A blood perfusion adsorbent for removing endotoxin and free radicals, and a preparation method and application thereof

Abstract

The application discloses a blood perfusion adsorbent for removing endotoxins and free radicals, and a preparation method and application thereof. The adsorbent (P-PEI Cu / TA) has simple preparation process, low cost, good blood compatibility, endotoxin adsorption performance and the ability of removing free radicals generated by oxidative stress, can remove endotoxins in blood of patients with sepsis, and remove free radicals generated by oxidative stress caused by endotoxins. The development of the blood perfusion adsorbent of the application is helpful to extracorporeal blood perfusion treatment of patients with sepsis and reduction of medical cost.

Description

Technical Field

[0001] This invention belongs to the field of blood perfusion adsorbent technology, specifically relating to a blood perfusion adsorbent for scavenging endotoxins and free radicals, its preparation method, and its application. Background Technology

[0002] Sepsis is a common and fatal disease. Endotoxin-induced sepsis is a major cause of multi-organ failure and death in patients, with uncontrolled oxidative stress being a key contributing factor to death. Polymyxin B adsorption columns (PMX) are one product used to treat sepsis. Their structure consists of α-chloroacetamide-methylpolystyrene fiber covalently bound to polymyxin B. They utilize the antibacterial activity of polymyxin B against Gram-negative bacteria and its specific binding to endotoxins to remove endotoxins. However, the application of these adsorption columns is limited by the fact that polymyxin B is an antibiotic and the columns themselves are expensive. A copolymer membrane of acrylonitrile and sodium methacrylate sulfonate is surface-modified with linear polyethyleneimine (PEI) and heparin. PEI has abundant amino groups with strong positive charge, which can adsorb endotoxins through electrostatic interaction. However, this membrane is expensive. A cytokine adsorption box, made of polystyrene-divinylbenzene copolymer beads, exhibits good biocompatibility and can adsorb pro-inflammatory mediators, anti-inflammatory mediators, myoglobin, bilirubin, bile acids, PAMPs, and DAMPs, but cannot adsorb endotoxins. Currently used medical adsorbents target endotoxins and some cytokines, but do not address the removal of free radicals generated by oxidative stress. Therefore, there is an urgent need for a low-cost blood perfusion product that can remove free radicals and endotoxins generated by oxidative stress in the blood. Summary of the Invention

[0003] To address the problem of the inability to simultaneously eliminate endotoxins in sepsis and free radicals generated by oxidative stress, the primary objective of this invention is to provide a blood perfusion adsorbent with good blood compatibility that adsorbs endotoxins in the blood and eliminates free radicals generated by oxidative stress in the blood.

[0004] Another objective of this invention is to provide a specific method for preparing the above-mentioned porous polystyrene divinylbenzene microspheres based on metal phenolic networks and branched polyethyleneimine functionalization.

[0005] Another objective of this invention is the application of the aforementioned metal-phenolic network and polyethyleneimine-functionalized polystyrene-divinylbenzene porous microsphere adsorbent in hemoperfusion. This polystyrene-divinylbenzene microsphere adsorbent, based on tannic acid and polyethyleneimine functionalization, is particularly useful in the field of biomedical materials for the extracorporeal hemoperfusion treatment of sepsis. Utilizing the excellent biocompatibility of the polystyrene-divinylbenzene porous structure, the strong positively charged adsorption capacity of branched polyethyleneimine for toxins, and the oxidative stress scavenging ability of the metal-phenolic network constructed from tannic acid and copper, a hemoperfusion adsorbent with excellent biocompatibility and endotoxin and oxidative stress scavenging capabilities is obtained.

[0006] The objective of this invention is achieved through the following scheme.

[0007] A method for preparing a hemoperfusion adsorbent for scavenging endotoxins and free radicals includes the following steps:

[0008] (1) Mix polystyrene divinylbenzene microspheres, photoinitiator, mercaptopropionic acid and solvent, and obtain carboxylated polystyrene divinylbenzene microspheres by ultraviolet light initiation reaction;

[0009] (2) The carboxylated polystyrene divinylbenzene microspheres, crosslinking agent and solvent were mixed and incubated. Then the incubated microspheres were mixed with branched polyethyleneimine aqueous solution to carry out grafting reaction to obtain polystyrene divinylbenzene microspheres grafted with branched polyethyleneimine.

