A porous ulinastatin adsorbent having a responsive shell and a method of preparing the same

Abstract

The present application relates to the technical field of new materials, in particular to a porous ulinastatin adsorbent with a responsive shell and a preparation method thereof.The porous ulinastatin adsorbent with a responsive shell is a full-synthetic polymer matrix, and has a solid structure.A high-crosslinking-degree polyvinyl alcohol / styrene / divinylbenzene core and a MOF nanoparticle-enhanced framework endow the porous ulinastatin adsorbent with a responsive shell with excellent mechanical strength, and the porous ulinastatin adsorbent with a responsive shell can withstand the pressure in an industrial chromatography column and is not prone to breakage.Because both recognition and desorption are physical processes, the replacement frequency and overall cost of the porous ulinastatin adsorbent with a responsive shell in the production process are greatly reduced, and a reliable guarantee is provided for industrial large-scale and continuous production.

Description

Technical Field

[0001] This invention relates to the field of new materials technology, and in particular to a porous ulinastatin adsorbent with a responsive shell and its preparation method. Background Technology

[0002] Ulinastatin is a glycoprotein with broad-spectrum protease inhibitory activity, widely used in the treatment of various critical illnesses such as acute pancreatitis and acute circulatory failure, resulting in high clinical demand. Currently, commercially available ulinastatin is mainly extracted and purified from the urine of healthy adult males. However, existing processes for extracting ulinastatin from urine suffer from numerous bottlenecks, leading to low production efficiency and high costs, severely restricting the accessibility of this drug.

[0003] Currently, the extraction of ulinastatin from urine typically relies on traditional chromatography techniques, primarily ion exchange chromatography and affinity chromatography, which depend on specific chromatographic adsorbent media. Existing adsorbents and processes suffer from the following prominent problems:

[0004] 1. The extraction process is cumbersome, resulting in low yields and activity of the target product. Urine is an extremely complex mixture, containing not only ulinastatin but also high concentrations of urea, salts, pigments, and a large number of other proteins with varying structures and properties (such as albumin and globulins). Existing adsorption materials, such as traditional ion exchange resins or gel filtration media, exhibit poor selectivity for ulinastatin. While adsorbing the target protein, a large amount of impurity proteins are also co-adsorbed. This necessitates multiple and cumbersome purification steps to achieve the required drug purity, with the overall yield of ulinastatin typically below 60%.

[0005] 2. Existing adsorbents struggle to balance selectivity and adsorption capacity. To improve selectivity, some studies have employed affinity chromatography. While this method offers high selectivity, the ligands themselves are extremely expensive, chemically unstable, and prone to degradation and inactivation in complex urine environments. Furthermore, effective cleaning and regeneration are difficult, failing to meet the cost and control requirements of large-scale industrial production. On the other hand, although some macroporous resins possess high specific surface area and physical adsorption capacity, their adsorption primarily relies on non-specific hydrophobic interactions or van der Waals forces, exhibiting almost no specific recognition ability, resulting in poor adsorption selectivity and low product purity.

[0006] 3. The regeneration difficulties and stability issues of physical adsorbents: Some materials based on physical adsorption, such as certain modified polymer microspheres, although exhibiting considerable adsorption capacity during initial use, show a significant decline in adsorption performance after multiple adsorption-desorption cycles. This is mainly due to irreversible blockage of the pore structure, loss or deactivation of surface functional groups, and biological contamination. These problems lead to short adsorbent lifespan, poor reusability, and increased production costs and environmental pressure.

[0007] In summary, there is an urgent need in this field to develop a novel ulinastatin adsorbent that simultaneously meets the requirements of high selectivity, high adsorption capacity and fast mass transfer rate, mild elution conditions and high recovery rate, as well as excellent physicochemical stability and reusability. Therefore, developing a novel ulinastatin adsorbent is of great scientific significance and practical application value for innovating its production process, reducing production costs, and ensuring drug supply. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention proposes a porous ulinastatin adsorbent with a responsive shell, its preparation method, and its application.

