Urea bioconversion removing material and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a urea bioconversion removing material and a preparation method thereof. The urea bioconversion scavenging material includes: a ZIF metal organic framework; the urease is connected with metal ions in the ZIF metal organic framework through electrostatic adsorption, and the urease is wrapped by the ZIF metal organic framework. The urea biotransformation removal material can efficiently remove urea in peritoneal dialysis liquid through a biotransformation method, almost has no toxic or side effect on a human body, meanwhile, the ZIF metal organic framework serving as a carrier also enables urease to have higher stability and bioavailability, and the urea biotransformation removal material can be used for removing urea in peritoneal dialysis liquid or blood in a portable artificial kidney and has good application prospects. And the cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a urea bioconversion removal material and a preparation method thereof. Background Art

[0002] The wearable artificial kidney (WAK), or portable artificial kidney, has a complete dialysis device and process, but is smaller than the traditional dialysis device, similar to a backpack, and can be worn on the body to achieve efficient and continuous removal of uremic toxins. Currently, most systems that meet the requirements of WAK are REcirculating DialYsis sorbent system (REDY) filters.

[0003] Due to factors such as decreased glomerular filtration rate and renal failure, toxins mainly composed of urea cannot be excreted from the body and accumulate in the body. When the concentration of urea is too high, reaching 20-30 mmol / L, it indicates the occurrence of end-stage kidney disease (ESKD). In clinical treatment, the urea clearance index (Kt / V) is a key indicator that can be used to assess whether peritoneal dialysis is sufficient. However, as a small molecule with high hydrophilicity, urea is difficult to remove using adsorbents and other methods, such as amino-functionalized adsorbents (amined SiO2), modified chitosan, and aldehyde-functionalized adsorbents. Therefore, the removal of urea in peritoneal dialysis fluid is extremely important.

[0004] The toxin removal part of the REDY filter is mainly composed of activated carbon, urease and zirconium oxide zirconium phosphate ion exchanger. First, the peritoneal dialysis fluid passes through the activated carbon part to remove most of the non-urea organic compounds; then urease decomposes urea into carbonate and ammonium ions, and then passes through the zirconium oxide zirconium phosphate ion exchanger to exchange the ammonium ions for Na + and H + , in order to achieve the purpose of removing urea. However, urease was initially loaded with materials such as alumina, which would lead to aluminum leakage and produce more serious toxic side effects on the body. In addition, there are also disadvantages such as low enzyme bioavailability and high cost. Summary of the invention

[0005] 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 urea bioconversion removal material, which can efficiently remove urea in peritoneal dialysis fluid by bioconversion, and has almost no toxic side effects on the human body. At the same time, the ZIF metal organic framework as a carrier also makes urease have higher stability and bioavailability, which can be used for urea removal in peritoneal dialysis fluid or blood in portable artificial kidneys, which is conducive to reducing costs.

[0006] In one aspect of the present invention, the present invention provides a urea bioconversion removal material, the urea bioconversion removal material comprising: ZIF metal-organic framework; Urease, the urease is connected to the metal ions in the ZIF metal-organic framework through electrostatic adsorption, and the ZIF metal-organic framework encapsulates the urease.

[0007] Furthermore, the ZIF metal organic framework includes MAF-7 or ZIF-8 or ZIF-90.

[0008] Further, based on the total mass of the urea bioconversion removal material, the particle size of the urea bioconversion removal material is 1-2 μm.

[0009] In another aspect of the present invention, the present invention provides a method for preparing the urea bioconversion removal material as described above, the preparation method comprising the following steps: The ZIF metal organic framework encapsulates the urease, and the urease is connected to the metal ions in the ZIF metal organic framework through electrostatic adsorption.

[0010] Furthermore, the preparation method comprises the following steps: S1, adding urease and ammonia water to the Hmtz 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 urea bioconversion removal material.

[0011] Furthermore, in the mixed solution, the concentration of urease is 0.33~1 g / L.

[0012] Furthermore, in the mixed solution, the concentration of urease is 0.5-0.7 g / L.

