Sodium polystyrenesulfonate modified chitosan as well as preparation method and application thereof

Abstract

The invention discloses sodium polystyrenesulfonate modified chitosan as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The modified chitosan provided by the invention can generate far better adsorption capacity with the histone by virtue of weak amino positive electricity, so that the adsorption of the chitosan on the damage-related molecular mode of the histone is realized, and the long-standing technical bottlenecks of poor selectivity, low efficiency and the like in the field of histone separation and adsorption are solved; and the preparation method is simple and high in biological safety, and has relatively strong technical frontier and market application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a sodium polystyrene sulfonate modified chitosan, its preparation method, and its application. Background Technology

[0002] Currently, histone adsorption has the following problems: (1) Poor selectivity: Existing adsorbents such as ion exchange resins are not specific enough for histone adsorption and are prone to co-adsorbing other basic proteins or impurities. (2) Low histone adsorption efficiency: Histones have weak binding force with the carrier, resulting in low adsorption capacity, difficulty in elution, and insufficient dynamic binding load. (3) Insufficient compatibility of adsorbents, making large-scale clinical application difficult: Histone neutralizers such as heparin can provide specific adsorption of histones through strong electrostatic efficiency. However, the significant anticoagulant properties of heparin itself make it difficult to use in critically ill patients with histone-mediated sepsis, thus limiting the clinical application of histone adsorption.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a sodium polystyrene sulfonate modified chitosan, its preparation method and its application, thereby providing a new solution for histone adsorption.

[0005] This invention is implemented as follows: In a first aspect, the present invention provides a sodium polystyrene sulfonate modified chitosan, comprising: chitosan microspheres and sodium polystyrene sulfonate and DNA within the pores of the chitosan microspheres; in the raw materials for preparing sodium polystyrene sulfonate modified chitosan, each 25g wet weight of chitosan microspheres is mixed with at least 50mL of sodium polystyrene sulfonate monomer solution, the concentration of the sodium polystyrene sulfonate monomer solution being 3% (m / v)-15% (m / v); the mixing mass ratio of the dry weight of the chitosan raw material to the dry weight of the DNA powder is 1-3:1, and the sodium polystyrene sulfonate monomer forms a sodium polystyrene sulfonate network structure within the pores of the chitosan microspheres through a cross-linking reaction.

[0006] Secondly, the present invention also provides a method for preparing sodium polystyrene sulfonate modified chitosan, which includes the following steps: i: Preparation of chitosan and DNA mixture: Mix the DNA-containing solution with the chitosan raw material in a certain proportion, with the mixing mass ratio of the dry weight of the chitosan raw material to the dry weight of the DNA powder being 1-3:1; ii: Add GDL acidifying agent to the mixture from step i and heat to react; iii: The reaction solution heated in step ii is dropped into a coagulation bath to prepare chitosan microspheres loaded with DNA; iv: After washing the DNA-loaded chitosan microspheres, they were contacted with a sodium styrene sulfonate monomer solution; through a cross-linking reaction, sodium styrene sulfonate-modified chitosan with sodium styrene sulfonate cross-linked in the chitosan microspheres was obtained.

[0007] Thirdly, the present invention also provides the application of sodium polystyrene sulfonate modified chitosan or sodium polystyrene sulfonate modified chitosan prepared by the above preparation method in the following (1) or (2): (1) Preparation of histone adsorption products; (2) Histone adsorption for non-disease treatment purposes; (3) Application in the preparation of drugs for the treatment of at least one of the following diseases: histone-mediated critical illnesses, thrombosis and coagulation disorders, autoimmune diseases and nervous system diseases.

[0008] Fourthly, the present invention also provides a histone adsorption product comprising: the above-mentioned sodium polystyrene sulfonate modified chitosan or sodium polystyrene sulfonate modified chitosan prepared by the above-mentioned preparation method.

[0009] Fifthly, the present invention also provides a medicament for treating at least one of the following diseases, wherein the disease is selected from at least one of the following: histone-mediated critical illness, thrombosis and coagulation dysfunction, autoimmune diseases, and nervous system diseases.

[0010] The present invention has the following beneficial effects: This invention involves initiating the polymerization reaction of sodium styrene sulfonate monomers within the porous structure of chitosan (CS) to generate a sodium styrene sulfonate network structure. The sodium styrene sulfonate then forms an interpenetrating network through in-situ crosslinking polymerization, thereby imbuing the chitosan with a large number of sulfonic acid groups (-SO3). - The abundant sulfonic acid groups in chitosan provide large negative charge centers. Histones are proteins rich in positively charged amino acids (especially lysine and arginine). Therefore, chitosan modified with sodium polystyrene sulfonate can electrostatically bind to histones rich in positively charged amino acids, with a binding force far exceeding that of unmodified chitosan relying solely on its weak amino positive charge. The function of chitosan microspheres changes from passive (due to its own weak amino positive charge) to active histone capture. Introducing sulfonic acid groups, a functional group, into the porous surface of chitosan allows for strong ion-pair interactions with the basic amino acid residues of histones, achieving near-affinity chromatographic selectivity. Therefore, modification with sodium polystyrene sulfonate significantly enhances the adsorption capacity of modified chitosan for histones. This enables the adsorption of histones, a damage-related molecular pattern, by chitosan, overcoming the long-standing technical bottlenecks of poor selectivity and low efficiency in the field of histone separation and adsorption. Furthermore, the preparation method provided by this invention is simple, biosafety-enhancing, and possesses strong technological advancement and market application potential.