[0010] (3) Grafted branched polyethyleneimine polystyrene divinylbenzene microspheres were placed in a container containing tannic acid (TA) and Cu 2+ The blood perfusion adsorbent was incubated in a mixed aqueous solution of ions and purified to obtain a blood perfusion adsorbent that removes endotoxins and free radicals.

[0011] Preferably, the Cu in the mixed aqueous solution in step (3) 2+ The molar ratio of ions to tannic acid is (1-10):(1-10), and more preferably (1-2):(1-2).

[0012] Preferably, in step (3), the ratio of the polystyrene divinylbenzene microspheres grafted with branched polyethyleneimine to the mixed aqueous solution is 1 g: (10-100 ml).

[0013] Preferably, the incubation temperature in step (3) is 10 to 40°C, and the incubation time is 1 min to 1 h, more preferably 10 min to 20 min.

[0014] Preferably, the Cu in step (3) 2+ The ions originate from at least one of copper sulfate, copper sulfate monohydrate, and copper sulfate pentahydrate.

[0015] Preferably, in step (1),

[0016] The surface of the polystyrene-divinylbenzene microspheres contains double bonds;

[0017] The polystyrene-divinylbenzene microspheres are porous microspheres;

[0018] The polystyrene-divinylbenzene microspheres described herein have nanoscale pore sizes;

[0019] The polystyrene divinylbenzene microspheres have a particle size of 100–1000 μm;

[0020] The photoinitiator is at least one of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and 2,2-dimethoxy-2-phenylacetophenone;

[0021] The solvent is N,N-dimethylformamide.

[0022] Step (1) involves first adding photoinitiator and polystyrene divinylbenzene microspheres, and then adding mercaptopropionic acid. This is because mercaptopropionic acid is unstable, and side reactions should be prevented to avoid hindering the grafting reaction.

[0023] Step (1) is carried out in the dark to protect the photoinitiator, and nitrogen gas is used to remove oxygen from the solvent and reduce the occurrence of side reactions.

[0024] Preferably, in step (1),

[0025] The mass ratio of the polystyrene divinylbenzene microspheres, mercaptopropionic acid, photoinitiator, and solvent is (0.1-1):(0.1-1):(0.1-0.12):30.

[0026] The reaction is a photo-initiated free radical addition reaction between the residual double bonds of polystyrene divinylbenzene microspheres and the thiol group of mercaptopropionic acid.

[0027] The reaction temperature is 20–50°C, more preferably 30–40°C; the reaction time is 30 min–2 h, more preferably 30 min–1 h.

[0028] Preferably, in step (2),

[0029] The crosslinking agent is at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS);

[0030] The branched polyethyleneimine contains secondary amine group structures, primary amine group structures, and tertiary amine group structures;

[0031] The branched polyethyleneimine has a molecular weight of 600 to 100,000, more preferably 1,800;

[0032] The solvent is a phosphate buffer solution with pH = 7.4 and a preferred concentration of 0.01 mM.

[0033] Preferably, in step (2),

[0034] The carboxyl group and crosslinking agent molar ratio of the carboxylated polystyrene divinylbenzene microspheres is 1:

[0035] (1~20);

[0036] The concentration range of branched polyethyleneimine in the aqueous solution is 10–40 mg / ml;

[0037] The ratio of the mass of the incubated microspheres to the volume of the branched polyethyleneimine solution is 1g:

[0038] (20-80ml);

[0039] The carboxylated polystyrene divinylbenzene microspheres were mixed with the crosslinking agent and incubated for 1–12 h at a temperature of 20–40 °C.

[0040] The grafting reaction takes 1 to 48 hours and is carried out at a temperature of 20 to 40°C.

[0041] A blood perfusion adsorbent prepared by any of the above preparation methods.

[0042] The blood perfusion adsorbent is a spherical particle with a particle size of 100μm to 1000μm.

[0043] The above-described blood perfusion adsorbents are used to remove endotoxins and free radicals.

[0044] This invention employs a novel method using polystyrene-divinylbenzene microspheres (PS-DVB) as a substrate. The method involves grafting carboxylated microspheres via a mercaptoolefin click reaction, followed by a amidation reaction to graft polyethyleneimine onto the microsphere surface. Finally, a copper tannin-phenolic network-polyethyleneimine functional layer is electrostatically self-assembled. This system, without altering the blood compatibility of the PS-DVB microspheres, endows them with excellent hydrophilicity, endotoxin adsorption capacity, and the ability to scavenge large amounts of free radicals generated by oxidative stress, demonstrating significant potential for the in vitro treatment of sepsis.