[0009] This invention provides a method for preparing a porous ulinastatin adsorbent with a responsive shell, comprising the following steps:

[0010] Step 1: Prepare an aqueous phase using 1–2 g agarose, 0.5–1 g polyvinyl alcohol, and 80–120 ml deionized water; prepare an oil phase using 6–8 g styrene, 1–2 g divinylbenzene, 0.5–1 g MOF nanoparticles, and 0.1–0.2 g octylphenol polyoxyethylene ether; add the oil phase dropwise to the aqueous phase and shear using a high-speed shear emulsifier to form a stable bicontinuous phase emulsion, followed by an ice-water bath to obtain a solidified emulsion;

[0011] Step 2: Add 0.1-0.2g of initiator to the cured emulsion obtained in Step 1, introduce high-purity nitrogen gas, stir and filter under water bath conditions to obtain a solid product, wash the solid product with hot water to obtain polymer microspheres;

[0012] Step 3: Place the polymer microspheres obtained in Step 2 into a 0.1M disodium ethylenediaminetetraacetate solution and stir to obtain polymer microspheres with mesoporous structures. Wash the polymer microspheres with mesoporous structures with deionized water and then treat them with hot water to obtain polymer microspheres with macropores and mesopores. Then, process them in a supercritical CO2 extraction device to obtain porous microspheres.

[0013] Step 4: After vacuum drying the porous microspheres obtained in Step 3 in a vacuum drying oven, disperse them in anhydrous toluene to obtain a reaction solution. After ultrasonic treatment, add 1-2 mL of monomer and 0.1-0.2 mL of ligand to the reaction solution, purge with nitrogen, and add 0.03-0.06 g of CuBr catalyst to obtain a reaction system. Seal the reaction system, place it in an oil bath and stir slowly with magnetic force. Wash the reaction system alternately with tetrahydrofuran and methanol to obtain the composite microsphere intermediate product.

[0014] Step 5: Disperse the composite microsphere intermediate obtained in Step 4 in a methanol-water mixed solvent, add 2-3g of monomer, purge with nitrogen gas, then add 0.03-0.06g of initiator, and react in a water bath to generate composite microspheres; after the reaction is complete, wash the composite microspheres sequentially with 1M NaCl solution and deionized water until the conductivity of the washing solution remains unchanged, and then place them in a vacuum drying oven for vacuum drying to obtain a porous ulinastatin adsorbent with a responsive shell.

[0015] Preferably, in step one, the polyvinyl alcohol is PVA-1788 type with a degree of alcoholysis of 87-89%; the styrene is washed with 5% NaOH solution to remove the polymerization inhibitor before use; the MOF nanoparticles are vinyl-modified MIL-100(Fe) with a particle size of 50-100 nm; the specific steps of adding the oil phase to the aqueous phase and shearing it with a high-speed shear emulsifier are as follows: in a constant temperature water bath of 50-70°C, the oil phase is added to the aqueous phase at a rate of 0.5-1 mL / min, while simultaneously shearing it with a high-speed shear emulsifier at a speed of 8000-10000 rpm for 5-8 minutes.

[0016] Preferably, in step two, the initiator is selected from one of azobisisobutyronitrile, azobisisobutyronitrile, or benzoyl peroxide; the specific steps for introducing high-purity nitrogen gas, stirring, filtering, and obtaining a solid product under water bath conditions, and washing the solid product with hot water to obtain polymer microspheres are as follows: introducing high-purity nitrogen gas to remove oxygen for 30-40 minutes, stirring slowly at 150-200 rpm in a constant temperature water bath at 55-65°C, and polymerizing for 20-24 hours. After the reaction is completed, filtering and collecting the solid product, and washing it with hot water at 50-60°C 3-4 times to obtain polymer microspheres.

[0017] Preferably, in step three, the specific conditions for hot water treatment are 80–90°C for 2–3 hours; the specific steps for stirring are stirring at 50–60°C for 10–12 hours; the pH of the disodium ethylenediaminetetraacetate solution is 8–8.5; and the specific conditions for placing the device in the supercritical CO2 extraction apparatus are a temperature of 40–50°C and a pressure of 30–40 MPa.

[0018] Preferably, in step four, the monomer is dimethylaminoethyl methacrylate, and the ligand is N,N,N',N'',N''-pentamethyldivinyltriamine; the dimethylaminoethyl methacrylate is treated with an alkaline alumina column to remove polymerization inhibitors before use; the nitrogen purging time is 30-40 minutes; the specific steps of the oil bath and slow magnetic stirring are as follows: react in an oil bath at 80-90°C for 5-6 hours, during which slow magnetic stirring at 200-220 rpm is maintained.

