Preparation method and application of a full-biomass-based hemoperfusion adsorption material
By using phase change lysozyme to coat cellulose microspheres, the problems of blood compatibility and bilirubin adsorption performance of cellulose-based hemoperfusion adsorbents were solved, resulting in a fully biomass-based hemoperfusion adsorbent material with high biocompatibility and high adsorption capacity, suitable for bilirubin adsorption in hemoperfusion.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN UNIV
- Filing Date
- 2024-03-28
- Publication Date
- 2026-06-19
AI Technical Summary
Existing cellulose-based hemoperfusion adsorbents have reduced blood compatibility after modification, which limits their clinical application, and it is difficult to achieve high biocompatibility and high bilirubin adsorption performance.
A phase change lysozyme-coated cellulose microsphere method was adopted. This method involves preparing an oil-in-water emulsion, reducing the lysozyme with a disulfide reducing agent to form a phase change lysozyme solution, and then coating the solution onto the cellulose microspheres. The uncoated portion was then washed away to obtain a fully biomass-based blood perfusion adsorbent material.
The biocompatibility of the cellulose adsorbent material was improved, and its adsorption capacity for bilirubin was significantly enhanced, achieving effective bilirubin adsorption at 37°C, which has broad potential for clinical application.
Smart Images
Figure CN118253290B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of functional polymer materials and relates to a method for preparing and applying a biomass-based blood perfusion adsorption material. Background Technology
[0002] Currently, adsorbents commonly used in hemoperfusion are mainly classified into activated carbon, synthetic resins, and polysaccharides, collectively referred to as hemoperfusion materials. Cellulose, as the most abundant natural polysaccharide, possesses advantages such as wide availability, non-toxicity, good blood compatibility, rich in active groups, and ease of chemical modification. It is frequently used as a carrier for affinity adsorbents, showing great potential in constructing hemoperfusion adsorbents with good blood compatibility, cell compatibility, and high adsorption performance. Cellulose-based hemoperfusion materials, through modification, grafting, and simple composite processes, are widely used for bilirubin adsorption. Among these, cellulose microspheres, due to their low preparation cost, large surface area, and high mechanical strength, have seen rapid development in the field of medical polymer adsorption and separation materials in recent years.
[0003] Cellulose-based bilirubin adsorbents are crucial for hemoperfusion technology, directly determining its effectiveness. The inherent structural characteristics of cellulose allow for selective adsorption through simple compounding and covalent bonding of various modifying groups, including immunomodulatory groups; this is currently a major research direction in hemoperfusion materials. Despite their unique advantage of high bilirubin adsorption, most cellulose-based adsorbents exhibit reduced blood compatibility and numerous side effects after compounding or modification, limiting their practical clinical application. Developing cellulose-based hemoperfusion adsorbents with high biocompatibility and high bilirubin adsorption performance remains a challenge. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings and deficiencies of existing blood perfusion materials and to provide a method for preparing and applying a fully biomass-based blood perfusion adsorbent material.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A method for preparing a fully biomass-based blood perfusion adsorbent material includes the following steps:
[0007] (1) Preparation of regenerated cellulose microspheres: An oily liquid was used as the oil phase and a cellulose solution as the aqueous phase. The mixture was emulsified with a surfactant to obtain a water-in-oil emulsion. Acid was added to the emulsion system to convert the cellulose solution in the aqueous phase into a cellulose sol gel. After removing the oil phase and washing and purifying, regenerated cellulose microspheres were obtained.
[0008] (2) Phase transition of lysozyme: The disulfide bonds in lysozyme are reduced by a disulfide reducing agent under neutral or alkaline conditions to obtain a phase transition lysozyme solution.
[0009] (3) The regenerated cellulose microspheres obtained in step (1) are soaked in the phase change lysozyme solution obtained in step (2) for incubation, so that the phase change lysozyme is coated on the cellulose microspheres. The uncoated phase change lysozyme is removed to obtain the whole biomass-based blood perfusion adsorbent material.