[0045] The metal phenolic network constructed from tannic acid and Cu and the polystyrene divinylbenzene microspheres functionalized with polyethyleneimine of the present invention, while maintaining good blood compatibility, have the ability to remove endotoxins and free radicals, and can be used for hemoperfusion therapy for patients with sepsis.

[0046] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0047] (1) The materials used in this invention are low in cost, easy to obtain, simple to prepare, and have low toxicity.

[0048] (2) The blood perfusion adsorbent of the present invention has good blood compatibility and has the dual functions of endotoxin adsorption and free radical scavenging. It can effectively remove endotoxins and a large number of free radicals generated by oxidative stress in the blood of sepsis patients. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the structure of the blood perfusion microspheres of the present invention;

[0050] Figure 2 The infrared spectra of the blood perfusion microspheres obtained in Examples 1-2 of this invention are shown below.

[0051] Figure 3 The X-ray photoelectron spectra of the blood perfusion microspheres obtained in Examples 1-2 of this invention are shown below.

[0052] Figure 4 This is a graph showing the hemolysis rate of the obtained blood perfusion microspheres in Example 3 of the present invention;

[0053] Figure 5 This is a diagram showing the coagulation results of the obtained blood perfusion microspheres in Example 4 of the present invention;

[0054] Figure 6 This is a graph showing the cytotoxicity results of the obtained hemoperfusion microspheres in Example 5 of the present invention;

[0055] Figure 7 This is a graph showing the DPPH free radical scavenging results of the obtained blood perfusion microspheres in Example 6 of the present invention;

[0056] Figure 8 This is a graph showing the ABTS free radical scavenging results of the obtained blood perfusion microspheres in Example 7 of the present invention;

[0057] Figure 9 This is a graph showing the superoxide anion scavenging results of the obtained blood perfusion microspheres in Example 8 of the present invention;

[0058] Figure 10 This is a graph showing the hydrogen peroxide removal results of the obtained blood perfusion microspheres in Example 9 of the present invention;

[0059] Figure 11 This is a diagram showing the endotoxin adsorption results of the obtained blood perfusion microspheres in Example 10 of the present invention. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the implementation of the present invention is not limited thereto.

[0061] In the following examples, both the raw materials and reagents are readily available.

[0062] An adsorbent for adsorbing endotoxins in blood and removing free radicals generated by oxidative stress in blood, its preparation method and application, wherein the adsorbent uses polyethylene divinylbenzene microspheres as a substrate, and polyethyleneimine, tannic acid and Cu metal phenolic network together constitute the functional layer.

[0063] In this embodiment of the invention, polyethylene divinylbenzene microspheres are first grafted with mercaptopropionic acid under ultraviolet light at ambient temperature, then grafted with polyethyleneimine through an amidation reaction, and then a functional layer of tannic acid metal phenolic network is constructed by electrostatic self-assembly. After repeated washing with sterile water and anhydrous ethanol, and vacuum drying, the sample is stored in a sealed and dry environment.

[0064] Example 1: Preparation of blood perfusion microspheres (P-PEI Cu / TA 0.5)

[0065] (1) Add 0.12g of photoinitiator I2959, 1g of polystyrene divinylbenzene microspheres and 1g of mercaptopropionic acid to 30ml of DMF. Use vortexing and stirring to fully dissolve mercaptopropionic acid and photoinitiator to obtain reaction solution.

[0066] (2) The reaction solution was sealed and protected from light and stirred with nitrogen for 30 min. Then, it was stirred and reacted under a UV lamp (200W) for 1 h. The reaction solution was then removed and the microspheres were repeatedly washed with sterile water and anhydrous ethanol. After that, they were placed in a vacuum drying oven and dried at 40 °C to obtain microsphere 1 (P-COOH).

[0067] (3) Immerse 500 mg of microsphere 1 in 20 ml of phosphate buffer (1×PBS) (pH = 7.4), then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of -COOH(microsphere 1):EDC:NHS = 1:20:20 in the phosphate buffer containing microsphere 1. Adjust the pH to 5.5 with hydrochloric acid solution to obtain solution 1.

[0068] (4) Stir Solution 1 at 37°C for 1 hour, then aspirate the liquid and wash the microspheres three times with sterile water.

[0069] (5) Dissolve branched polyethyleneimine (MW=1800) at a concentration of 20 mg / ml in PBS with pH=7.41, and adjust the pH to 7.4 with hydrochloric acid solution to obtain solution 2.