[0019] Preferably, in step five, the volume ratio of the methanol-water mixed solvent is 1:1; the monomer is sulfobetaine methacrylate; the nitrogen purging time is 20-30 minutes; the initiator is 2,2'-azobis(2-methylpropanediamine) dihydrochloride; the water bath reaction conditions are 60-80°C for 3-4 hours; and the vacuum drying treatment in a vacuum drying oven is 40-50°C for 8-12 hours.

[0020] The present invention also provides a porous ulinastatin adsorbent with a responsive shell prepared according to the method.

[0021] The present invention also provides the application of the porous ulinastatin adsorbent with the responsive shell in the adsorption of ulinastatin.

[0022] The beneficial effects of this invention are:

[0023] This invention achieves multi-level synergistic recognition and efficient loading of ulinastatin by constructing a three-tiered interconnected pore structure of macropores, mesopores, and micropores, and designing a pH / ionic strength dual-responsive mixed-charge shell. The mixed-charge shell precisely matches the surface properties of ulinastatin through electrostatic complementarity, hydrophobic synergy, and a hydrogen bond network, while exhibiting weak adsorption of impurity proteins. The three-tiered interconnected pore structure ensures rapid diffusion and high-capacity adsorption of ulinastatin molecules. This allows for the single extraction of high-purity, high-concentration ulinastatin from urine, simplifying subsequent purification steps.

[0024] The adsorption-desorption process of this invention is entirely based on physical action, requiring no destructive chemical reagents such as strong acids, strong bases, or organic solvents. Only a NaCl buffer solution at pH 6.0 is needed to achieve efficient desorption of ulinastatin by shielding electrostatic interactions and triggering conformational changes in the polymer chains, resulting in a high elution recovery rate. These mild conditions maximize the protection of ulinastatin's biological activity and avoid chemical damage to the material. After elution, the material quickly returns to its initial adsorption state, completing regeneration. This process is energy-saving and environmentally friendly, significantly reducing eluent costs and wastewater treatment costs.

[0025] This invention presents a porous ulinastatin adsorbent with a responsive shell, based on a fully synthetic polymer matrix. Its robust structure and excellent stability ensure continuous and economical production. The highly cross-linked polyvinyl alcohol / styrene / divinylbenzene core and MOF nanoparticle-reinforced framework endow the porous ulinastatin adsorbent with a responsive shell with superior mechanical strength, capable of withstanding the pressure in industrial chromatography columns and resisting breakage. Since both recognition and desorption are physical processes, and the porous ulinastatin adsorbent with a responsive shell has a stable structure, the frequency of adsorbent replacement and overall cost during production are significantly reduced, providing a reliable guarantee for large-scale, continuous industrial production. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the preparation process of the porous ulinastatin adsorbent with a responsive shell in this invention.

[0027] Figure 1 In the middle: 1: aqueous phase; 2: oil phase; 3: cured emulsion; 4: polymer microspheres; 5: composite microspheres; 51: mesoporous structure; 52: macroporous structure; 53: microporous structure; 6: composite microsphere intermediate; 7: porous ulinastatin adsorbent with responsive shell. Detailed Implementation

[0028] Example 1:

[0029] To better illustrate the preparation process involved in this invention and its advantages over the prior art, it will be based on... Figure 1 Further explanation will follow.

[0030] according to Figure 1 As shown, a method for preparing a porous ulinastatin adsorbent with a responsive shell includes the following steps:

[0031] Step 1: Dissolve 2g agarose and 0.5g polyvinyl alcohol in 100mL deionized water, heat to 60°C and stir until completely dissolved, as the aqueous phase. The polyvinyl alcohol is PVA-1788 type with a degree of alcoholysis of 89%. Styrene is washed with 5% NaOH solution to remove polymerization inhibitors before use. Mix 8g styrene, 2g divinylbenzene, 1g metal-organic framework nanoparticles (MOF nanoparticles), and 0.1g octylphenol polyoxyethylene ether evenly as the aqueous phase. The oil phase, in which the MOF nanoparticles are vinyl-modified MIL-100(Fe) with a particle size of 80 nm, is added dropwise to the aqueous phase at a rate of 1 mL / min in a 60°C constant temperature water bath. At the same time, a high-speed shear emulsifier is used to shear the emulsion at 10,000 rpm for 5 minutes to form a stable bicontinuous phase emulsion. The bicontinuous phase emulsion is then immediately transferred to an ice-water bath and rapidly cooled to below 10°C to gel the agarose and fix the emulsion structure to obtain a solidified emulsion.