[0010] In step (1), the oily liquid preferably includes any one or any combination of kerosene, transformer oil, pump oil, 200# gasoline, turbine oil, liquid paraffin, petroleum ether, rapeseed oil, etc.
[0011] In step (1), the cellulose solution is preferably obtained by dissolving the cellulose material in a sodium hydroxide / urea low-temperature aqueous solution system; the cellulose material includes cotton, flax, taro, jute, and other materials containing a large amount of high-quality cellulose. Further, the cellulose solution is prepared by a method comprising the following steps: weighing sodium hydroxide, urea, and water in a mass ratio of 7:12:81, mixing them evenly, and pre-cooling to -13 to -10°C; quickly adding cotton linter cellulose into the pre-cooled solution, stirring thoroughly, centrifuging to degas, removing impurities, and obtaining the cellulose solution. The preferred stirring conditions are 1000–1500 rpm for 4–8 min; the preferred centrifugation conditions are 4000–8000 rpm for 5–15 min; and the mass ratio of cotton linter cellulose, sodium hydroxide, urea, and water is 2–8:7:12:81.
[0012] In step (1), the volume ratio of cellulose solution to oil phase is preferably 1 / 2 to 1 / 6; the volume of surfactant is preferably 1% to 5% of the volume of oil phase.
[0013] In step (1), the surfactant is preferably Span 80; the acid is preferably hydrochloric acid.
[0014] Step (1) is preferably as follows: 1–15 mL of Span 80 is mixed with 60–600 mL of soybean oil and stirred at 200–900 rpm for 1–2 hours; 20–200 mL of cellulose solution is added dropwise to the well-mixed soybean oil over 5–15 minutes, and stirring is continued for 1–4 hours to fully emulsify, resulting in a water-in-oil emulsion; 1–6 M hydrochloric acid is added to the emulsion system to slowly change the pH of the system to neutral, and the emulsified cellulose sol is converted into cellulose sol gel. Stirring is stopped, the solution is allowed to stand and separate into layers, the upper layer is discarded, and the lower layer of precipitated regenerated cellulose microspheres is taken. These microspheres are washed 2–5 times with anhydrous ethanol and then with deionized water, each time for 1–4 hours, to obtain regenerated cellulose microspheres. The emulsification conditions are preferably room temperature stirring for 1–4 hours, and the stirring speed is preferably 200–900 rpm.
[0015] In step (2), the disulfide bond reducing agent is preferably tris(2-carbonylethyl)phosphine (TCEP), and the alkaline conditions are preferably in NaOH solution.
[0016] Step (2) is preferably performed as follows: lysozyme and TCEP are separately dispersed in 10 mmol / L HEPES buffer, and the pH of the solution is adjusted to 7.4-11.4 using 2-6 M NaOH, ensuring that the pH of the lysozyme solution and the TCEP solution are the same; the two solutions are mixed for 5-60 min to obtain a phase-change lysozyme solution. The final concentration of lysozyme in the mixture is preferably 1-25 g / L, and the final concentration of TCEP is preferably 5-50 mM.
[0017] In step (3), the incubation conditions are preferably 5 to 60 minutes of standing.
[0018] In step (3), the method for removing uncoated phase change lysozyme includes ultrapure water sedimentation 3-5 times and ultrapure water washing. Ultrapure water sedimentation (immersion washing, using a large amount of water to remove well-dispersed phase change lysozyme) and ultrapure water washing (placing the coated microspheres in a nylon bag with a specific pore size for ultrapure water washing to remove aggregated phase change lysozyme that cannot disperse well in water) ensure that excess phase change lysozyme is washed away.
[0019] A biomass-based blood perfusion adsorbent material is obtained by the above preparation method.
[0020] The aforementioned biomass-based hemoperfusion adsorption material can be used for hemoperfusion adsorption of bilirubin in blood.
[0021] The above-mentioned biomass-based hemoperfusion adsorbent material is used in the preparation of bilirubin adsorbents for hemoperfusion.
