Preparation method of protein binding uremic toxin adsorption resin

Abstract

The invention discloses a preparation method of protein binding uremic toxin adsorption resin, and belongs to the technical field of adsorption resin for blood perfusion. Comprising the following steps: preparing a dispersion phase, wherein the dispersion phase is chlorobenzene, and a dispersing agent is Span-80; preparing a polymerization phase, wherein the polymerization phase is composed of sodium alginate, a chitosan solution and a glutaraldehyde aqueous solution; polymerization: adding the polymerization phase into the dispersion phase, adjusting the stirring speed, and carrying out polymerization reaction; and modifying. Reverse suspension polymerization increases hydrophilic functional groups of the resin, and better biocompatibility can be provided. The selected amino functional group has positive charges, and can be electrostatically combined with negatively charged indoxyl sulfate and p-cresol sulfate, so that the adsorption capacity is higher, the adsorption efficiency is higher, and the combination of toxin and resin is firmer. And few types of organic matters are used in actual production, so that toxicological verification is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of medical adsorption resins, and in particular relates to a resin for adsorbing indoxyl sulfate and p-cresol sulfate. Background Art

[0002] Most protein-bound uremic toxins (hereinafter referred to as "protein-bound toxins") are enteric toxins, which are generated by amino acids in food through the action of intestinal bacteria. In patients with impaired renal function, protein-bound toxins accumulate in the kidneys. Protein-bound toxins represented by indoxyl sulfate and p-cresol sulfate in uremic patients are organic anionic compounds with negative charges. P-cresol sulfate and indoxyl sulfate not only accelerate the progression of renal failure, but are also closely related to the occurrence and development of cardiovascular events, the primary cause of death in patients with chronic kidney disease. Uremic levels of p-cresol sulfate and indoxyl sulfate can seriously damage the kidneys and cardiovascular system by mediating oxidative stress, activating inflammatory response pathways, and inhibiting the expression of anti-aging genes. They are independent risk factors for predicting the occurrence and development of cardiovascular complications in patients with chronic kidney disease. How to effectively remove protein-bound toxins from the serum of hemodialysis patients has also become a research hotspot in the field of blood purification. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a method for preparing a protein-bound uremic toxin adsorption resin. Reverse suspension polymerization increases the hydrophilic functional groups of the resin, which can provide better biocompatibility. The product can be electrostatically bonded with negatively charged indoxyl sulfate and p-cresol sulfate, resulting in a higher adsorption amount, higher adsorption efficiency, and a firmer bond between the toxin and the resin.

[0004] The preparation method comprises the following steps:

[0005] (1) Preparation of dispersed phase: Add dispersed phase and dispersant into a reaction vessel and stir evenly; the dispersed phase is chlorobenzene and the dispersant is SPan-80; add 20-40 g of dispersant per liter of chlorobenzene.

[0006] (2) Preparation of polymerization phase: The polymerization phase is composed of sodium alginate, chitosan solution and glutaraldehyde aqueous solution; the mass ratio of sodium alginate, chitosan solution and glutaraldehyde aqueous solution in the polymerization phase is 1-3:3-4:1; the chitosan solution is prepared by dissolving chitosan in 5% acetic acid solution with a mass fraction of 2 times; the mass fraction of glutaraldehyde aqueous solution is 25%.

[0007] (3) Polymerization: Add the polymer phase to the dispersed phase, adjust the stirring speed, and after the polymer phase is dispersed into beads of 0.15-1.25 mm, heat it to 58-62°C, add 0.2 mol / L hydrochloric acid aqueous solution to the polymerization system until the pH value of the system is less than 1, and after the beads are fixed, raise the temperature to 70-78°C, then add 0.2 mol / L NaOH aqueous solution to neutralize and continue the reaction until completion. After the reaction is completed, filter out the resin, wash it with methanol to remove free organic matter, soak it in 0.2 mol / L alkaline solution to remove free organic matter, and then wash it with distilled water until neutral, extract unreacted polymers and oligomers with methanol, and vacuum dry to obtain chitosan polymer beads; the mass ratio of dispersed phase to polymer phase is 10-15:1.

[0008] (4) Modification: Add chitosan polymer beads to a 0.5 mol / L NaOH solution, then add epichlorohydrin dropwise, stir at 70°C until the reaction is complete, wash with ethanol and water in sequence, and then dry; add the dried product to a 0.5 mol / L NaOH solution, then add ethylenediamine dropwise, stir at 60°C until the reaction is complete, wash with ethanol and water in sequence until the eluate is neutral, and then dry.