[0032] Before use, styrene is washed with a 5% NaOH solution to remove the polymerization inhibitor. The procedure for removing the polymerization inhibitor is as follows: Pour PVA-1788 styrene into a separatory funnel, the volume of which should not exceed half the volume of the separatory funnel. Add an equal volume of 5% NaOH solution to the separatory funnel. Tightly stopper the separatory funnel. Holding the separatory funnel, invert it and shake vigorously for 2 minutes to ensure sufficient contact between the two phases. Let it stand for 15 minutes until the organic phase (upper layer, styrene) and the aqueous phase (lower layer, NaOH solution) are completely and clearly separated. Open the stopcock and discharge the lower yellow NaOH waste liquid into a waste container. To ensure complete removal of the polymerization inhibitor, repeat the above steps twice using fresh 5% NaOH solution until the aqueous phase becomes very light in color or almost colorless. Finally, add an equal volume of deionized water to the separatory funnel, shake vigorously to wash, and remove residual alkali. After standing and separating, discard the lower aqueous phase. This washing step should be repeated 3 times until the washing aqueous phase is neutral (tested with pH paper). Add sufficient lumps of anhydrous sodium sulfate or anhydrous magnesium sulfate, gently shake until the powder no longer clumps and remains loose and fluid. The drying process should continue for 30 minutes to obtain polyvinyl alcohol with the polymerization inhibitor removed.

[0033] Step 2: Add 0.2g of initiator azobisisobutyronitrile to the cured emulsion obtained in Step 1, purge with high-purity nitrogen to remove oxygen for 30 minutes, and polymerize at 200rpm in a constant temperature water bath at 65°C for 24 hours. After the reaction is complete, filter to obtain a solid product. Wash the solid product three times with 60°C hot water to remove residual surfactant octylphenol polyoxyethylene ether and unreacted styrene and divinylbenzene monomers, obtaining polymer microspheres.

[0034] Step 3: Place the polymer microspheres obtained in Step 2 into 200 mL of 0.1 M disodium ethylenediaminetetraacetate solution (pH 8.5), and stir at 60°C for 12 hours to remove Fe from the MOF nanoparticles. 3+ Ions are used to form a mesoporous structure of 2-50 nm, resulting in polymer microspheres with mesoporous structures. These microspheres are then washed with deionized water and placed in 90°C hot water for 2 hours to dissolve agarose, forming a macroporous structure. This yields polymer microspheres with both macropores and mesopores. Finally, these microspheres are placed in a supercritical CO2 extraction apparatus and treated at 40°C and 30 MPa for 4 hours to extract the small amount of unreacted styrene and divinylbenzene monomers remaining from the polymerization process, forming a microporous structure and resulting in porous microspheres with three-level interconnected channels. These are referred to as porous microspheres.

[0035] Step 4: After vacuum drying the porous microspheres obtained in Step 3 at 60°C for 2 hours in a vacuum drying oven, disperse them in 50 mL of anhydrous toluene to obtain a reaction solution. Sonicate the reaction solution for 30 minutes to ensure dispersion of the porous microspheres. Add 2 mL of dimethylaminoethyl methacrylate monomer to the reaction solution. Before use, dimethylaminoethyl methacrylate is treated with an alkaline alumina column to remove polymerization inhibitors. Then add 0.1 mL of N,N,N',N'',N''-pentamethyldivinyltriamine ligand, purge with nitrogen for 30 minutes to remove oxygen, and add 0.05 g of CuBr catalyst under nitrogen protection to obtain the reaction system. Seal the reaction system and react in a 90°C oil bath for 6 hours, maintaining slow magnetic stirring at 200 rpm. After the reaction, wash three times alternately with tetrahydrofuran and methanol to remove unreacted monomers and catalyst, obtaining a composite microsphere intermediate with a poly(N,N-dimethylaminoethyl methacrylate) shell, referred to as: composite microsphere intermediate.