[0011] Secondly, by incorporating DNA into chitosan, the DNA binds to chitosan through hydrogen bonds and electrostatic forces, which can improve the stability of the microspheres. At the same time, the phosphate groups of DNA can adsorb positively charged histones through electrostatic interactions, which helps to improve the adsorption performance of sodium polystyrene sulfonate modified chitosan for histones.

[0012] Secondly, the sodium polystyrene sulfonate modified chitosan prepared in this invention retains the initial morphology of chitosan microspheres and exhibits good biocompatibility. The raw materials for the microspheres, including chitosan, sodium polystyrene sulfonate, and DNA, do not possess significant anticoagulant activity. This transforms the application of chitosan-based materials from controlled-release carriers in clinical treatment to specific adsorbents for core damaging molecules (i.e., histones) during the development of critical illnesses such as sepsis. Utilizing sodium polystyrene sulfonate modified chitosan as a supplement to circulating histone adsorbents provides a foundation for immunomodulatory therapy in critically ill patients with sepsis and improves their survival rate. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 Electron microscopy images of microspheres show porous polymer structures; Figure 2 Figure 1 shows the results of static adsorption efficiency detection of histones in CS, C3D1, and C3D1-PSS microspheres. Figure 3 Figure 1 shows the effects of CS, C3D1, and C3D1-PSS on complement activation. Figure 4 The results of the hemolysis rate analysis of CS, C3D1, and C3D1-PSS microspheres are shown in the figure. Figure 5 Figure 1 shows the effects of CS, C3D1, and C3D1-PSS microspheres on blood routine tests. Figure 6 Figure 1 shows the flow cytometry results showing that CS, C3D1, and C3D1-PSS can significantly reduce histone-induced endothelial cell apoptosis. Figure 7 The figure shows the effect of C3D1-PSS microspheres on the in vitro adsorption of histones on kidney and lung tissue damage. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0016] In a first aspect, the present invention provides a sodium polystyrene sulfonate modified chitosan, comprising: chitosan microspheres and sodium polystyrene sulfonate and DNA within the pores of the chitosan microspheres; in the raw materials for preparing sodium polystyrene sulfonate modified chitosan, each 25g wet weight of chitosan microspheres is mixed with at least 50mL of sodium polystyrene sulfonate monomer solution, the concentration of the sodium polystyrene sulfonate monomer solution being 3% (m / v)-15% (m / v); the mixing mass ratio of the dry weight of the chitosan raw material to the dry weight of the DNA powder is 1-3:1, and the sodium polystyrene sulfonate monomer forms a sodium polystyrene sulfonate network structure within the pores of the chitosan microspheres through a cross-linking reaction.

[0017] In one embodiment, 25g of wet-weight chitosan microspheres are mixed with 50mL-100mL of sodium styrene sulfonate monomer solution.

[0018] Setting the sodium styrene sulfonate monomer solution in excess relative to the chitosan microspheres helps to more fully fill the pores of the chitosan microspheres with sodium styrene sulfonate monomer material, which facilitates the formation of a three-dimensional network structure within the pores of chitosan through subsequent polymerization.

[0019] For example, each 25g wet weight chitosan microspheres is mixed with 100-10000mL of sodium styrene sulfonate monomer solution.

[0020] The mixing ratio of chitosan raw material and DNA is crucial. If the proportion of DNA exceeds one-quarter, the chitosan will not be able to form spheres. If the mass proportion of chitosan raw material is less than two-thirds, the adsorption performance of sodium polystyrene sulfonate modified chitosan will be poor.

[0021] This invention generates a polystyrene sulfonate network structure within the porous structure of chitosan (CS) by initiating the polymerization reaction of sodium styrene sulfonate monomers. This results in chitosan carrying a large number of sulfonic acid groups (-SO3). -The abundant sulfonic acid groups in chitosan provide large negative charge centers. Histones are proteins rich in positively charged amino acids (especially lysine and arginine). Therefore, chitosan modified with sodium polystyrene sulfonate can electrostatically bind to histones rich in positively charged amino acids, and its binding force is far greater than that provided by the weak amino positive charge of unmodified chitosan. The function of chitosan microspheres changes from passive (due to its own weak amino positive charge) to active capture of histones. The introduction of sulfonic acid groups as functional groups on the porous surface of chitosan allows for strong ion-pair interactions with the basic amino acid residues of histones. Therefore, modification with sodium polystyrene sulfonate significantly enhances the adsorption capacity of modified chitosan for histones. This achieves the adsorption of histones, a damage-related molecular model, by chitosan, solving the long-standing technical bottlenecks in histone adsorption such as poor selectivity and low efficiency. Furthermore, the preparation method provided by this invention is simple, biosafety-enhancing, does not activate complement, does not cause hemolysis, and maintains normal blood cell counts, demonstrating strong technological advancement and market application potential. The sodium polystyrene sulfonate modified chitosan prepared in this invention retains the initial morphology of chitosan microspheres and has good biocompatibility.