[0009] In step (4), 0.5-1.5 mL of epichlorohydrin and 0.1-0.3 mL of ethylenediamine are added dropwise per gram of chitosan polymer beads.

[0010] The invention also provides application of the prepared resin in adsorbing indoxyl sulfate and p-cresol sulfate.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The reverse suspension polymerization of the present invention increases the hydrophilic functional groups of the resin and can provide better biocompatibility.

[0013] The selected amino functional group carries a positive charge, and can electrostatically bond with the negatively charged indoxyl sulfate and p-cresol sulfate, resulting in a higher adsorption amount, higher adsorption efficiency, and a firmer bond between the toxin and the resin.

[0014] In actual production, fewer types of organic matter are used, which reduces toxicological verification. DETAILED DESCRIPTION

[0015] The present invention is further described below by means of specific examples. However, those skilled in the art should be aware that the specific examples of the present invention do not limit the present invention in any way, and any equivalent replacements made on the basis of the present invention fall within the protection scope of the present invention.

[0016] The concentrations of the compounds in the examples are all mass concentrations.

[0017] Example 1

[0018] (1) Preparation of dispersed phase: In a three-necked flask equipped with a stirrer, a thermometer, and a reflux condenser, add 1000 ml of chlorobenzene as dispersed phase and 30 g of SPan-80 as dispersant, and stir to mix well.

[0019] (2) Preparation of polymer phase: 30 g of sodium alginate (crosslinker 1), 55 g of chitosan solution (monomer, 50 g of chitosan dissolved in 100 g of 5% acetic acid solution), and 15 g of glutaraldehyde (25%; crosslinker 2) aqueous solution were stirred and mixed to obtain a polymer phase.

[0020] (3) Polymerization: Add the polymer phase to the dispersed phase, adjust the stirring speed, wait until the aqueous phase is dispersed into beads of appropriate size, raise the temperature to 58-62°C, add 20ml of 0.2mol / L hydrochloric acid aqueous solution to the polymerization system, raise the temperature to 70-78°C after the beads are formed, and then add 20ml of 0.2mol / L NaOH aqueous solution to react for 3h. After the reaction is completed, filter out the resin, wash it with methanol, soak it in 0.2mol / L alkaline solution, wash it with distilled water until it is neutral, extract it with methanol for 24h, and vacuum dry it to obtain chitosan polymer beads.

[0021] The effect of cross-linking agent glutaraldehyde concentration on chitosan polymer beads:

[0022] Glutaraldehyde concentration Chitosan polymer beads 6％ Light yellow, brittle. 25％ Dark brown, smooth resin surface, good strength. 50％ Dark brown, hollow and brittle resin.

[0023] (4) Modification: Weigh 20g chitosan polymer beads, add to a 0.5mol / L NaOH solution until completely immersed, then add 20mL epichlorohydrin dropwise, react at 70℃ with magnetic stirring for 2h, then wash with ethanol and water in turn, and dry at 60℃. Add the above product to a 0.5mol / L NaOH solution until completely immersed, then add 4mL ethylenediamine dropwise, react at 60℃ with magnetic stirring for 4h, then wash with ethanol and water in turn, and dry at 60℃.

[0024] Example 1 Characterization of the prepared resin:

[0025] Test items Test results granularity 0.15-0.3mm≥90% Ball rate after grinding 42.0％ Moisture 25％ Amino content 10.39mmol / g Specific surface area 300㎡ / g

[0026] Adsorption performance test:

[0027] Adsorption performance of indoxyl sulfate

[0028] Prepare an aqueous solution of indoxyl sulfate at a concentration of 25 mg / L, take 25 mL and place it in a 50 mL stoppered conical flask, weigh 1.0 g of the resin adsorbent prepared in Example 1 (dry weight, the wet state can be converted into dry weight or directly weigh the adsorbent after constant weight by atmospheric pressure drying method) and put it into the flask, place it in a reciprocating constant temperature water bath oscillator at (37±1)°C and oscillate at a rate of (60±10) times / min for 2 h, measure and calculate the concentration of indoxyl sulfate solution before and after adsorption by ultraviolet spectrophotometry at 216 nm, and calculate the decline rate according to formula (1):

[0029]

[0030] Where:

[0031] c r1 ——Decrease rate of indoxyl sulfate solution, %;