[0036] The specific steps for removing the polymerization inhibitor from dimethylaminoethyl methacrylate (DME) using an alkaline alumina column before use are as follows: First, close the stopcock at the bottom of the glass column and add approximately 1 / 3 volume of anhydrous diethyl ether (or n-hexane). Slowly and in batches, add 150-mesh alkaline alumina powder into the column through a funnel, while gently tapping the column wall to ensure uniform settling of the alumina particles, removing air bubbles and forming a tight, crack-free stationary phase bed. The alumina packing height should be approximately 2 / 3 of the column height. Open the stopcock at the bottom of the column, allowing the solvent to flow slowly under gravity until the liquid level just reaches the top surface of the alumina packing. Next, mix the DME to be purified with an equal volume of anhydrous diethyl ether and gently shake to ensure homogeneity. Use a dropper to draw up the mixed sample and slowly and evenly add it along the column wall to the top surface of the alumina packing, avoiding impact on the packing. Further open the stopcock at the bottom of the column, controlling the flow rate at 3 mL / min (2 drops per second), to allow the sample to completely penetrate the packing. Subsequently, a large amount of anhydrous diethyl ether was continuously added as eluent for elution. The colorless, transparent eluent was collected in a round-bottom flask. The yellow polymerization inhibitor component reacted strongly with the basic alumina and was retained at the top of the column, forming a distinct yellow band. The collected eluent was then carefully distilled under reduced pressure using a rotary evaporator at ≤35°C in a water bath to remove the anhydrous diethyl ether solvent. This yielded dimethylaminoethyl methacrylate with the polymerization inhibitor removed by the basic alumina column.

[0037] Step 5: The composite microsphere intermediate obtained in Step 4 was redispersed in 50 ml of a methanol-water mixed solvent with a methanol-water volume ratio of 1:1. Then, 3 g of monomer, namely sulfobetaine methacrylate, was added. Nitrogen gas was purged for 20 minutes to remove oxygen. Then, 0.05 g of initiator, namely 2,2'-azobis(2-methylpropanediamine) dihydrochloride, was added. The reaction was carried out in a 60°C water bath for 4 hours to generate poly(sulfobetaine methacrylate) composite microspheres, referred to as: composite microspheres. After the reaction, the composite microspheres were washed sequentially with 1M NaCl solution and deionized water until the conductivity of the washing solution remained unchanged. Subsequently, they were vacuum dried at 40°C for 12 hours to obtain: a porous ulinastatin adsorbent with a responsive shell.

[0038] Example 2:

[0039] Except for the addition of 0.6g of MOF nanoparticles in step one, the other steps are the same as in Example 1.

[0040] Example 3:

[0041] Except for the addition of 0.8g of MOF nanoparticles in step one, the other steps are the same as in Example 1.

[0042] Example 4:

[0043] Except for the addition of 1.2g of MOF nanoparticles in step one, the other steps are the same as in Example 1.

[0044] Comparative Example 1:

[0045] Except for step one, which does not include MOF nanoparticles, and steps four and five, the remaining steps are the same as in Example 1, resulting in porous microspheres without MOF and responsive shell.

[0046] Effect test:

[0047] 1. Specific surface area calculation method:

[0048] This invention employs a low-temperature nitrogen adsorption method, using a fully automated specific surface area and pore size analyzer to determine the specific surface area of ​​the adsorbent. Before testing, the sample is degassed under vacuum at 120°C for 6 hours. Nitrogen adsorption-desorption isotherms are collected at -196°C, and the specific surface area is calculated using the BET (Brunauer-Emmett-Teller) model within a relative pressure range of 0.05-0.35. This is the most commonly used and universally accepted standard method for characterizing porous materials.

[0049] 2. Specific steps for ulinastatin adsorption:

[0050] Step 1: Take 5.0 g of the porous ulinastatin adsorbent with a responsive shell prepared in Example 1 and pack it into a chromatography column with a diameter of 1.5 cm and a height of 15 cm to form a uniform adsorption bed. Equilibrate the adsorbent by passing 7 column volumes of pre-cooled PBS buffer (0.01 M, pH 7.4) through the column at a flow rate of 1.0 mL / min to bring it to its optimal initial adsorption state.

[0051] Step 2: Slowly pump the urine sample into the adsorption column at a low flow rate of 1.0 mL / min. This process is best performed in a 4°C cold chamber to maximize the bioactivity of ulinastatin. Collect the flow-through solution, which can be retained for adsorption capacity analysis.

[0052] Step 3: Elute the porous ulinastatin adsorbent with the responsive shell using 15 column volumes of PBS buffer (pH 7.4) containing 0.15 M NaCl. This moderately ionic strength buffer effectively elutes impurities without eluting specifically bound ulinastatin. Continue eluting until the UV absorption baseline (at 280 nm) of the effluent returns to a stable state, indicating that the impurities have been washed away.