[0022] In one embodiment, the microspheres of sodium polystyrene sulfonate modified chitosan have a diameter of 1.3-1.8 mm.

[0023] In a preferred embodiment of the present invention, the DNA is selected from calf thymus DNA or salmon DNA sodium salt.

[0024] In a preferred embodiment of the present invention, when preparing chitosan microspheres, the DNA raw material is first mixed with the chitosan raw material, GDL acidifying agent is added, and the mixture is heated to obtain a reaction solution. The reaction solution is then subjected to phase separation and coagulation in an anionic or alkaline environment to obtain chitosan microspheres.

[0025] Chitosan dissolves (with a positive charge) in acidic solutions (such as GDL acidifier). When the dissolved chitosan encounters anionic or alkaline environments, the cross-linking between molecular chains is enhanced, forming a three-dimensional network structure that is insoluble in water, thereby solidifying and shaping.

[0026] In a preferred embodiment of the present invention, the reaction solution is in an alkaline environment, which causes chitosan to change from a dissolved state to an insoluble state. In a preferred embodiment of the present invention, the alkaline environment is in sodium hydroxide, potassium hydroxide, ammonia, or ethanol-NaOH solution.

[0027] When preparing chitosan microspheres, a cross-linking agent may or may not be added, and those skilled in the art can choose according to their needs. Adding a cross-linking agent helps to improve the sphericity and strength of the chitosan microspheres.

[0028] In a preferred embodiment of the present invention, the reaction solution is added dropwise to a coagulation bath containing a crosslinking agent and an alkaline solution, and coagulates into spheres. In a preferred embodiment of the present invention, the crosslinking agent is an anionic crosslinking agent. In a preferred embodiment of the present invention, the anionic crosslinking agent is selected from phosphates, citrates, sulfates, oxalates, sodium alginate, sodium carboxymethyl cellulose, or xanthan gum. In a preferred embodiment of the present invention, the phosphate is selected from sodium tripolyphosphate or sodium hexametaphosphate; The coagulation bath is a mixture of sodium tripolyphosphate and sodium hydroxide with a mass concentration ratio of 2-6:4; In a preferred embodiment of the present invention, the reaction solution is added dropwise to a mixed coagulation bath containing 2-6% (m / v) sodium tripolyphosphate and 4% (m / v) sodium hydroxide, and coagulates into spheres.

[0029] The above mixing ratio results in better microsphere sphericity.

[0030] Secondly, the present invention also provides a method for preparing sodium polystyrene sulfonate modified chitosan, which includes the following steps: i: Preparation of chitosan and DNA mixture: Mix the DNA-containing solution with the chitosan raw material in a certain proportion, with the mixing mass ratio of the dry weight of the chitosan raw material to the dry weight of the DNA powder being 1-3:1; ii: Add GDL acidifying agent to the mixture from step i and heat to react; iii: The reaction solution heated in step ii is dropped into a coagulation bath to prepare chitosan microspheres loaded with DNA; iv: After washing the DNA-loaded chitosan microspheres, they were contacted with a sodium styrene sulfonate monomer solution; through a cross-linking reaction, sodium styrene sulfonate-modified chitosan with sodium styrene sulfonate cross-linked in the chitosan microspheres was obtained.

[0031] The preparation method provided by this invention is simple, has high biosafety, and possesses strong technological advancement and market application potential.

[0032] In a preferred embodiment of the present invention, the mass fraction of DNA in the DNA solution is 1%-1.5%.

[0033] In a preferred embodiment of the present invention, the chitosan raw material is chitosan powder with a degree of deacetylation of 50%-90%. The degree of deacetylation may affect the viscosity of chitosan, the sphericity of the droplets, and the number of hydrogen bonds when cross-linking with DNA; it can be 50%, 70%, or 90%.

[0034] In a preferred embodiment of the present invention, 50-200 mg of GDL acidifier is added to every 5 mL of the mixture from step i, and the mixture is stirred and reacted at 50℃±0.5℃.

[0035] In a preferred embodiment of the present invention, the crosslinking reaction in step iv involves blending chitosan microspheres containing sodium styrene sulfonate monomer with a crosslinking agent and an initiator, and then performing the crosslinking reaction at 50°C ± 0.5°C for 6-8 hours under nitrogen atmosphere.

[0036] In a preferred embodiment of the present invention, the crosslinking agent is selected from N,N'-methylenebisacrylamide, and the initiator is selected from ammonium persulfate, potassium persulfate, or a water-soluble azo initiator.

[0037] Water-soluble azo initiators, such as azobisisobutyramidine hydrochloride (AIBA), can be used.

[0038] In a preferred embodiment of the present invention, a reaction system is obtained by blending chitosan microspheres containing sodium styrene sulfonate monomer, N,N'-methylenebisacrylamide solution, and ammonium persulfate. The final concentration of the N,N'-methylenebisacrylamide solution in the reaction system is 0.5% (m / m), and the final concentration of the ammonium persulfate is 0.13% (m / m). The crosslinking reaction is carried out at 50℃ ± 0.5℃ for 6-8 hours under nitrogen atmosphere. The 0.5% (w / w) N,N'-methylenebisacrylamide solution is used to mediate the copolymerization reaction.