[0070] (6) Mix 20 ml of solution 2 with all the cleaned microspheres, stir and react overnight at 37°C in the dark, then remove the liquid and wash repeatedly with sterile water and anhydrous ethanol, and dry in a vacuum drying oven at 40°C to obtain microsphere 2 (P-PEI).

[0071] (7) Dissolve tannic acid (TA) and copper sulfate pentahydrate (CuSO4·5H2O) in deionized water at a molar ratio of TA:CuSO4·5H2O = 2:1 to obtain solution 3.

[0072] (8) Microspheres 2 were immersed in solution 3 at a concentration of 0.01 mg / ml for 5 minutes, and then 10×PBS was added to adjust the pH to 7.4. After immersion for another 10 minutes, the liquid was removed, and the microspheres were repeatedly washed with sterile water and anhydrous ethanol and dried in a vacuum drying oven at 40℃ to obtain microspheres (P-PEI Cu / TA 0.5).

[0073] Example 2: Preparation of blood perfusion microspheres (P-PEI Cu / TA 1)

[0074] (1) Add 0.12g of photoinitiator I2959, 1g of polystyrene diethylene microspheres and 1g of mercaptopropionic acid to 30ml of DMF. Use vortexing and stirring to fully dissolve mercaptopropionic acid and photoinitiator to obtain reaction solution.

[0075] (2) The reaction solution was sealed and protected from light and stirred with nitrogen for 30 min. Then, it was stirred and reacted under a UV lamp (200W) for 1 h. The reaction solution was then removed and the microspheres were repeatedly washed with sterile water and anhydrous ethanol. After that, they were placed in a vacuum drying oven and dried at 40 °C to obtain microsphere 1 (P-COOH).

[0076] (3) Immerse 500 mg of microsphere 1 in 20 ml of phosphate buffer (1×PBS) (pH = 7.4), then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of -COOH(microsphere 1):EDC:NHS = 1:20:20 in the phosphate buffer containing microsphere 1. Adjust the pH to 5.5 with hydrochloric acid solution to obtain solution 1.

[0077] (4) Stir Solution 1 at 37°C for 1 hour, then aspirate the liquid and wash the microspheres three times with sterile water.

[0078] (5) Dissolve branched polyethyleneimine (MW=1800) at a concentration of 20 mg / ml in PBS with pH=7.41, and adjust the pH to 7.4 with hydrochloric acid solution to obtain solution 2.

[0079] (6) Then mix 20 ml of solution 2 with all the cleaned microspheres, stir and react overnight at 37°C in the dark, then remove the liquid and wash repeatedly with sterile water and anhydrous ethanol, and dry in a vacuum drying oven at 40°C to obtain microsphere 2 (P-PEI).

[0080] (7) Dissolve tannic acid (TA) and copper sulfate pentahydrate (CuSO4·5H2O) in deionized water at a molar ratio of TA:CuSO4·5H2O = 1:1 to obtain solution 3.

[0081] (8) Microspheres 2 were immersed in solution 3 at a concentration of 0.01 mg / ml for 5 min, and then 10×PBS was added to adjust the pH to 7.4. After that, they were immersed for 10 min, and then the liquid was removed and the microspheres were repeatedly washed with sterile water and anhydrous ethanol. They were dried in a vacuum drying oven at 40℃ to obtain microspheres (P-PEI Cu / TA 1).

[0082] Appropriate amounts of dried PS-DVB, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 powders were thoroughly mixed with KBr solid to prepare sample thin films. The chemical bond composition of the microspheres before and after grafting modification was investigated at room temperature. The scanning range was 4000–400 cm⁻¹. The results are shown in the table below. Figure 2 .

[0083] Figure 2 Compared to PS-DVB, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 groups exhibit broad absorption peaks at wavelengths of 3200–3700 nm, originating from the hydroxyl groups in TA and the NH groups in polyethyleneimine. They also show absorption peaks at 1714 nm, originating from the C=O group in the carboxyl group of mercaptopropionic acid, at 1645 nm, and at 1550 nm, originating from the NH groups in polyethyleneimine. Furthermore, they show absorption peaks at 1200 nm and 1045 nm, originating from the CO groups in tannic acid. These characteristics indicate the successful introduction of mercaptopropionic acid, polyethyleneimine, and tannic acid into PS-DVB microspheres.

[0084] The elemental composition and content of PS-DVB, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 microspheres were analyzed using X-ray photoelectron spectroscopy (XPS). The X-ray source was an Al K monochromator (hν = 1486.71 eV, 5 mA, 15 kV) with a vacuum degree less than 5 x 10⁻⁶. -8 mbar, results are shown Figure 3 .