[0022] Advantages and beneficial effects of the present invention:
[0023] The present invention uses phase change lysozyme to coat cellulose, which not only improves the biocompatibility of cellulose adsorbent materials, but also greatly enhances the adsorption capacity of cellulose adsorbent materials for bilirubin.
[0024] The materials used in this invention are widely available, and the prepared all-biomass-based blood perfusion adsorbent material has extremely high biocompatibility. This work provides a simple and effective strategy for improving the biocompatibility of microsphere adsorbents for blood perfusion.
[0025] The biomass-based blood perfusion adsorption material of the present invention can effectively adsorb bilirubin at 37°C.
[0026] The biomass-based hemoperfusion adsorbent of the present invention can effectively adsorb bilirubin, demonstrating the potential application of this hemoperfusion adsorbent in clinical hemoperfusion bilirubin removal. Attached Figure Description
[0027] Figure 1 These are comparison images of regenerated cellulose microspheres and biomass-based hemoperfusion adsorbent materials, along with their scanning electron microscope (SEM) images. (a, b) SEM images of regenerated cellulose microspheres; (c, d) SEM images of the biomass-based hemoperfusion adsorbent material.
[0028] Figure 2 This is a diagram illustrating the actual operation of the biomass-based hemoperfusion adsorption material for bilirubin adsorption. (a, b) Dynamic adsorption of bilirubin by the biomass-based hemoperfusion adsorption material, (c) Static adsorption of bilirubin by the biomass-based hemoperfusion adsorption material. Detailed Implementation
[0029] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of the present invention. Unless otherwise stated, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0030] This invention provides a method for preparing a fully biomass-based blood perfusion adsorbent material, which specifically includes the following steps:
[0031] (1) Preparation of cellulose solution: Weigh sodium hydroxide, urea and water in a mass ratio of 7:12:81 and mix them evenly. Place the solution in a -40℃ cold trap to pre-cool to -13 to -10℃. Quickly add cotton linter cellulose and stir at 1000 to 1500 rpm for 4 to 8 minutes. Centrifuge at 4000 to 8000 rpm for 5 to 15 minutes at room temperature to remove bubbles and impurities. After centrifugation, place the cellulose solution in a refrigerator at 2 to 8℃ for later use.
[0032] In this step, the preferred mass ratio of cotton linter cellulose raw material, sodium hydroxide, urea, and water is 2-8:7:12:81.
[0033] (2) Preparation of regenerated cellulose microspheres: Mix 1-15 mL of Span80 with 60-600 mL of soybean oil and stir for 1-2 h; add 20-200 mL of the cellulose solution obtained in step (1) dropwise to the well-mixed soybean oil within 5-15 min, continue stirring for 1-4 h to emulsify, and slowly add 1-6 M hydrochloric acid to adjust the pH of the solution to neutral. Stop stirring and let the solution stand for 5-20 min to separate into layers. Pour off the upper layer of liquid and wash with anhydrous ethanol and deionized water 2-5 times, 1-4 h each time, to remove the oil phase, surfactant and impurities in the regenerated cellulose. The resulting regenerated cellulose microspheres can be stored in water.
[0034] In this step, the volume ratio of cellulose solution to soybean oil is preferably 1 / 2 to 1 / 6; the volume of surfactant is preferably 1% to 5% of the oil phase volume; and the mechanical stirring speed is preferably 200 to 900 rpm.
[0035] (3) Phase transition of lysozyme: Lysozyme and TCEP were separately dispersed in 10 mmol / L HEPES buffer. The pH of the solution was adjusted to 7.4-11.4 using 2-6 M NaOH, so that the pH of the lysozyme solution and the TCEP solution were the same. The two solutions were mixed for 5-60 min to obtain the phase-transition lysozyme solution.
[0036] In this step, the final concentration of lysozyme in the mixture is preferably 1-25 g / L, and the final concentration of TCEP reducing agent is preferably 5-50 mM.