[0039] Thirdly, the present invention also provides the application of sodium polystyrene sulfonate modified chitosan or sodium polystyrene sulfonate modified chitosan prepared by the above preparation method in the following (1) or (2): (1) Preparation of histone adsorption products; (2) Histone adsorption for non-disease treatment purposes; (3) Application in the preparation of drugs for the treatment of at least one of the following diseases: histone-mediated critical illnesses, thrombosis and coagulation disorders, autoimmune diseases and nervous system diseases.

[0040] Experiments showed that chitosan modified with sodium styrene sulfonate could antagonize histone-induced apoptosis in human umbilical vein endothelial cells: the histone-induced apoptosis rate was significantly reduced, alleviating histone-induced endothelial barrier dysfunction. Simultaneously, these microspheres could reduce the release of histone-induced inflammatory factors such as interleukin-6 in the blood of an animal sepsis model, reducing histone-mediated excessive immune responses in sepsis-affected blood and exerting an immunomodulatory effect. The sodium styrene sulfonate-modified chitosan provided by this invention significantly improved liver and kidney function damage in a sepsis model, alleviated multi-organ dysfunction during sepsis, and exerted an organ-protective effect.

[0041] In a preferred embodiment of the present invention, the histone-mediated critical illnesses are selected from sepsis, septic shock, severe trauma and traumatic coagulopathy, acute respiratory distress syndrome, severe acute pancreatitis, severe burns or ischemia-reperfusion injury. In a preferred embodiment of the present invention, the autoimmune disease is selected from systemic lupus erythematosus and rheumatoid arthritis; Neurological diseases are selected from acute brain injury, stroke, and neurodegenerative diseases.

[0042] In a preferred embodiment of the present invention, the histone adsorption product is selected from histone adsorbents, histone adsorption columns, blood purification products, histone antagonists, or human umbilical vein endothelial cell apoptosis antagonists.

[0043] The sodium polystyrene sulfonate modified chitosan provided by this invention can antagonize apoptosis of human umbilical vein endothelial cells and has at least one of the following uses: stabilizing atherosclerotic plaques, preventing restenosis, improving vascular endothelial function, treating ischemia / reperfusion injury, reducing the vascular toxicity of chemotherapy drugs, regulating tumor vascular normalization, preventing and treating diabetic vascular complications, and promoting vascularization and wound healing.

[0044] Assembling the sodium polystyrene sulfonate-modified chitosan provided by this invention into a histone adsorption column for histone adsorption is a method that can be easily implemented by those skilled in the art.

[0045] In a preferred embodiment of the invention, the blood-purifying product is selected from a blood purifier or a perfusion adsorption column. In one embodiment, sodium polystyrene sulfonate-modified chitosan is used as the blood purification membrane.

[0046] Fourthly, the present invention also provides a histone adsorption product comprising: the above-mentioned sodium polystyrene sulfonate modified chitosan or sodium polystyrene sulfonate modified chitosan prepared by the above-mentioned preparation method.

[0047] In a preferred embodiment of the present invention, the histone adsorption product is selected from histone adsorbents, histone adsorption columns, blood purification products, histone antagonists, or human umbilical vein endothelial cell apoptosis antagonists.

[0048] In a preferred embodiment of the present invention, the blood purification product is selected from a blood purifier or a perfusion adsorption column.

[0049] Fifthly, the present invention also provides a medicament for treating at least one of the following diseases, wherein the disease is selected from at least one of the following: histone-mediated critical illness, thrombosis and coagulation dysfunction, autoimmune diseases and nervous system diseases; In a preferred embodiment of the present invention, the histone-mediated critical illnesses are selected from sepsis, septic shock, severe trauma and traumatic coagulopathy, acute respiratory distress syndrome, severe acute pancreatitis, severe burns or ischemia-reperfusion injury.

[0050] Ischemia-reperfusion injury includes, but is not limited to, myocardial ischemia-reperfusion injury, cerebral ischemia-reperfusion injury, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, intestinal ischemia-reperfusion injury, or limb ischemia-reperfusion injury.

[0051] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0052] Example 1 This embodiment provides a sodium polystyrene sulfonate-modified chitosan and its preparation method. The preparation method is as follows: (1) Dissolve salmon DNA sodium salt in reverse osmosis water to prepare a DNA solution with a mass fraction of 1%; (2) Chitosan powder with a degree of deacetylation of 70% is uniformly dispersed in the salt solution of step (1) to obtain a 3% mass fraction chitosan-DNA solution; the mass ratio of chitosan powder to DNA solution is 3:1. (3) Add 116 mg of GDL acidifier to each 5 mL of mixed solution obtained in step (2), heat at 50 °C and stir at high speed overnight.

[0053] (4) After the reaction is complete, the sample obtained in step (3) is dropped into a mixed coagulation bath with a final concentration of 6% sodium tripolyphosphate and 4% sodium hydroxide, and soaked for 12 h to stabilize into spheres (to obtain chitosan microspheres C3D1 containing DNA). (5) After repeatedly washing the microspheres obtained in step (4) with a certain amount of reverse osmosis water, the microspheres are immersed in a 15% (m / v) sodium styrene sulfonate solution and reacted overnight to allow the sodium styrene sulfonate solution to fully enter the pores of the microspheres; the ratio of the amount of microspheres to the amount of sodium styrene sulfonate monomer solution is 25 g wet weight: 100 mL.