[0053] Step 4: Use PBS buffer (pH 6.0) containing 1.0 M NaCl as the elution buffer. Elute at a flow rate of 1.0 mL / min and begin collecting the eluent. Monitor with a UV detector and collect the eluent fraction showing a protein peak at 280 nm. This fraction is the solution rich in high-purity ulinastatin.

[0054] Step 5: First, thoroughly wash with 5 column volumes of pH 6.0 PBS buffer containing 1.0 M NaCl to ensure no residue remains. Then, wash with at least 10 column volumes of PBS buffer (0.01 M, pH 7.4) until the effluent pH returns to 7.4 and the conductivity drops to match the initial equilibration solution. At this point, the adsorption column is fully regenerated and can be used immediately for the next purification cycle.

[0055] The formula for calculating the adsorption capacity of ulinastatin is:

[0056] ;

[0057] in, Ulinastatin adsorption capacity (mg / g). The initial concentration (mg / L) of the ulinastatin solution before adsorption begins. The concentration of ulinastatin remaining in the solution (mg / L) after adsorption reaches equilibrium. The total volume (L) of the solution used in the adsorption experiment. The dry weight (g) of the porous ulinastatin adsorbent with a responsive shell that participated in the adsorption experiment.

[0058] 3. Calculation method for elution recovery rate:

[0059] The formula for calculating the elution recovery rate is:

[0060] ;

[0061] in, Eluent recovery rate (%) The concentration of ulinastatin remaining in the solution (mg / L) after adsorption reaches equilibrium. The total volume (L) of the ulinastatin-rich eluent collected. Ulinastatin adsorption capacity (mg / g). The dry weight (g) of the porous ulinastatin adsorbent with a responsive shell that participated in the adsorption experiment.

[0062] 4. Calculation method for capacity retention rate after 50 reuses:

[0063] The formula for calculating the capacity retention rate after 50 reuses is as follows:

[0064] ;

[0065] in, The capacity retention rate (%) after 50 reuses. This represents the adsorption capacity after the 50th cycle. This represents the initial adsorption capacity. ,in, The initial concentration (mg / L) of the ulinastatin solution before adsorption begins. The concentration of ulinastatin remaining in the solution (mg / L) after adsorption reaches equilibrium. The total volume (L) of the solution used in the adsorption experiment. The dry weight (g) of the porous ulinastatin adsorbent with a responsive shell that participated in the adsorption experiment.

[0066] The performance of Examples 1-4 and Comparative Example 1 of this invention is shown in Table 1.

[0067] Table 1. Performance test results of Examples 1-4 and Comparative Example 1:

[0068]

[0069] The principle of this invention:

[0070] The synergistic mass transfer principle of the three-tiered interconnected pore structure in step three is as follows: Macropores (1-10 μm) allow for rapid urine permeation and flow, significantly reducing fluid resistance. When urine flows through the porous ulinastatin adsorbent with a responsive shell, the macropore structure ensures uniform liquid distribution, avoids channeling, and improves treatment efficiency. Ulinastatin molecules with a hydrated diameter of approximately 5-8 nm can freely enter the interior of the porous ulinastatin adsorbent with a responsive shell. Mesopores of 2-50 nm serve as the main adsorption sites, providing the primary specific surface area and specific binding sites. The mesopores formed by MOF nanoparticles have a narrow pore size distribution, with a size well-matched to the ulinastatin molecule size, effectively blocking larger impurity proteins (such as albumin, approximately 7 nm in diameter) from entering the deep adsorption sites. Micropores smaller than 2 nm serve as capacity-enhancing regions, further improving adsorption capacity through micropore filling. The strong van der Waals forces generated by micropores enable the physical adsorption of ulinastatin molecules. This effect is particularly important under low concentration conditions, ensuring a wide dynamic adsorption range for porous ulinastatin adsorbents with responsive shells. The three-level interconnected channels form a "diffusion-adsorption" synergistic system. Macropores ensure rapid transport, mesopores provide selective channels, and micropores enhance the overall capacity, resolving the contradiction between "slow mass transfer" and "low capacity" in traditional adsorbents.