[0085] Figure 3Compared to PS-DVB, the P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 groups showed the addition of binding energy peaks for Cu, N, and S, demonstrating the successful introduction of Cu, polyethyleneimine, and mercaptopropionic acid. The O element exhibited a stronger binding energy peak, characterizing the introduction of tannic acid and the construction of a copper-tannic acid complex network.

[0086] In this invention example, three groups were set up: PS-DVB, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1, to evaluate the coagulation, hemolysis, endotoxin adsorption performance, free radical scavenging ability, and other properties of this invention.

[0087] Comparative Example 1: Preparation of blood perfusion microspheres (P-PEI)

[0088] (1) Add 0.12g of photoinitiator I2959, 1g of polystyrene diethylene microspheres and 1g of mercaptopropionic acid to 30ml of DMF. Use vortexing and stirring to fully dissolve mercaptopropionic acid and photoinitiator to obtain reaction solution.

[0089] (2) The reaction solution was sealed and protected from light and stirred with nitrogen for 30 min. Then, it was stirred and reacted under a UV lamp (200W) for 1 h. The reaction solution was then removed and the microspheres were repeatedly washed with sterile water and anhydrous ethanol. After that, they were placed in a vacuum drying oven and dried at 40 °C to obtain microsphere 1 (P-COOH).

[0090] (3) Immerse 500 mg of microsphere 1 in 20 ml of phosphate buffer (1×PBS) (pH = 7.4), then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of -COOH(microsphere 1):EDC:NHS = 1:20:20 in the phosphate buffer containing microsphere 1. Adjust the pH to 5.5 with hydrochloric acid solution to obtain solution 1.

[0091] (4) Stir Solution 1 at 37°C for 1 hour, then aspirate the liquid and wash the microspheres three times with sterile water.

[0092] (5) Dissolve branched polyethyleneimine (MW=1800) at a concentration of 20 mg / ml in PBS with pH=7.41, and adjust the pH to 7.4 with hydrochloric acid solution to obtain solution 2.

[0093] (6) Then mix 20 ml of solution 2 with all the cleaned microspheres, stir and react overnight at 37°C in the dark, then remove the liquid and wash repeatedly with sterile water and anhydrous ethanol, and dry in a vacuum drying oven at 40°C to obtain microsphere 2 (P-PEI).

[0094] Comparative Example 2: Preparation of blood perfusion microspheres (P-PEI TA)

[0095] (1) Add 0.12g of photoinitiator I2959, 1g of polystyrene diethylene microspheres and 1g of mercaptopropionic acid to 30ml of DMF. Use vortexing and stirring to fully dissolve mercaptopropionic acid and photoinitiator to obtain reaction solution.

[0096] (2) The reaction solution was sealed and protected from light, and nitrogen gas was introduced and stirred for 30 min. Then, the reaction was carried out under a UV lamp (200W) with stirring for 1 h. The reaction solution was then aspirated, and the microspheres were repeatedly washed with sterile water and anhydrous ethanol. After that, they were placed in a vacuum drying oven and dried at 40 °C to obtain microsphere 1 (P-COOH).

[0097] (3) Immerse 500 mg of microsphere 1 in 20 ml of phosphate buffer (1×PBS) (pH = 7.4), then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of -COOH(microsphere 1):EDC:NHS = 1:20:20 in the phosphate buffer containing microsphere 1. Adjust the pH to 5.5 with hydrochloric acid solution to obtain solution 1.

[0098] (4) Stir Solution 1 at 37°C for 1 hour, then aspirate the liquid and wash the microspheres three times with sterile water.

[0099] (5) Dissolve branched polyethyleneimine (MW=1800) at a concentration of 20 mg / ml in PBS with pH=7.41, and adjust the pH to 7.4 with hydrochloric acid solution to obtain solution 2.

[0100] (6) Then mix 20 ml of solution 2 with all the cleaned microspheres, stir and react overnight at 37°C in the dark, then remove the liquid and wash repeatedly with sterile water and anhydrous ethanol, and dry in a vacuum drying oven at 40°C to obtain microsphere 2 (P-PEI).

[0101] (7) Dissolve tannic acid (TA) in deionized water at a concentration of 2 mg / ml to obtain solution 3.