[0037] (4) Soak the regenerated cellulose microspheres obtained in step (2) in the phase change lysozyme solution obtained in step (3), mix them and let them stand for 5 to 60 minutes so that the phase change lysozyme is coated on the regenerated cellulose microspheres. After sedimentation in ultrapure water 3 to 5 times and washing with ultrapure water, the wet biomass-based blood perfusion adsorbent material is finally obtained and can be stored in deionized water.
[0038] Example 1
[0039] (1) Weigh 100g of sodium hydroxide, urea and water in a mass ratio of 7:12:81, dissolve them by sonication and place them in a -40℃ cold trap to cool to -10℃; quickly put 2g of cotton linter cellulose sample into the pre-cooled solution, mechanically stir at 1000rpm for 4min, and then centrifuge at 5000rpm for 8min at room temperature to remove air bubbles in the system; after centrifugation, place the cellulose solution in a 4℃ refrigerator for later use.
[0040] (2) Mix 3 mL of Span80 with 300 mL of soybean oil (Span80 is 1% of the volume of soybean oil) and stir mechanically at 300 rpm for 2 h. Add 150 mL of cellulose solution dropwise to the mixture of Span80 and soybean oil within 15 min (the volume ratio of cellulose solution to soybean oil is 1 / 2). Continue stirring for 1 h to emulsify, then slowly add 2 M hydrochloric acid until the system is neutral. After stirring stops, let the solution stand to separate into layers, pour off the upper layer of liquid, collect the bottom layer of regenerated cellulose microspheres, wash them three times with anhydrous ethanol and then with deionized water for 1 h each time, and remove the water in the system using a sand core filter to obtain wet regenerated cellulose microspheres with a solid content of 7.2%.
[0041] (3) Lysozyme and TCEP were separately dispersed in 10 mmol / L HEPES buffer to prepare 5 mg / mL lysozyme solution and 50 mM TCEP solution, respectively. The pH of both solutions was adjusted to 7.4 using 2 M NaOH solution. 5 mL of lysozyme solution and 5 mL of TCEP solution were mixed together in a 15 cm diameter petri dish for 20 min to obtain a phase change lysozyme solution with pH 7.4.
[0042] (4) Weigh 1.4g of the wet regenerated cellulose microspheres obtained in step (2) (solid content of about 100mg) and soak them in 10mL of phase change lysozyme solution obtained in step (3) (the mass ratio of the solid content of regenerated cellulose microspheres and lysozyme is about 4:1). After standing and incubating for 40min, the microspheres are settled three times with ultrapure water and washed with ultrapure water to remove the easily detached phase change lysozyme. The water in the system is removed by a sand core filtration device. Finally, a wet biomass-based blood perfusion adsorbent material with a solid content of 7.6% is obtained.
[0043] (5) 526 mg (solid content approximately 40 mg) of wet biomass-based hemoperfusion adsorbent material was used to conduct an adsorption experiment on 10 mL of 100 ppm bilirubin solution. The adsorbent material and bilirubin solution were mixed in a 25 mL Erlenmeyer flask and subjected to static adsorption in a constant temperature shaker at 37℃ and 100 rpm for 4 hours. After adsorption, the color of the bilirubin solution became significantly lighter, and the biomass-based hemoperfusion adsorbent material changed from white to orange after adsorbing bilirubin. The concentration of bilirubin after adsorption was detected using a bilirubin reagent kit. The calculated adsorption capacity of the biomass-based hemoperfusion adsorbent material for bilirubin was 7.21 mg / g.
[0044] Example 2
[0045] (1) Weigh 100g of sodium hydroxide, urea and water in a mass ratio of 7:12:81, dissolve them by sonication and place them in a -40℃ cold trap to cool to -11℃; quickly put 4g of cotton linter cellulose sample into the pre-cooled solution, mechanically stir at 1200rpm for 5min, and then centrifuge at 6000rpm for 10min at room temperature to remove air bubbles in the system; after centrifugation, place the cellulose solution in a 4℃ refrigerator for later use.