[0032] c0——concentration of indoxyl sulfate solution before adsorption, in milligrams per liter (mg / L);

[0033] c t ——Concentration of indoxyl sulfate solution after 2 hours of adsorption, in milligrams per liter (mg / L);

[0034] Adsorption performance of p-cresol sulfate

[0035] Prepare a 25 mg / L aqueous solution of p-cresol sulfate, take 25 mL and place it in a 50 mL conical flask with a stopper, weigh 1.0 g of adsorbent (dry weight, wet state can be converted to dry weight or directly weigh the adsorbent after constant weight by atmospheric pressure drying method) and put it into the flask, place it in a reciprocating constant temperature water bath oscillator at (37±1)℃ and oscillate at a rate of (60±10) times / min for 2 hours, measure and calculate the concentration of p-cresol sulfate solution before and after adsorption by ultraviolet spectrophotometry at 210 nm, and calculate the decline rate according to formula (2):

[0036]

[0037] Where:

[0038] c r2 ——Decrease rate of sulfuric acid p-cresol solution, %;

[0039] c0——concentration of p-cresol sulfate solution before adsorption, in milligrams per liter (mg / L);

[0040] c t ——Concentration of p-cresol sulfate solution after 2 hours of adsorption, in milligrams per liter (mg / L);

[0041] Test results:

[0042] Adsorbed substances Adsorption rate (%) Indoxyl sulfate 98.6 p-Cresol Sulfate 99.1

[0043] Example 2

[0044] (1) Preparation of dispersed phase: In a three-necked flask equipped with a stirrer, a thermometer, and a reflux condenser, add 1000 ml of chlorobenzene as dispersed phase and 20 g of SPan-80 as dispersant, and stir to mix well.

[0045] (2) Preparation of polymer phase: 15 g of sodium alginate (crosslinker 1), 45 g of chitosan solution (monomer, 50 g of chitosan dissolved in 100 g of 5% acetic acid solution), and 15 g of glutaraldehyde (25%; crosslinker 2) aqueous solution were stirred and mixed to obtain a polymer phase.

[0046] (3) Polymerization: Add the polymer phase to the dispersed phase, adjust the stirring speed, wait until the aqueous phase is dispersed into beads of appropriate size, raise the temperature to 58-62°C, add 20ml of 0.2mol / L hydrochloric acid aqueous solution to the polymerization system, raise the temperature to 70-78°C after the beads are formed, and then add 20ml of 0.2mol / L NaOH aqueous solution to react for 3h. After the reaction is completed, filter out the resin, wash it with methanol, soak it in 0.2mol / L alkaline solution, wash it with distilled water until it is neutral, extract it with methanol for 24h, and vacuum dry it to obtain chitosan polymer beads.

[0047] (4) Modification: Weigh 20g chitosan polymer beads, add to a 0.5mol / L NaOH solution until completely immersed, then add 10mL epichlorohydrin dropwise, react at 70℃ with magnetic stirring for 2h, then wash with ethanol and water in turn, and dry at 60℃. Add the above product to a 0.5mol / L NaOH solution until completely immersed, then add 2mL ethylenediamine dropwise, react at 60℃ with magnetic stirring for 4h, then wash with ethanol and water in turn, and dry at 60℃.

[0048] Example 2 Characterization of prepared resin:

[0049] Test items Test results granularity 0.15-0.3mm≥90% After grinding 40.3％ Moisture 27.6％ Amino content 10.51mmol / g Specific surface area 273㎡ / g

[0050] Adsorption performance detection: The detection method is the same as in Example 1.

[0051] Test results:

[0052] Adsorbed substances Adsorption rate (%) Indoxyl sulfate 95.3 p-Cresol Sulfate 96.8

[0053] Example 3

[0054] (1) Preparation of dispersed phase: In a three-necked flask equipped with a stirrer, a thermometer, and a reflux condenser, add 1000 ml of chlorobenzene as dispersed phase and 40 g of SPan-80 as dispersant, and stir to mix well.

[0055] (2) Preparation of polymer phase: 45 g of sodium alginate (crosslinker 1), 60 g of chitosan solution (monomer, 50 g of chitosan dissolved in 100 g of 5% acetic acid solution), and 15 g of glutaraldehyde (25%; crosslinker 2) aqueous solution were stirred and mixed to obtain a polymer phase.