[0102] (8) Microspheres 2 were immersed in solution 3 at a concentration of 0.01 mg / ml for 5 min, and then 10×PBS was added to adjust the pH to 7.4. After immersion for 10 min, the liquid was removed and the microspheres were repeatedly washed with sterile water and anhydrous ethanol and dried in a vacuum drying oven at 40℃ to obtain microspheres (P-PEI TA).

[0103] Example 3: Hemolysis Experiment

[0104] The following hemolysis test is performed according to the People's Republic of China Pharmaceutical Industry Standard (YY / T 1651.1-2019). The specific operating procedure for the hemolysis test is as follows:

[0105] (1) Add 8 mL of 4% red blood cell solution (Hongquan Bio) to 10 mL of 0.9% sodium chloride solution to prepare experimental blood, and place it in a constant temperature water bath at 37℃ for later use.

[0106] (2) Weigh 10 mg each of PS-DVB, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 and place them in test tubes. Three parallel samples were prepared.

[0107] (3) Add 1 mL of experimental blood and let it stand in a 37°C constant temperature water bath for 60 min.

[0108] (4) 1 ml of deionized water was mixed with 0.8 ml of 4% rabbit red blood cell solution and experimental blood as positive and negative controls, respectively;

[0109] (5) After incubation, centrifuge at 800g for 5 minutes, aspirate the supernatant and place it into a 96-well plate, and use an enzyme-linked immunosorbent assay (ELISA) reader to test the 545nm OD value of the sample.

[0110] (6) Calculate the hemolysis rate. If the hemolysis rate is <5%, the material is considered safe and meets the requirements for hemolysis testing. If the hemolysis rate is >5%, the material can induce the rupture of red blood cells and is not suitable for clinical use. Results are shown below. Figure 4 .

[0111] PS-DVB, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 are all less than 2%, meeting the national standard ISO 10993-5, which requires that the hemolysis rate of biomedical materials in contact with blood be less than 5%.

[0112] Example 4: Coagulation Test

[0113] (1) Weigh 20 mg each of PS-DVB, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 into test tubes, and make three parallel samples.

[0114] (2) Take 9 mL of fresh blood from New Zealand rabbits and mix it with 1 mL of 3.8% sodium citrate. Then centrifuge at 4000 rpm for 10 min to obtain anemic platelet plasma.

[0115] (3) Take 0.5 mL of plasma and 20 mg of microspheres and incubate them at 37 °C for 30 min. The control group is fresh rabbit plasma that has not been incubated with microspheres.

[0116] (4) Serum was collected, and the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) were measured using a coagulation analyzer. The control group consisted of fresh rabbit plasma that was not co-incubated with the microspheres. Results are shown below. Figure 5 .

[0117] Figure 5 In the control group, the APTT, PT, TT, and FIB values ​​of P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 were not significantly different from those of the control group, indicating that P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 have good blood compatibility.

[0118] Example 5: Cytotoxicity Experiment

[0119] (1) Weigh 10 mg each of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 and add them to a centrifuge tube.

[0120] (2) Add 1 mL of complete culture medium (DMEM:FBS:double antibiotic = 90:10:1), place it in a constant temperature air shaker, and incubate at 37℃ and 100 r / min for 24 h.

[0121] (3) Take out the DMEM solution and filter it three times with a 0.22μm sterile filter membrane to remove bacteria, and obtain the sterile extract of the corresponding microspheres for later use.

[0122] (4) L929 cells were seeded in 96-well plates at a density of 10,000 cells / well, and 100 μL of extract of different microspheres and 100 μL of complete culture medium were added respectively. The plates were then incubated in a 5% carbon dioxide, 37°C constant temperature cell culture incubator for 24 h.

[0123] (5) Gently aspirate the complete culture medium from the well plate and rinse twice with PBS solution.

[0124] (6) Add 200 μL of CCK-8 working solution (complete culture medium: CCK-8 stock solution = 10:1) to each well, wrap it with aluminum foil to protect it from light, place it in 5% carbon dioxide, and culture the cells at 37°C for 2 hours before taking it out.

[0125] (7) Gently aspirate 100 μL of solution from each well and transfer it to a new plate. Measure the absorbance of each well at 450 nm using a microplate reader. Perform three replicates for each sample. Results are shown below. Figure 6 .

[0126] Figure 6 In the study, the cell viability of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 were all greater than 90%, and there were no significant differences among them, demonstrating that the adsorbent has good biocompatibility.

[0127] Example 6: DPPH free radical scavenging experiment

[0128] (1) Dissolve a certain amount of DPPH in anhydrous ethanol solution and vortex mix.