[0046] (2) Mix 6 mL of Span80 with 300 mL of soybean oil (Span80 is 2% of the volume of soybean oil) and mechanically stir at 450 rpm for 1.5 h. Add 150 mL of cellulose solution dropwise to the mixture of Span80 and soybean oil within 10 min (the volume ratio of cellulose solution to soybean oil is 1 / 2). Continue stirring for 2 h to emulsify, then slowly add 3M hydrochloric acid until the system is neutral. After stirring stops, let the solution stand to separate into layers, pour off the upper layer of liquid, collect the bottom layer of regenerated cellulose microspheres, wash them three times with anhydrous ethanol and then with deionized water for 1 h each time, and remove the water in the system using a sand core filter to obtain wet regenerated cellulose microspheres with a solid content of 8.1%.
[0047] (3) Lysozyme and TCEP were separately dispersed in 10 mmol / L HEPES buffer to prepare 10 mg / mL lysozyme solution and 50 mM TCEP solution, respectively. The pH of both solutions was adjusted to 9.4 using 4 M NaOH solution. 5 mL of lysozyme solution and 5 mL of TCEP solution were mixed together in a 15 cm diameter petri dish for 10 min to obtain a phase change lysozyme solution with a pH of 9.4.
[0048] (4) Weigh 1.23g of the wet regenerated cellulose microspheres obtained in step (2) (solid content of about 100mg) and soak them in 10mL of phase change lysozyme solution obtained in step (3) (the mass ratio of solid content of regenerated cellulose microspheres and lysozyme is about 2:1). After standing and incubating for 30min, the microspheres are settled in ultrapure water 4 times and washed with ultrapure water to clean the easily detachable phase change lysozyme. The water in the system is removed by sand core filtration device. Finally, a wet whole biomass-based blood perfusion adsorbent material with a solid content of 8.8% is obtained.
[0049] (5) 454 mg (solid content approximately 40 mg) of wet biomass-based hemoperfusion adsorbent material was used to conduct an adsorption experiment on 10 mL of 200 ppm bilirubin solution. The adsorbent material and bilirubin solution were mixed in a 25 mL Erlenmeyer flask and subjected to static adsorption in a constant temperature shaker at 37℃ and 200 rpm for 4 hours. After adsorption, the color of the bilirubin solution became significantly lighter, and the biomass-based hemoperfusion adsorbent material changed from white to orange after adsorbing bilirubin. The concentration of bilirubin after adsorption was detected using a bilirubin reagent kit. The calculated adsorption capacity of the biomass-based hemoperfusion adsorbent material for bilirubin was 13.2 mg / g.
[0050] Example 3
[0051] (1) Weigh 100g of sodium hydroxide, urea and water in a mass ratio of 7:12:81, dissolve them by sonication and place them in a -40℃ cold trap to cool to -12℃; quickly put 6g of cotton linter cellulose sample into the pre-cooled solution, mechanically stir at 1500rpm for 6min, and then centrifuge at 8000rpm for 12min at room temperature to remove air bubbles in the system; after centrifugation, place the cellulose solution in a 4℃ refrigerator for later use.
[0052] (2) Mix 9 mL of Span80 with 300 mL of soybean oil (Span80 is 3% of the volume of soybean oil) and stir mechanically at 500 rpm for 2 h. Add 100 mL of cellulose solution dropwise to the mixture of Span80 and soybean oil within 5 min (the volume ratio of cellulose solution to soybean oil is 1 / 3). Continue stirring for 3 h to emulsify, then slowly add 4 M hydrochloric acid until the system is neutral. After stirring stops, let the solution stand to separate into layers, pour off the upper layer of liquid, collect the regenerated cellulose microspheres at the bottom layer, wash them 4 times with anhydrous ethanol and then with deionized water for 1 h each time, and remove the water in the system with a sand core filter to obtain wet regenerated cellulose microspheres with a solid content of 9.1%.
[0053] (3) Lysozyme and TCEP were dispersed in 10 mmol / L HEPES buffer to prepare 20 mg / mL lysozyme solution and 50 mM TCEP solution, respectively. The pH of both solutions was adjusted to 11.4 using 4 M NaOH solution. 5 mL of lysozyme solution and 5 mL of TCEP solution were mixed together in a 15 cm diameter petri dish for 30 min to obtain a phase-change lysozyme solution with a pH of 11.4.