[0013] 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.

[0014] 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.

[0015] Furthermore, the freeze-drying is carried out under vacuum conditions at a temperature of -60 to -40°C for 12 to 24 hours.

[0016] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: In the urea bioconversion removal material of the present invention, urease can decompose urea and then remove urea, and the ZIF metal organic framework is used as a carrier to load urease. The urea bioconversion removal material has good enzyme stability, high specificity, good thermal stability and pH stability, can be stored for a long time, and has good biological and blood compatibility, providing a new strategy and method for removing excess urea in dialysate. The urea bioconversion removal material can achieve high-efficiency removal of peritoneal dialysis fluid urea in a portable artificial kidney device, has high enzyme bioavailability, and solves the problems of large volume and weight in the REDY system.

[0017] In addition, 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

[0018] 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.

[0019] Figure 1 These are the results of the removal of urea in the buffer by various urea bioconversion removal materials in Experimental Example 1 of the present invention.

[0020] Figure 2 This is the result of the clearance of urea from the patient's peritoneal dialysis fluid by U@MAF-7-2 in Experimental Example 2 of the present invention.

[0021] Figure 3 This is the biocompatibility evaluation result in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] The terms “first”, “second” and “third” are used for descriptive purposes only and should not be understood as indicating or implying relative importance.

[0024] 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.

[0025] In one aspect of the present invention, the present invention provides a urea bioconversion removal material, the urea bioconversion removal material comprising: ZIF metal-organic framework; Urease, the urease is connected to the metal ions in the ZIF metal-organic framework through electrostatic adsorption, and the ZIF metal-organic framework encapsulates the urease.

[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. Urease and metal ions are connected together through electrostatic interaction, and there is also a certain coordination effect between urease and metal ions.

[0027] In the urea bioconversion removal material of the present invention, urease can decompose urea and then remove urea, and the ZIF metal organic framework is used as a carrier to load urease. The urea bioconversion removal material has good enzyme stability, high specificity, good thermal stability and pH stability, can be stored for a long time, and has good biological and blood compatibility, providing a new strategy and method for removing excess urea in the dialysate. The urea bioconversion removal material of the present invention is conducive to achieving high-efficiency removal of peritoneal dialysis fluid urea in a portable artificial kidney device and can be repeatedly recycled, has high enzyme bioavailability, reduces the amount of adsorbent used and the volume weight of the device, and further improves the urea treatment efficiency; solves the problems of large volume and weight in the REDY system.

[0028] It is understandable that the ZIF metal-organic framework has a rich pore structure, which can prevent some foreign matter from contacting urease and improve the specificity of the enzyme to a certain extent.

[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] It should be noted that, in the present invention, MAF-7 is used as an example for explanation, and the action mechanism of ZIF-8 or ZIF-90 can be referred to in detail with respect to MAF-7, which will not be described in detail.

[0031] In some embodiments of the present invention, the particle size of the urea bioconversion scavenging material is 1-2 μm based on the total mass of the urea bioconversion scavenging material.

[0032] In another aspect of the present invention, the present invention provides a method for preparing the urea bioconversion removal material as described above, the preparation method comprising the following steps: encapsulating urease in a ZIF metal-organic framework, and connecting the urease to the metal ions in the ZIF metal-organic framework through electrostatic adsorption.

[0033] In some embodiments of the present invention, the preparation method includes the following steps: S1, adding urease and ammonia water to Hmtz (3-methyl-1,2,4-triazole) solution to obtain a mixed solution; S2, adding the mixed solution dropwise to a zinc salt solution to react 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 urea bioconversion removal material. Thus, the preparation method of the urea bioconversion removal material is simple and easy to implement, so that the urease is in situ loaded into the ZIF metal organic framework, and the urease is immobilized by biomimetic mineralization.

[0034] In some embodiments of the present invention, in the mixed solution, the concentration of urease is 0.33~1 g / L.

[0035] In some preferred embodiments of the present invention, the concentration of urease in the mixed solution is 0.5-0.7 g / L.