[0129] (2) Weigh 10 mg each of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 into centrifuge tubes and set up three parallel samples.

[0130] (3) Add 4 ml of DPPH ethanol diluent to each sample and incubate in a shaker at 37°C for 8 min in the dark. The positive control is DPPH diluent and the negative control is deionized water.

[0131] (4) Pipette 100 μL into a 96-well plate and measure its absorbance at 517 nm. Calculate the clearance rate. See the results below. Figure 7 .

[0132] Figure 7 Among the samples, the P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 groups showed stronger DPPH radical scavenging effects compared to the PS-DVB and P-PEI groups. Specifically, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 achieved DPPH radical scavenging rates of 85.5% and 60.8%, respectively. This demonstrates that the adsorbent possesses DPPH radical scavenging capabilities.

[0133] Example 7: ABTS Free Radical Scavenging Experiment

[0134] (1) Mix equal volumes of 0.35 mg / ml K2S2O6 and 2.05 mg / ml ABTS aqueous solution and react overnight in the dark to obtain ABTS free radical solution.

[0135] (2) Weigh 10 mg each of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 into centrifuge tubes, and perform three parallel sets for each sample.

[0136] (3) Add 4 mL of ABTS diluent and incubate in a shaker at 37°C for 3 hours in the dark. The positive control is ABTS free radical diluent and the negative control is deionized water.

[0137] (4) Pipette 100 μL into a 96-well plate and measure its absorbance at 734 nm. Calculate the clearance rate. See the results below. Figure 8 .

[0138] Figure 8 Among the samples, the P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 groups showed stronger ABTS radical scavenging effects compared to the PS-DVB and P-PEI groups. Specifically, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 achieved ABTS radical scavenging rates of 90% and 65.3%, respectively. This demonstrates the free radical scavenging ability of the adsorbent.

[0139] Example 8: Superoxide Anion Scavenging Experiment

[0140] (1) Prepare a riboflavin-containing solution in PBS (25 mM, pH 7.4). 83-88-5 A solution of 20 μM L-methionine (12.5 mM) and 75 μM (MW = 817.6) (61.32 mg / L) nitrotetrazole blue (NBT) (20 μM), L-methionine (12.5 mM), and nitrotetrazole blue (NBT) (75 μM, MW = 817.6) (61.32 mg / L).

[0141] (2) Weigh 10 mg each of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 into centrifuge tubes, and perform three parallel sets for each sample.

[0142] (3) Add the microspheres to the prepared 300 μL mixed solution. Irradiate the mixture with ultraviolet (UV) light at a constant intensity of 25 °C for 10 minutes.

[0143] (4) The negative control was defined as a group containing riboflavin, L-methionine and NBT but placed in a dark environment. The positive control was defined as a group containing riboflavin, L-methionine and NBT, irradiated with ultraviolet light for a period of time, and the treated sample was defined as a UV-illuminated group containing riboflavin, L-methionine, NBT and microspheres.

[0144] (5) Take 200 μL of each sample and add it to a 96-well plate. Measure the absorbance at 534 nm. Calculate the clearance rate. See the results below. Figure 9 .

[0145] Figure 9Among the samples, the P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 groups showed stronger superoxide anion scavenging effects compared to the PS-DVB and P-PEI groups. Specifically, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 achieved superoxide anion scavenging rates of 55% and 43.6%, respectively. This demonstrates the superoxide anion scavenging ability of this adsorbent.

[0146] Example 9: Hydrogen peroxide scavenging experiment

[0147] (1) Weigh 10 mg each of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 into centrifuge tubes, and perform three parallel sets for each sample.

[0148] (2) Add 300 μL of 2.5 mM hydrogen peroxide solution to each centrifuge tube.

[0149] (3) The mixed solution was incubated at 37°C in the dark for 12 hours.

[0150] (4) Prepare the colorimetric solution using 1.33 mL Ti(SO4)2 (24 wt%), 8.33 mL H2SO4 (98 wt%), and 50 mL deionized water. Add 50 μL of the incubated mixture to 100 μL of the colorimetric solution.

[0151] (5) The blank control is defined as the group without microspheres.

[0152] (6) Pipette 100 μL into each well of a 96-well plate and measure the absorbance at 405 nm. Calculate the clearance rate. See the results below. Figure 10 .