[0071] The mechanism of responsive shell recognition and controlled release of ulinastatin molecules is as follows: In a urinary environment (pH 7.4), the tertiary amino group (pKa ≈ 7.5) of N,N-dimethylaminoethyl methacrylate (NMA) is partially deprotonated, resulting in an electrically neutral and moderately extended molecular chain. The hydrophobic methyl group of the NMA backbone interacts with the hydrophobic region of ulinastatin for adsorption. Under acidic elution conditions (pH 6.0), NMA is fully protonated and becomes positively charged. The molecular chain is highly extended due to electrostatic repulsion. Simultaneously, NMA and the positively charged region on the ulinastatin surface generate electrostatic repulsion, promoting desorption.

[0072] In urine (a low ionic strength environment), the zwitterionic groups of polymethyl methacrylate sulfonate betaine undergo intramolecular positive and negative charge interactions, causing the chain conformation to coil up and form a dense hydrophilic layer, allowing small molecules of ulinastatin to pass through but blocking large molecular impurities. In the eluent (a high ionic strength environment), salt ions shield the intramolecular electrostatic interactions of polymethyl methacrylate sulfonate betaine, causing the chain conformation to extend, altering the surface topology, disrupting the original binding equilibrium, and promoting desorption.

[0073] Hydrophobic synergy and hydrogen bond network synergy promote the adsorption of ulinastatin. The hydrophobic backbone of N,N-dimethylaminoethyl methacrylate interacts with the hydrophobic regions of ulinastatin. At the same time, the tertiary amine group of N,N-dimethylaminoethyl methacrylate and the carboxyl group of polymethyl methacrylate sulfobetaine form hydrogen bonds with the glycosyl group and peptide chain of ulinastatin.

[0074] When eluting with NaCl buffer at pH 6.0, changes in pH and ionic strength lead to a synergistic conformational transition in the two responsive polymers, N,N-dimethylaminoethyl methacrylate and polymethyl methacrylate sulfobetaine. The high-salt environment first disrupts the electrostatic interactions and the conformation of polymethyl methacrylate sulfobetaine, while the acidic environment subsequently induces the protonation and chain extension of N,N-dimethylaminoethyl methacrylate. These two changes work together to completely alter the surface properties of the material, achieving gentle yet complete elution.

[0075] The functional mechanism of MOF nanoparticles:

[0076] The crystal structure of MIL-100(Fe) provides a template for mesoporous formation. Its 3-5 nm cage-like structure, after etching, forms uniform mesopores. MOF particles act as crosslinking points during polymerization, enhancing the mechanical strength of the polymer network. Residual ligands after etching may bind to histidine residues of ulinastatin through coordination. Fe 3+ The vacancies left after the ions are removed can serve as additional adsorption sites.

[0077] The steps in the method of this invention can be adjusted, combined, or deleted according to actual needs. In this invention, the descriptions of each embodiment have their own emphasis; parts not detailed or described in a certain embodiment can be referred to in the relevant descriptions of other embodiments. The technical features of this invention can be combined arbitrarily. To keep the description concise, not all possible combinations of the technical features in the embodiments are described; however, as long as these combinations of technical features do not contradict each other, they should be considered within the scope of this invention.

[0078] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A method for preparing a porous ulinastatin adsorbent having a responsive shell, characterized by, It comprises the following steps: Step one: preparing an aqueous phase with 1-2 g of agarose, 0.5-1 g of polyvinyl alcohol, and 80-120 mL of deionized water; preparing an oil phase with 6-8 g of styrene, 1-2 g of divinylbenzene, 0.5-1 g of MOF nanoparticles, and 0.1-0.2 g of octylphenol polyoxyethylene ether; adding the oil phase into the aqueous phase drop by drop and shearing with a high-speed shearing emulsifier to form a stable bicontinuous phase emulsion, and then obtaining a solidified emulsion in an ice water bath; the MOF nanoparticles are vinyl-modified MIL-100(Fe), and the particle size of the vinyl-modified MIL-100(Fe) is 50-100 nm; Step two: adding 0.1-0.2 g of an initiator to the solidified emulsion obtained in step one, purging high-purity nitrogen, stirring under water bath conditions, filtering, obtaining a solid product, and washing the solid product with hot water to obtain polymer microspheres; Step three: placing the polymer microspheres obtained in step two in a 0.1M ethylenediaminetetraacetic acid disodium solution, stirring, obtaining polymer microspheres with mesopores, washing with deionized water first, then treating with hot water, obtaining polymer microspheres with macropores and mesopores, and placing the polymer microspheres in a supercritical CO2 extraction device for treatment to obtain porous microspheres; Step four: placing the porous microspheres obtained in step three in a vacuum drying oven for vacuum drying, dispersing the porous microspheres in anhydrous toluene to obtain a reaction solution, ultrasonic treatment, adding 1-2 mL of dimethylaminoethyl methacrylate and 0.1-0.2 mL of N,N,N',N'',N''-pentamethyldivinyltriamine to the reaction solution, purging nitrogen, adding 0.03-0.06 g of CuBr catalyst to obtain a reaction system, sealing the reaction system, oil bath and slow magnetic stirring, and washing the reaction system with tetrahydrofuran and methanol alternately to obtain a composite microsphere intermediate product; Step five: dispersing the composite microsphere intermediate product obtained in step four in a methanol-water mixed solvent, adding 2-3 g of sulfobetaine methacrylate, purging nitrogen, adding 0.03-0.06 g of an initiator, and water bath reaction to generate composite microspheres; after the reaction is completed, the composite microspheres are washed with 1M NaCl solution and deionized water in sequence until the conductivity of the washing liquid is unchanged, and the composite microspheres are placed in a vacuum drying oven for vacuum drying treatment to obtain a porous ulinastatin adsorbent with a responsive shell.