[0056] (3) Polymerization: Add the polymer phase to the dispersed phase, adjust the stirring speed, wait until the aqueous phase is dispersed into beads of appropriate size, raise the temperature to 58-62°C, add 20ml of 0.2mol / L hydrochloric acid aqueous solution to the polymerization system, raise the temperature to 70-78°C after the beads are formed, and then add 20ml of 0.2mol / L NaOH aqueous solution to react for 3h. After the reaction is completed, filter out the resin, wash it with methanol, soak it in 0.2mol / L alkaline solution, wash it with distilled water until it is neutral, extract it with methanol for 24h, and vacuum dry it to obtain chitosan polymer beads.

[0057] (4) Modification: Weigh 20g chitosan polymer beads, add to a 0.5mol / L NaOH solution until completely immersed, then add 30mL epichlorohydrin dropwise, react at 70℃ with magnetic stirring for 2h, then wash with ethanol and water in turn, and dry at 60℃. Add the above product to a 0.5mol / L NaOH solution until completely immersed, then add 6mL ethylenediamine dropwise, react at 60℃ with magnetic stirring for 4h, then wash with ethanol and water in turn, and dry at 60℃.

[0058] Example 3 Characterization of prepared resin:

[0059] Test items Test results granularity 0.15-0.3mm≥90% After grinding 42.1％ Moisture 25.6％ Amino content 10.55mmol / g Specific surface area 267㎡ / g

[0060] Adsorption performance detection: The detection method is the same as in Example 1.

[0061] Test results:

[0062] Adsorbed substances Adsorption rate (%) Indoxyl sulfate 93.8 p-Cresol Sulfate 95.6

Claims

1. A method for preparing a protein-bound uremic toxin adsorption resin, characterized in that: The following steps are involved: (1) Preparing the dispersed phase: Add the dispersed phase and the dispersant into the reaction vessel and stir evenly; the dispersed phase is chlorobenzene and the dispersant is SPan-80; (2) preparing a polymer phase: the polymer phase is composed of sodium alginate, chitosan solution and glutaraldehyde aqueous solution; (3) Polymerization: Add the polymer phase to the dispersed phase, adjust the stirring speed, and after the polymer phase is dispersed into beads of 0.15-1.25 mm, raise the temperature to 58-62°C, add 0.2 mol / L hydrochloric acid aqueous solution to the polymerization system until the pH value of the system is less than 1. After the beads are formed, raise the temperature to 70-78°C, add 0.2 mol / L NaOH aqueous solution to neutralize and continue the reaction until completion. After the reaction is completed, filter out the resin, wash with methanol to remove free organic matter, soak with 0.2 mol / L alkaline solution to remove free organic matter, and then wash with distilled water until neutral. Extract unreacted polymers and oligomers with methanol, and vacuum dry to obtain chitosan polymer beads; (4) Modification: Add the chitosan polymer beads to a 0.5 mol / L NaOH solution until they are completely immersed, then add epichlorohydrin dropwise, stir at 70°C until the reaction is complete, wash with ethanol and water in sequence, and then dry; add the dried product to a 0.5 mol / L NaOH solution until they are completely immersed, then add ethylenediamine dropwise, stir at 60°C until the reaction is complete, wash with ethanol and water in sequence until the eluate is neutral, and then dry.

2. The method for preparing the protein-bound uremic toxin adsorption resin according to claim 1, characterized in that: Add 20-40 g of dispersant to each liter of chlorobenzene in the dispersed phase.

3. The method for preparing the protein-bound uremic toxin adsorption resin according to claim 1, characterized in that: The mass ratio of sodium alginate, chitosan solution and glutaraldehyde aqueous solution in the polymer phase is 1-3:3-4:1; the chitosan solution is prepared by dissolving chitosan in 2 times the mass of 5% acetic acid solution; the mass fraction of glutaraldehyde aqueous solution is 25%.

4. The method for preparing the protein-bound uremic toxin adsorption resin according to claim 1, characterized in that: In step (3), the mass ratio of the dispersed phase to the polymerized phase is 10-15:

1.

5. The method for preparing the protein-bound uremic toxin adsorption resin according to claim 1, characterized in that: In step (4), 0.5-1.5 mL of epichlorohydrin and 0.1-0.3 mL of ethylenediamine are added dropwise per gram of chitosan polymer beads.

6. Use of the resin prepared according to any one of claims 1 to 5 in the adsorption of indoxyl sulfate and p-cresol sulfate.

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