[0054] (6) The modified microspheres were added to a 0.5% N,N'-methylenebisacrylamide solution and a 0.13% ammonium persulfate solution, and heated under a nitrogen atmosphere at 50 °C for 6 h in the absence of oxygen to crosslink the microspheres, so that the sodium styrene sulfonate solution was stably crosslinked in the microspheres. (7) After the reaction is complete, the microspheres are removed, washed repeatedly in reverse osmosis water, and stored in PBS solution (pH 7.4) for later use.

[0055] Sodium polystyrene sulfonate modified chitosan (C3D1-PSS) was prepared.

[0056] Comparative Example 1 Compared to Example 1, the only difference is in step (2), where the mass ratio of chitosan powder to DNA solution is 4:1. As a result, the mixture could not form spheres, and chitosan microspheres could not be prepared.

[0057] Comparative Example 2 Compared to Example 1, the only difference is in step (2), where the mass ratio of chitosan powder to DNA solution is 2:1. The resulting sodium polystyrene sulfonate-modified chitosan exhibited poor mechanical properties.

[0058] Comparative Example 3 Compared with Example 1, chitosan powder was directly dropped into a mixed coagulation bath containing 6% sodium tripolyphosphate and 4% sodium hydroxide by mass, and soaked for 12 h to stabilize into spheres, thereby obtaining DNA-free chitosan microspheres (CS).

[0059] Experimental Example 1 The CS, C3D1, and C3D1-PSS microspheres obtained in Example 1 and Comparative Example 3 were freeze-dried, then sputter-coated with gold. The ultrastructure of the microsphere surface was observed using a scanning electron microscope (Phenom Pure electron microscope, Thermo Scientific). The results are as follows: Figure 1 As shown.

[0060] Figure 1 The microspheres have a diameter of 1.55 mm, and the volume of the C3D1-PSS microspheres has increased, proving that the in-situ crosslinking polymerization introduced a large amount of sodium polystyrene sulfonate.

[0061] Experiment Example 2 Evaluation of blood compatibility of sodium polystyrene sulfonate modified chitosan histone adsorbent microspheres.

[0062] 1. Blood collection In this embodiment, the blood used for the biocompatibility evaluation of CS, C3D1, and C3D1-PSS microspheres was derived from healthy male blood donors. In this embodiment, three types of 5 mL vacuum tubes containing different anticoagulants were used for blood collection: blood collected from purple-tipped blood collection tubes (containing EDTA anticoagulant) was used for complete blood count and hemolysis tests; blood collected from blue-tipped blood collection tubes (containing citrate anticoagulant, with an anticoagulant-to-blood volume ratio of 1:9) was used for clotting time determination; and blood collected from red-tipped blood collection tubes (containing hirudin anticoagulant, with an anticoagulant concentration of 40 μg / mL) was used for complement activation tests.

[0063] 2. Histone adsorption 2.1 Static histone adsorption (1) Dissolve histones in physiological saline to prepare histone solutions with concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL and 0 μg / mL respectively.

[0064] (2) Use a UV spectrophotometer to detect the OD value of the above standard histone solution at 280 nm and plot a standard curve.

[0065] (3) Soak 100 mg of wet CS, C3D1, and C3D1-PSS microspheres in a 24-well plate at 37 °C overnight using PBS.

[0066] (4) After aspirating the PBS, add 1000 μL of histone solution with a concentration of 200 μg / ml and incubate in a constant temperature incubator at 37 ℃ for 4 h.

[0067] (5) After incubation, the OD value of the adsorbed histone solution at a wavelength of 280 nm was detected by ultraviolet spectrophotometer, and the histone concentration was calculated according to the standard curve.

[0068] (6) Use the histone solution that has not come into contact with the material as a blank control. Repeat the experiment three times to reduce measurement error. The histone loss rate in the solution is calculated using the following formula: Histone Loss (%) = (C control C sample ) / C control × 100 Among them, C control The concentration of histones in the control group, C sample This refers to the protein concentration in each sample.

[0069] Histone adsorption efficiency, such as Figure 2 As shown in the results, the chitosan microspheres (CS) without DNA exhibited the lowest histone reduction rate, indicating the worst histone adsorption effect. The modified chitosan, modified with sodium polystyrene sulfonate, showed the highest adsorption efficiency.

[0070] 2.2 Complement activation In this embodiment, enzyme-linked immunosorbent assay (ELISA) was used to evaluate the complement activation effect of CS, C3D1, and C3D1-PSS microspheres. The levels of complement C3a and C5a in plasma before and after material contact with blood were measured using a human C3a (Thermo Fisher Scientific BMS2089) kit and a C5a ELISA kit (Thermo Fisher Scientific BMS2088).

[0071] (1) Using PBS, 50 mg of wet weight CS, C3D1, and C3D1-PSS microspheres were soaked overnight at 37 °C in a 48-well plate. (2) After the PBS was completely absorbed, 300 μL of fresh, healthy whole blood from a blood donor with hirudin anticoagulation was added to the well plate and incubated at 37 °C for 1 h.