[0153] Figure 10 Among the groups, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 showed stronger hydrogen peroxide removal effects compared to the PS-DVB group. P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 achieved hydrogen peroxide removal rates of 27.6% and 21.6%, respectively. This demonstrates the hydrogen peroxide removal capability of this adsorbent.

[0154] Example 10: Endotoxin Adsorption Capacity Experiment

[0155] (1) Take an appropriate amount of bacterial endotoxin standard, add it to the test water, reconstitute it, shake it on a vortex mixer for 15 minutes, and then dilute it to an endotoxin aqueous solution of 8 EU / mL.

[0156] (2) Weigh 10 mg of PS-DVB, P-PEI, P-PEI TA, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 adsorbent into a pyrogen-free test tube. Each sample is set up in three parallel groups. The positive control group is the one without sample, and the negative sample is the water used for endotoxin testing.

[0157] (3) Add 1 ml of endotoxin solution and allow it to adsorb statically at 37°C for 2 h.

[0158] (4) Then, the endotoxin content was detected using Beyotime's endotoxin detection kit. The results are shown in the figure. Figure 11 .

[0159] Figure 11 Among them, P-PEI, P-PEI Cu / TA 0.5, and P-PEI Cu / TA 1 showed higher endotoxin removal capacity compared to the PS-DVB group. P-PEI Cu / TA 0.5 and P-PEI Cu / TA 1 achieved endotoxin removal rates of 90.6% and 91.4%, respectively, proving that the adsorbent has endotoxin adsorption capacity.

[0160] The above specific embodiments are all feasible implementations of the present invention, and further describe in detail the purpose, technical solution and beneficial effects of the present invention. However, the implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals, characterized in that, Includes the following steps: (1) Mix polystyrene divinylbenzene microspheres, photoinitiator, mercaptopropionic acid and solvent, and obtain carboxylated polystyrene divinylbenzene microspheres by ultraviolet light initiation reaction; (2) Carboxylated polystyrene divinylbenzene microspheres, crosslinking agent and solvent are mixed and incubated, and then the incubated microspheres are mixed with branched polyethyleneimine aqueous solution to carry out grafting reaction to obtain polystyrene divinylbenzene microspheres grafted with branched polyethyleneimine. (3) Grafted branched polyethyleneimine polystyrene divinylbenzene microspheres were placed in a container containing tannic acid and Cu 2+ The blood perfusion adsorbent was incubated in a mixed aqueous solution of ions and purified to obtain a blood perfusion adsorbent that removes endotoxins and free radicals.

2. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, In step (3), Cu in the mixed aqueous solution 2+ The molar ratio of ions to tannic acid is (1-10):(1-10), and the ratio of the amount of the grafted branched polyethyleneimine polystyrene divinylbenzene microspheres to the mixed aqueous solution is 1g:(10-100ml).

3. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, The incubation temperature in step (3) is 10-40℃, and the incubation time is 1 min-1 h.

4. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, The Cu described in step (3) 2+ The ions originate from copper sulfate.

5. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, In step (1), The polystyrene divinylbenzene microspheres have a particle size of 100–1000 μm; The photoinitiator is at least one of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and 2,2-dimethoxy-2-phenylacetophenone; The solvent is N,N-dimethylformamide.

6. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, In step (1), The mass ratio of the polystyrene divinylbenzene microspheres, mercaptopropionic acid, photoinitiator, and solvent is (0.1~1):(0.1~1):(0.1~0.12):

30. The reaction temperature is 20–50°C, and the reaction time is 30 min–2 h.

7. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, In step (2), The crosslinking agent is at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The branched polyethyleneimine contains secondary amine group structures, primary amine group structures, and tertiary amine group structures; The molecular weight of the branched polyethyleneimine is 600 to 100,000; The solvent is phosphate buffer.

8. The method for preparing a blood perfusion adsorbent for scavenging endotoxins and free radicals according to claim 1, characterized in that, In step (2), The carboxyl group and crosslinking agent of the carboxylated polystyrene divinylbenzene microspheres have a molar ratio of 1:(1~20); The concentration range of branched polyethyleneimine in the aqueous solution is 10~40 mg / ml; The ratio of the mass of the incubated microspheres to the volume of the branched polyethyleneimine solution is 1g:(20~80ml). The carboxylated polystyrene divinylbenzene microspheres were mixed with the crosslinking agent and incubated for 1–12 h at a temperature of 20–40 °C. The grafting reaction takes 1 to 48 hours and is carried out at a temperature of 20 to 40°C.

9. A blood perfusion adsorbent prepared by the preparation method according to any one of claims 1-8.

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