[0054] (4) Weigh 1.1g of the wet regenerated cellulose microspheres obtained in step (2) (solid content of about 100mg) and soak them in 10mL of phase change lysozyme solution obtained in step (3) (the mass ratio of the solid content of the regenerated cellulose microspheres and lysozyme is about 1:1). After standing and incubating for 60min, the microspheres are settled in ultrapure water 5 times and washed with ultrapure water to clean the easily detachable phase change lysozyme. The water in the system is removed by sand core filtration device, and finally a wet whole biomass-based blood perfusion adsorbent material with a solid content of 9.7% is obtained.
[0055] (5) 412 mg (solid content approximately 40 mg) of wet biomass-based hemoperfusion adsorbent material was used to conduct an adsorption experiment on 10 mL of 150 ppm bilirubin solution. The adsorbent material and bilirubin solution were mixed in a 25 mL Erlenmeyer flask and subjected to static adsorption in a constant temperature shaker at 37℃ and 150 rpm for 4 hours. After adsorption, the color of the bilirubin solution became significantly lighter, and the biomass-based hemoperfusion adsorbent material changed from white to orange after adsorbing bilirubin. The bilirubin concentration after adsorption was detected using a bilirubin reagent kit. The calculated adsorption capacity of the biomass-based hemoperfusion adsorbent material for bilirubin was 20.21 mg / g.
[0056] Example 4
[0057] (1) Weigh 100g of sodium hydroxide, urea and water in a mass ratio of 7:12:81, dissolve them by sonication and place them in a -40℃ cold trap to cool to -12.5℃; quickly put 4g of cotton linter cellulose sample into the pre-cooled solution, mechanically stir at 1200rpm for 5min, and then centrifuge at 5000rpm for 8min at room temperature to remove air bubbles in the system; after centrifugation, place the cellulose solution in a 4℃ refrigerator for later use.
[0058] (2) Mix 9 mL of Span80 with 300 mL of soybean oil (Span80 is 3% of the volume of soybean oil) and stir mechanically at 300 rpm for 1 h. Add 100 mL of cellulose solution dropwise to the mixture of Span80 and soybean oil within 10 min (the volume ratio of cellulose solution to soybean oil is 1 / 3). Continue stirring for 2 h to emulsify, then slowly add 3M hydrochloric acid until the system is neutral. After stirring stops, let the solution stand to separate into layers, pour off the upper layer of liquid, collect the regenerated cellulose microspheres at the bottom layer, wash them three times with anhydrous ethanol and then with deionized water for 2 h each time, and remove the water in the system using a sand core filter to obtain wet regenerated cellulose microspheres with a solid content of 9%.
[0059] (3) Lysozyme and TCEP were dispersed in 10 mmol / L HEPES buffer to prepare 20 mg / mL lysozyme solution and 50 mM TCEP solution, respectively. The pH of both solutions was adjusted to 7.4 using 2 M NaOH solution. 5 mL of lysozyme solution and 5 mL of TCEP solution were mixed together in a 15 cm diameter petri dish for 40 min to obtain a phase-change lysozyme solution with pH 7.4.
[0060] (4) Weigh 1.1g of the wet regenerated cellulose microspheres obtained in step (2) (solid content of about 100mg) and soak them in 10mL of phase change lysozyme solution obtained in step (3) (the mass ratio of the solid content of the regenerated cellulose microspheres and lysozyme is about 1:1). After standing and incubating for 40min, the microspheres are settled three times with ultrapure water and washed with ultrapure water to clean the easily detachable phase change lysozyme. The water in the system is removed by a sand core filtration device, and finally a wet whole biomass-based blood perfusion adsorbent material with a solid content of 9.8% is obtained.