[0072] (3) After incubation, centrifuge at 6000 r / min for 15 min to obtain the plasma to be tested. Use fresh plasma that has not come into contact with the material as a blank control, and use fresh plasma obtained by stimulation with 50 mg of cellulose powder as a positive control. Repeat the experiment 3 times to reduce measurement error.

[0073] (4) Add 100 μL of the diluted standard to the reaction well; dilute the sample to be tested 1000 times with the sample diluent and add 100 μL to the reaction well. Immediately add 50 μL of biotin-labeled antibody. Cover the plate, gently shake to mix, and incubate at 37 °C for 2 h.

[0074] (5) Shake off the liquid in the well, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat 5 times.

[0075] (6) Add 50 μL of avidin-HRP to each well, gently shake to mix, and incubate at 37 °C for 1 h.

[0076] (7) Shake off the liquid in the well, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat 5 times.

[0077] (8) Add 50 μL of substrate A and B to each well, gently shake to mix, and incubate at 37 °C in the dark for 10 min.

[0078] (9) Quickly add 50 μL of stop solution, and immediately use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of each well at a wavelength of 450 nm and record the experimental results.

[0079] The effect of microspheres on complement activation is as follows: Figure 3 As shown, the results indicate that sodium polystyrene sulfonate-modified chitosan does not affect complement activation.

[0080] 2.3 Hemolysis rate.

[0081] (1) Soak 100 mg wet weight of CS, C3D1, and C3D1-PSS microspheres in a 1.5 mL centrifuge tube at 37 °C overnight using PBS.

[0082] (2) Add 10 mL of PBS to 5 mL of fresh, healthy whole blood anticoagulated with EDTA, mix well, and centrifuge at 2000 rpm for 10 min to separate red blood cells. Repeat 5 times to obtain a red blood cell suspension. Dilute the red blood cell suspension 20 times in PBS to prepare 100 mL of diluted red blood cell suspension for later use.

[0083] (3) Aspirate all PBS from each centrifuge tube and add 1 mL of diluted red blood cell suspension. Incubate the mixture at 37 °C for 3 h.

[0084] (4) After incubation, centrifuge at 2000 rpm for 3 min to separate red blood cells from supernatant.

[0085] (5) The absorbance of the supernatant of each sample, the positive control, and the negative control at a wavelength of 545 nm was measured using a UV spectrophotometer to determine the absorbance value of the hemoglobin released in the solution, and the experimental results were recorded. The experiment was repeated 3 times to reduce measurement error. The hemolysis ratio of the microspheres was calculated using the following formula: ; ABSs are the absorbance values ​​of the supernatant of each sample, while ABSp and ABSn are the absorbance values ​​of the positive control (red blood cell suspension obtained by deionized water treatment) and the negative control (red blood cell suspension treated with PBS), respectively.

[0086] The results of the effect of the above microspheres on hemolysis are as follows: Figure 4 As shown, the results indicate that sodium polystyrene sulfonate-modified chitosan does not affect cell hemolysis.

[0087] 2.4 Blood cell count A complete blood count (CBC) was performed to assess the effect of the microspheres on blood cells by measuring changes in red blood cell count, white blood cell count, and platelet count before and after 30 minutes of contact with CS, C3D1, and C3D1-PSS microspheres. Fresh plasma that had not been in contact with the materials served as a blank control. The specific experimental procedures are as follows: (1) Using PBS, place 50 mg of wet weight CS, C3D1, C3D1-PSS microspheres in a 48-well plate and soak overnight at 37 °C.

[0088] (2) After aspirating all the PBS from each well, add 500 μL of fresh, healthy whole blood from a donor that has been anticoagulated with EDTA. Incubate the material and plasma at 37 °C for 30 min.

[0089] (3) After incubation, 500 μL of whole blood from each well was transferred to a dedicated test cup for the complete blood count (CBC) analyzer. The CBC analyzer was used to automatically perform CBC analysis on different samples, and the experimental results were recorded. Hematocrit was used to correct other CBC count indicators to reduce experimental errors caused by varying degrees of blood concentration during the isothermal incubation process in vitro. The experiment was repeated 3 times to reduce measurement errors.

[0090] The blood cell count results for each microsphere are as follows: Figure 5 As shown, CS, C3D1, and C3D1-PSS microspheres have no effect on blood routine tests.

[0091] Experiment Example 2 Evaluation of the adsorption efficiency of sodium polystyrene sulfonate-modified chitosan histones on microspheres.

[0092] The toxic effects of the microspheres on endothelial cells after adsorbing histones were evaluated using flow cytometry. The specific experimental steps are as follows: (1) Histone was dissolved in starvation medium without fetal bovine serum to prepare histone stimulation solutions with a concentration of 50 ug / mL.

[0093] (2) Use PBS to soak 100 mg of CS, C3D1, and C3D1-PSS microspheres in a 24-well plate at 37 °C overnight.

[0094] (3) After aspirating the PBS, add 1 mL of starvation medium to the well as a control group and a blank + microsphere control group; add the above histone stimulation solution to the well to form a microsphere pre-adsorption stimulation solution, and place the well plate in a 37 ℃ constant temperature incubator for 3 h.