[0036] 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 and the time is 5-10 min.

[0037] 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.

[0038] 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.

[0039] In some specific embodiments of the present invention, the method for preparing the urea bioconversion removal 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 and dissolve it in water; (3) adding a certain amount of urease to the solution obtained in step (2) and adding a small amount of aqueous ammonia, and mixing; (4) adding the mixed solution obtained in step (3) dropwise to the solution obtained in step (1), stirring at room temperature for a period of time; (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 precipitate, centrifuge it again at high speed, discard the supernatant, and repeat the operation several times; (7) freeze-drying the obtained precipitate to obtain the urea bioconversion removal material.

[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 urea bioconversion removal 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 urease, 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 urea bioconversion removal material, which was labeled as U@MAF-7-1.

[0042] Example 2 The preparation method of urea bioconversion removal material comprises the following steps: 0.357 g of Zn(NO3)2·6H2O was weighed and dissolved in 15 mL of distilled water for later use; 0.299 g of Hmtz was weighed and dissolved in 10 mL of distilled water, 20 mg of urease was added, and 600 μL of 10% ammonia water was added, mixed, and the volume was fixed to 15 mL; the above mixed solution was added dropwise to the Zn(NO3)2 solution, stirring while adding and stirring at room temperature for 24 h. After that, the mixture was centrifuged at 10000 rpm for 6 min, the supernatant was discarded, and 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 urea bioconversion removal material, which was labeled as U@MAF-7-2.

[0043] Example 3 The preparation method of urea bioconversion removal 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 30 mg of urease, 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, and 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 urea bioconversion removal material, which was labeled as U@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 material.

[0045] Experimental Example 1 Urea clearance assay in buffer Weigh 50 mg of each of the urea bioconversion removal materials in Examples 1-3 and Comparative Example 1 into a centrifuge tube, add 10 mL of pH = 7-8 urea buffer (urea concentration is 25 mmoL / L), cover tightly and seal with a 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 10000 rpm (revolutions / minute) for 5 minutes, take the supernatant to detect the urea removal amount at different reaction time periods, and the specific results are shown in Figure 1 .

[0046] Experimental Example 2 Determination of urea clearance in peritoneal dialysis fluid of patients Weigh 50 mg of U@MAF-7-2 in Example 2 into a centrifuge tube, add 10 mL of peritoneal dialysis patient's peritoneal dialysis fluid (the urea concentration in the sample is about 20 mmoL / L), then add 0.3315 g of NADH and 0.073 g of α-ketoglutaric acid, 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 10000 rpm for 5 min, take the supernatant to detect the urea clearance at different reaction time periods, and refer to the specific results. Figure 2 .

[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 U@MAF-7 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 proliferation rate of cells in the positive control group was 25.06%, and the toxicity level was level 4, which met the standard that the toxicity level of the positive control group exceeded level 3. However, the cell activity of the MAF-7 and U@MAF-7 groups in comparative example 1 was higher than 95%, and there was no significant difference with the negative control group. Figure 3 It can be seen that the toxicity levels of the three materials are all less than or equal to level 1, which is consistent with 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 urea bioconversion removal material, characterized in that: include: ZIF metal-organic framework; Urease, the urease is connected to the metal ions in the ZIF metal-organic framework through electrostatic adsorption, and the ZIF metal-organic framework encapsulates the urease.

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

3. The urea bioconversion removal material according to claim 1, characterized in that: The particle size of the urea bioconversion scavenging material is 1-2 μm based on the total mass of the urea bioconversion scavenging material.

4. A method for preparing a urea bioconversion removal 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 the urease, and the urease is connected to the metal ions in the ZIF metal organic framework through electrostatic adsorption.

5. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: S1, adding urease and ammonia water to the Hmtz 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 urea bioconversion removal material.

6. The preparation method according to claim 5, characterized in that: In the mixed solution, the concentration of urease is 0.33-1 g / L.

7. The preparation method according to claim 5, characterized in that: In the mixed solution, the concentration of urease is 0.5-0.7 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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