution was centrifuged at 10000 rpm for 6 min, the supernatant was discarded, the precipitate was resuspended in 30 mL of distilled water, ultrasonically treated for 6 min, and then centrifuged at 10000 rpm for 6 min again, the supernatant was discarded, and the washing was repeated 3 times. The obtained precipitate was freeze-dried overnight to obtain the coenzyme regeneration material, which was labeled as GL@MAF-7-2.

[0043] Example 3 The preparation method of the coenzyme regeneration material comprises the following steps: Weigh 0.357 g of Zn(NO3)2·6H2O and dissolve it in 15 mL of distilled water for later use; weigh 0.299 g of Hmtz and dissolve it in 10 mL of distilled water, add 20 mg of glucose dehydrogenase, and then add 600 μL of 10% ammonia water, mix well, and make up to 15 mL; add the above mixed solution dropwise to the Zn(NO3)2 solution, stirring while adding and stirring at room temperature for 24 h. Then, the mixture was centrifuged at 11000 rpm for 5 min, the supernatant was discarded, the precipitate was resuspended in 30 mL of distilled water, ultrasonically treated for 5 min, and then centrifuged at 11000 rpm for 5 min again, the supernatant was discarded, and the washing was repeated 3 times. The obtained precipitate was freeze-dried overnight to obtain the coenzyme regeneration material, which was labeled as GL@MAF-7-3.

[0044] Comparative Example 1 The preparation method of MAF-7 comprises the following steps: Weigh 0.357 g of Zn(NO3)2·6H2O and dissolve it in 15 mL of distilled water for later use; weigh 0.299 g of Hmtz and dissolve it in 10 mL of distilled water, add 600 μL of 10% ammonia water, mix well, and make up to 15 mL; add the above mixed solution dropwise to the Zn(NO3)2 solution, stirring while adding, and stir at room temperature for 24 h. Then, centrifuge the mixture at 10000 rpm for 5 min, discard the supernatant, resuspend the precipitate in 30 mL of distilled water, ultrasonically treat for 5 min, centrifuge again at 10000 rpm for 5 min, discard the supernatant, and repeat the washing 3 times. The obtained precipitate was freeze-dried overnight to obtain MAF-7.

[0045] Experimental Example 1 Coenzyme regeneration efficiency determination Weigh 0.01 g of each coenzyme regeneration material in Examples 1-3 above into a centrifuge tube, add 10 mL NAD + and 50 mmol / L glucose buffer, mix well, cover tightly, seal with sealing film, place in air shaker, shake at 37°C for 5, 10, 20, 30, 60, 120 min. Then centrifuge at 10,000 rpm for 5 min, take the supernatant to detect NADH regeneration rate at different reaction time periods, the results are as follows Figure 1 shown.

[0046] Experimental Example 2 Determination of the efficiency of coenzyme regeneration in peritoneal dialysis fluid of patients Weigh 0.01 g of GL@MAF-7-2 material in Example 2 into a centrifuge tube and add 10 mL of NAD +and 50 mmol / L glucose concentration of the patient's peritoneal dialysis fluid, mix well, cover tightly, seal with sealing film, place in an air shaker, and shake at 37°C for 5, 10, 20, 30, 60, and 120 min. Then take out and centrifuge at 10,000 rpm for 5 min, take the supernatant to detect the NADH regeneration rate at different reaction time periods, the results are as follows Figure 2 shown.

[0047] Experimental Example 3 Biocompatibility evaluation (1) Cell culture: Human renal epithelial 293T cells were cultured in DMEM complete medium (containing DMEM basal medium, 10% fetal bovine serum (FBS) and 1% double antibody) at 37°C in a 5% CO2 cell culture incubator for 4-5 generations. Cell viability and status were stabilized before plating. 293T cells were seeded in a 96-well plate at a density of 1000 cells per well, and 100 μL of DMEM complete medium was added to each well and cultured for 24 h until the cell status was stable.

[0048] (2) Replace DMEM complete medium with different conditioned medium: A: Positive control: add 100 μL DMEM complete medium; B: Negative control: add 100 μL of DMEM complete medium containing 5% dimethyl sulfoxide (DMSO); C: Experimental group 1: 100 μL of DMEM complete medium containing 1 mg of MAF-7 material was added; D: Experimental group 2: Add 100 μL of DMEM complete medium containing 1 mg of GL@MAF-7-2 material.