[0061] Scanning electron microscopy images of the microstructure of freeze-dried biomass-based blood perfusion adsorbent and regenerated cellulose microspheres, for example... Figure 1 As shown, Figure 1 b and Figure 1 The microstructure comparison of d proves the successful encapsulation of phase change lysozyme on regenerated cellulose microspheres, and that the phase change lysozyme is a submicron-sized spherical particle.
[0062] (5) Take 408 mg (solid content approximately 40 mg) of wet biomass-based hemoperfusion adsorption material and conduct an adsorption experiment on 10 mL of 100 ppm bilirubin solution. The adsorption material and bilirubin solution are mixed in a 25 mL Erlenmeyer flask and subjected to static adsorption experiment in a constant temperature shaker at 37℃ and 150 rpm for 4 hours. Figure 2 As shown in the left figure (c), the color of the bilirubin solution becomes significantly lighter after adsorption, and the all-biomass-based hemoperfusion adsorption material changes from white to orange after adsorbing bilirubin. Figure 2 Figure c on the right shows a comparison of the actual images of the biomass-based hemoperfusion adsorption material before and after bilirubin adsorption.
[0063] A column (15 cm high, 1.1 cm inner diameter) was perfused with 500 mg of all-biomass-based hemoperfusion adsorbent material for dynamic adsorption experiments. The flow rate was 1 mL / min, and the concentration of bilirubin used for perfusion was 100 ppm. Samples were taken every 10 minutes, and the color change was as follows. Figure 2 As shown in b, with the increase of the number of samplings, the adsorption of bilirubin by the all-biomass-based blood perfusion adsorbent material in the perfusion column gradually reaches saturation, and the color of the solution also gradually changes from colorless at the beginning to close to the color of the original bilirubin solution.
[0064] The concentration of bilirubin after adsorption was detected using a bilirubin reagent kit. The calculated adsorption capacity of the all-biomass-based hemoperfusion adsorption material for bilirubin was 23.42 mg / g.
[0065] Example 5
[0066] (1) Weigh 100g of sodium hydroxide, urea and water in a mass ratio of 7:12:81, dissolve them by sonication and place them in a -40℃ cold trap to cool to -13℃; quickly put 8g of cotton linter cellulose sample into the pre-cooled solution, mechanically stir at 1500rpm for 8min, and then centrifuge at 8000rpm for 15min at room temperature to remove air bubbles from the system; after centrifugation, place the cellulose solution in a 4℃ refrigerator for later use.
[0067] (2) Mix 12 mL of Span80 with 300 mL of soybean oil (Span80 is 4% of the volume of soybean oil) and stir mechanically at 700 rpm for 2 h. Add 50 mL of cellulose solution dropwise to the mixture of Span80 and soybean oil within 5 min (the volume ratio of cellulose solution to soybean oil is 1 / 6). Continue stirring for 2 h to emulsify, then slowly add 4 M hydrochloric acid until the system is neutral. After stirring stops, let the solution stand to separate into layers, pour off the upper layer of liquid, collect the regenerated cellulose microspheres at the bottom layer, wash them three times with anhydrous ethanol and then with deionized water for 2 h each time, and remove the water in the system using a sand core filter to obtain wet regenerated cellulose microspheres with a solid content of 9.5%.
[0068] (3) Lysozyme and TCEP were dispersed in 10 mmol / L HEPES buffer to prepare 40 mg / mL lysozyme solution and 50 mM TCEP solution, respectively. The pH of both solutions was adjusted to 9.4 using 5 M NaOH solution. 5 mL of lysozyme solution and 5 mL of TCEP solution were mixed together in a 15 cm diameter petri dish for 50 min to obtain a phase change lysozyme solution with a pH of 9.4.
[0069] (4) Weigh 1.05g of the wet regenerated cellulose microspheres obtained in step (2) (solid content of about 100mg) and soak them in 10mL of phase change lysozyme solution obtained in step (3) (the mass ratio of the solid content of regenerated cellulose microspheres and lysozyme is about 1:2). After standing and incubating for 60min, the microspheres are settled in ultrapure water 5 times and washed with ultrapure water to clean the easily detachable phase change lysozyme. The water in the system is removed by sand core filtration device, and finally a wet whole biomass-based blood perfusion adsorbent material with a solid content of 10% is obtained.