[0095] (4) After incubation, transfer 1 mL of supernatant from each well to a six-well plate containing HUVEC cells and incubate at 37 ℃ for 12 h.

[0096] (5) After incubation, aspirate the HUVEC cell culture supernatant into a sterile centrifuge tube, digest the adherent cells with EDTA-free trypsin, and centrifuge at 600 ×g for 5 min at low temperature to ensure maximum recovery of all cells in the culture well.

[0097] (6) Wash the cell pellet twice with PBS pre-cooled at 4°C, and collect the cells under the same centrifugation conditions after each wash.

[0098] (7) Dilute the 4× concentration binding buffer with ultrapure water at a volume ratio of 1:3 to prepare a 1× working binding buffer.

[0099] (8) Resuspend the cells in freshly prepared 1× binding buffer and adjust the cell density to (1–5)×10⁻⁶.6 per mL. (9) Fluorescent labeling: Add 100 μL of cell suspension to a flow cytometer, add 5 μL of Annexin V-FITC fluorescent label, and react for 10 min at room temperature in the dark.

[0100] (10) Immediately after the reaction is complete, add 10 μL of propidium iodide (PI) staining solution and 400 μL of 1× binding buffer, mix gently, and then immediately run the instrument for detection to analyze the apoptosis status of each group. Figure 6 The results showed that, compared to CS and C3D1, C3D1-PSS microspheres could alleviate histone-induced endothelial cell apoptosis.

[0101] Experimental Example 3 In vitro circulatory anticoagulation animal experiments using sodium polystyrene sulfonate modified chitosan histone adsorbed microspheres.

[0102] Rabbits were randomly assigned to either a histone stimulation group or a histone stimulation + blood perfusion group, with 3 rabbits in each group. C3D1-PSS was filled into a pre-customized blood perfusion device shell to approximately 70% of its volume, forming a histone-adsorbed blood perfusion column. The column was then sterilized, sealed, and stored for later use.

[0103] Histone stimulation group: Rabbits were stimulated by intravenous injection of 50 mg / kg of histone at the ear margin. During the experiment, 200 mL of physiological saline was administered via peripheral intravenous fluid to maintain the volume balance of the experimental animals. Twenty-four hours after stimulation, the animals were euthanized by overdose anesthesia, and lung and kidney tissues were harvested, fixed in 4% polymethanol at room temperature, and subsequently subjected to HE staining for histological evaluation.

[0104] Histone stimulation + blood perfusion group (Hemodialysis): A C3D1-PSS-adsorbed blood perfusion column was connected to the extracorporeal circulation system, and the tubing was pre-filled with 500 mL of physiological saline for later use. Rabbits were stimulated according to step (1). After 20 min of stimulation, the extracorporeal circulation system was connected to the temporary catheter vascular access, and blood perfusion treatment was immediately performed using a self-made wearable artificial kidney prototype (mainly composed of a blood pump and pressure monitoring system). The blood flow rate was set to 5 mL / min, and hirudin was used as the initial anticoagulant by intravenous injection. Hirudin was then supplemented every 30 min thereafter. During the experiment, 200 mL of physiological saline was supplemented via peripheral vein to maintain the volume balance of the experimental animals. The animals were removed from the machine after 2 hours of circulatory stimulation. After 24 h of histone stimulation, the experimental animals were euthanized by overdose anesthesia, and lung and kidney tissues of 2×2 cm size were taken and fixed in 4% paraformaldehyde at room temperature. HE staining was then performed for histological evaluation.

[0105] Figure 7The results showed that C3D1-PSS, when introduced into extracorporeal circulation via a hemoperfusion column, could reduce the damage to kidney and lung tissues caused by histone stimulation.

[0106] In summary, the modified chitosan provided by this invention can adsorb histones with an adsorption capacity far exceeding that provided by chitosan's weak amino positive charge, thereby realizing the adsorption of histones, a damage-related molecular pattern, by chitosan. This solves the long-standing technical bottlenecks in the field of histone separation and adsorption, such as poor selectivity and low efficiency. Furthermore, the preparation method is simple, the biosafety is high, and it has strong technological advancement and market application potential.

[0107] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A sodium polystyrene sulfonate modified chitosan, characterized in that, It includes: Chitosan microspheres and sodium polystyrene sulfonate and DNA within the pores of the chitosan microspheres; in the raw materials for preparing the sodium polystyrene sulfonate modified chitosan, each 25g wet weight chitosan microsphere is mixed with at least 50mL of sodium polystyrene sulfonate monomer solution, the concentration of the sodium polystyrene sulfonate monomer solution being 3% (m / v)-15% (m / v); the mixing mass ratio of the dry weight of the chitosan raw material to the dry weight of the DNA powder is 1-3:1, and the sodium polystyrene sulfonate monomer undergoes a cross-linking reaction to form a sodium polystyrene sulfonate network structure within the pores of the chitosan microspheres.

2. The sodium polystyrene sulfonate modified chitosan according to claim 1, characterized in that, The DNA was selected from calf thymus DNA or salmon DNA sodium salt.