[0049] (3) After replacing the culture medium, the cells were cultured in a cell culture incubator for 72 h.

[0050] (4) Detect the cytotoxicity of the material using a cell proliferation-toxicity detection kit (Cell Counting Kit-8, CCK-8): After taking out the 96-well plate, the culture medium was aspirated, and 100 μL of DMEM basal culture medium containing 10% CCK-8 solution was added to each well. After incubation in a cell culture incubator for 3 h, the absorbance at 450 nm was detected using an ELISA reader.

[0051] (5) Calculate the relative proliferation rate (RGR) of 293T cells according to the following formula: RGR =X / X0×100% In the above formula, X is the absorbance value of each experimental group, and X0 is the absorbance value of the negative control group.

[0052] The evaluation criteria for cytotoxicity are: the cytotoxicity level of the positive control group is at least level 3 to ensure the accuracy of the experimental operation. If the cytotoxicity level of the positive control group is less than level 3, it needs to be retested to ensure the accuracy of the experiment. The cytotoxicity grading table is shown in Table 1. The results are shown in Figure 3 shown.

[0053] Table 1. Cytotoxicity grading table

[0054] Depend on Figure 3 It can be seen that the relative cell proliferation rate of the positive control group is 23.13%, and the toxicity level is level 4, which meets the standard that the toxicity level of the positive control group exceeds level 3. The cell activity of the MAF-7 and GL@MAF-7 groups is above 95%, and there is no significant difference from the negative control group. Therefore, it can be seen from the figure that the toxicity level of the three materials is less than or equal to level 1, which meets the cytotoxicity of biomedical materials.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A coenzyme regeneration material, characterized in that: include: ZIF metal-organic framework; Glucose dehydrogenase, wherein the glucose dehydrogenase is connected to the metal ions in the ZIF metal organic framework through electrostatic adsorption, and the ZIF metal organic framework encapsulates the glucose dehydrogenase.

2. The coenzyme regeneration material according to claim 1, characterized in that: The ZIF metal organic framework includes MAF-7 or ZIF-8 or ZIF-90.

3. The coenzyme regeneration material according to claim 1, characterized in that: The size of the coenzyme regeneration material is 1-2 μm based on the total mass of the coenzyme regeneration material.

4. A method for preparing the coenzyme regeneration material according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: The ZIF metal organic framework encapsulates glucose dehydrogenase, and the glucose dehydrogenase is connected to the metal ions in the ZIF metal organic framework through chemical bonds.

5. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: S1. Adding glucose dehydrogenase and aqueous ammonia to Hmtz solution, 2-methylimidazole solution or 2-formylimidazole solution to obtain a mixed solution; S2, adding the mixed solution dropwise into the zinc salt solution, and reacting under stirring; S3, performing solid-liquid separation on the product obtained after the reaction in step S2; S4. freeze-drying the solid obtained after the solid-liquid separation to obtain the coenzyme regeneration material.

6. The preparation method according to claim 5, characterized in that: In the mixed solution, the concentration of glucose dehydrogenase is 0.17-0.67 g / L.

7. The preparation method according to claim 5, characterized in that: In the mixed solution, the concentration of glucose dehydrogenase is 0.3-0.5 g / L.

8. The preparation method according to claim 5, characterized in that: In step S3, the solid-liquid separation includes a first centrifugal treatment, wherein the rotation speed of the first centrifugal treatment is 9000-11000 rpm and the time is 5-10 min.

9. The preparation method according to claim 5, characterized in that: In step S4, before freeze drying, the solid obtained after the solid-liquid separation is cleaned, and the cleaning process includes: S41, dispersing the solid obtained after the solid-liquid separation in water and subjecting it to ultrasonic treatment for 5 to 10 min to obtain a suspension solution; S42, subjecting the suspension solution to a second centrifugation treatment; The solid obtained by the second centrifugation treatment is dispersed in water and steps S41 and S42 are repeated.

10. The preparation method according to claim 5, characterized in that: The freeze drying is carried out under vacuum conditions at a temperature of -60 to -40°C for 12 to 24 hours.

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