2. The method for preparing a porous ulinastatin adsorbent with a responsive shell as described in claim 1, characterized in that, In step one, the polyvinyl alcohol is PVA-1788 type with an alcoholysis degree of 87-89%; the styrene is washed with a 5% NaOH solution before use to remove the polymerization inhibitor; and the specific steps of adding the oil phase into the aqueous phase and shearing with a high-speed shearing emulsifier are as follows: adding the oil phase into the aqueous phase at a rate of 0.5-1 mL / min in a 50-70°C constant temperature water bath, and shearing with a high-speed shearing emulsifier at a speed of 8000-10000 rpm for 5-8 minutes.

3. The method for preparing a porous ulinastatin adsorbent with a responsive shell as described in claim 2, characterized in that, The initiator in the second step is selected from one of azobisisheptyronitrile, azobisisobutyronitrile or dibenzoyl peroxide; the high-purity nitrogen is introduced to remove oxygen for 30-40 minutes, the solid product is obtained by stirring in a water bath, filtering, and washing the solid product with hot water; the specific steps for obtaining the polymer microspheres are as follows: the high-purity nitrogen is introduced to remove oxygen for 30-40 minutes, the polymerization is carried out at 55-65°C in a constant-temperature water bath with slow stirring at 150-200 rpm for 20-24 hours, the solid product is collected by filtering after the reaction, and the polymer microspheres are obtained by washing the solid product with hot water at 50-60°C for 3-4 times.

4. The method for preparing a porous ulinastatin adsorbent with a responsive shell as described in claim 3, characterized in that, In the third step, the pH of the ethylenediaminetetraacetic acid disodium solution is 8-8.5; the specific stirring step is stirring at 50-60°C for 10-12 hours; the specific conditions for the hot water treatment are 80-90°C for 2-3 hours; and the specific conditions for placing in the supercritical CO2 extraction device are a temperature of 40-50°C and a pressure of 30-40 MPa.

5. The method for preparing a porous ulinastatin adsorbent with a responsive shell as described in claim 4, characterized in that, In the fourth step, the dimethylaminoethyl methacrylate is removed from the polymerization inhibitor by passing through an alkaline alumina column before use; the nitrogen is introduced for 30-40 minutes; and the specific steps for the oil bath and slow magnetic stirring are as follows: the reaction is carried out in an oil bath at 80-90°C for 5-6 hours, and the slow magnetic stirring is maintained at 200-220 rpm during the reaction.

6. The method of claim 5, wherein the porous ulinastatin adsorbent having a responsive shell is prepared by the steps of: In the fifth step, the volume ratio of methanol to water in the methanol-water mixed solvent is 1:1; the nitrogen is introduced for 20-30 minutes; the initiator is 2,2'-azobis(2-methylpropylamidine) dihydrochloride; the conditions for the water bath reaction are 60-80°C for 3-4 hours; and the conditions for the vacuum drying treatment in the vacuum drying oven are 40-50°C for 8-12 hours.

7. The porous ulinastatin adsorbent with a responsive shell prepared by the preparation method according to any one of claims 1-6.

8. The application of the porous ulinastatin adsorbent with a responsive shell according to claim 7 in adsorbing ulinastatin.

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