[0070] (5) 400 mg (solid content approximately 40 mg) of wet biomass-based hemoperfusion adsorbent material was used to conduct an adsorption experiment on 10 mL of 150 ppm bilirubin solution. The adsorbent material and bilirubin solution were mixed in a 25 mL Erlenmeyer flask and subjected to static adsorption in a constant temperature shaker at 37℃ and 150 rpm for 3 hours. After adsorption, the color of the bilirubin solution became significantly lighter, and the biomass-based hemoperfusion adsorbent material changed from white to orange after adsorbing bilirubin. The concentration of bilirubin after adsorption was detected using a bilirubin reagent kit. The calculated adsorption capacity of the biomass-based hemoperfusion adsorbent material for bilirubin was 25.6 mg / g.
Claims
1. A method for preparing a fully biomass-based blood perfusion adsorbent material, characterized in that: Includes the following steps: (1) Preparation of regenerated cellulose microspheres: an oily liquid was used as the oil phase and a cellulose solution was used as the aqueous phase. The mixture was emulsified with a surfactant to obtain a water-in-oil emulsion. Adding acid to the emulsion system converts the cellulose solution in the aqueous phase into a cellulose sol gel. After removing the oil phase and washing and purifying, regenerated cellulose microspheres are obtained. (2) Phase transition of lysozyme: The disulfide bonds in lysozyme are reduced by a disulfide reducing agent under neutral or alkaline conditions to obtain a phase transition lysozyme solution; the disulfide reducing agent is tris(2-carbonylethyl)phosphine; lysozyme and TCEP are dispersed in 10 mmol / L HEPES buffer, and the pH of the two solutions is adjusted to 7.4-11.4 using 2-6M NaOH; the two solutions are mixed for 10-60 min to obtain a phase transition lysozyme solution; (3) Soak the regenerated cellulose microspheres obtained in step (1) in the phase change lysozyme solution obtained in step (2) to incubate, so that the phase change lysozyme is coated on the cellulose microspheres. Remove the uncoated phase change lysozyme to obtain the whole biomass-based blood perfusion adsorbent material.
2. The method of claim 1, wherein the method is characterized by: In step (1), the cellulose solution is obtained by dissolving the cellulose material in a sodium hydroxide / urea low-temperature aqueous solution system.
3. The method of claim 1, wherein the method further comprises: In step (1), the volume ratio of cellulose solution to oil phase is 1 / 2 to 1 / 6; the volume of surfactant is 1% to 5% of the volume of oil phase.
4. The preparation method of the all-biomass-based blood perfusion adsorbent material according to claim 1, characterized in that: Step (1) is as follows: Mix 1-15 mL of Span80 with 60-600 mL of soybean oil and stir at 200-900 rpm for 1-2 hours; add 20-200 mL of cellulose solution dropwise to the well-mixed soybean oil within 5-15 minutes, and continue stirring for 1-4 hours to fully emulsify and obtain a water-in-oil emulsion. Add 1-6M hydrochloric acid to the emulsion system to slowly change the pH of the system to neutral, and the emulsified cellulose sol is converted into cellulose sol gel. Stop stirring, let the solution stand to separate into layers, pour off the upper layer of liquid, take the lower layer of precipitated regenerated cellulose microspheres, and wash them with anhydrous ethanol and deionized water respectively to obtain regenerated cellulose microspheres.
5. The method of claim 1, wherein: In step (3), the incubation conditions are to let it stand for 5 to 60 minutes.
6. A fully biomass-based hemoperfusion adsorbent material, characterized by: It is obtained by the preparation method according to any one of claims 1-5.
7. The application of the all-biomass-based hemoperfusion adsorbent material according to claim 6 in the adsorption of bilirubin.
8. The use of the all-biomass-based hemoperfusion adsorbent material according to claim 6 in the preparation of bilirubin adsorbents for hemoperfusion.