3. The sodium polystyrene sulfonate modified chitosan according to claim 1, characterized in that, In preparing the chitosan microspheres, the DNA raw material is first mixed with the chitosan raw material, GDL acidifying agent is added, and the mixture is heated to obtain a reaction solution. The reaction solution is then subjected to phase separation and coagulation in an anionic or alkaline environment to obtain chitosan microspheres. Preferably, the reaction solution is in an alkaline environment, which causes chitosan to change from a dissolved state to an insoluble state; Preferably, the alkaline environment is in sodium hydroxide, potassium hydroxide, ammonia, or ethanol-NaOH solution; Preferably, the reaction solution is added dropwise to a coagulation bath containing a crosslinking agent and an alkaline solution, and coagulates into spheres; Preferably, the crosslinking agent is an anionic crosslinking agent, and more preferably, the anionic crosslinking agent is selected from phosphates, citrates, sulfates, oxalates, sodium alginate, sodium carboxymethyl cellulose, or xanthan gum; Preferably, the phosphate is selected from sodium tripolyphosphate or sodium hexametaphosphate; Preferably, the coagulation bath is a mixture of sodium tripolyphosphate and sodium hydroxide with a mass concentration ratio of 2-6:4; Preferably, the reaction solution is added dropwise to a mixed coagulation bath containing 2-6% (m / v) sodium tripolyphosphate and 4% (m / v) sodium hydroxide, and coagulates into spheres.

4. The method for preparing sodium polystyrene sulfonate modified chitosan according to any one of claims 1-3, characterized in that, It includes the following steps: i: Preparation of chitosan and DNA mixture: Mix the DNA-containing solution with the chitosan raw material in a certain proportion, with the mixing mass ratio of the dry weight of the chitosan raw material to the dry weight of the DNA powder being 1-3:1; ii: Add GDL acidifying agent to the mixture from step i and heat to react; iii: The reaction solution heated in step ii is dropped into a coagulation bath to prepare chitosan microspheres loaded with DNA; iv: After washing the DNA-loaded chitosan microspheres, contact them with a sodium styrene sulfonate monomer solution; through a cross-linking reaction, obtain sodium styrene sulfonate-modified chitosan with sodium styrene sulfonate cross-linked in the chitosan microspheres.

5. The method for preparing sodium polystyrene sulfonate modified chitosan according to claim 4, characterized in that, The DNA solution contains 1%-1.5% DNA by mass.

6. The method for preparing sodium polystyrene sulfonate modified chitosan according to claim 4, characterized in that, The chitosan raw material is chitosan powder with a degree of deacetylation of 50%-90%; Preferably, 50-200 mg of GDL acidifying agent is added to every 5 mL of the mixture from step i, and the mixture is stirred and reacted at 50℃±0.5℃.

7. The method for preparing sodium polystyrene sulfonate modified chitosan according to claim 4, characterized in that, The crosslinking reaction in step iv involves blending chitosan microspheres containing sodium styrene sulfonate monomer with a crosslinking agent and an initiator, and then performing the crosslinking reaction at 50℃±0.5℃ for 6-8 hours under nitrogen atmosphere. Preferably, the crosslinking agent is selected from N,N'-methylenebisacrylamide, and the initiator is selected from ammonium persulfate, potassium persulfate, or a water-soluble azo initiator; Preferably, a reaction system is obtained by blending chitosan microspheres containing sodium styrene sulfonate monomer, N,N'-methylenebisacrylamide solution, and ammonium persulfate. The final concentration of N,N'-methylenebisacrylamide solution in the reaction system is 0.5% (m / m), and the final concentration of ammonium persulfate is 0.13% (m / m). The crosslinking reaction is carried out at 50℃±0.5℃ for 6-8 hours under nitrogen atmosphere.

8. The use of sodium polystyrene sulfonate modified chitosan according to any one of claims 1-3 or the preparation method according to any one of claims 4-7 in the following (1) or (2): (1) Preparation of histone adsorption products; (2) Histone adsorption for non-disease treatment purposes; (3) Application in the preparation of drugs for the treatment of at least one of the following diseases: histone-mediated critical illnesses, thrombosis and coagulation disorders, autoimmune diseases and nervous system diseases; Preferably, the histone-mediated critical illnesses are selected from sepsis, septic shock, severe trauma and traumatic coagulopathy, acute respiratory distress syndrome, severe acute pancreatitis, severe burns or ischemia-reperfusion injury; Preferably, the autoimmune disease is selected from systemic lupus erythematosus and rheumatoid arthritis; The neurological diseases mentioned are selected from acute brain injury, stroke, and neurodegenerative diseases; Preferably, the histone adsorption product is selected from histone adsorbents, histone adsorption columns, blood purification products, histone antagonists, or human umbilical vein endothelial cell apoptosis antagonists. Preferably, the blood-purifying product is selected from a blood purifier or a perfusion adsorption column.

9. A histone adsorption product, characterized in that, It includes: sodium polystyrene sulfonate modified chitosan as described in any one of claims 1-3 or sodium polystyrene sulfonate modified chitosan prepared by the preparation method described in any one of claims 4-7; Preferably, the histone adsorption product is selected from histone adsorbents, histone adsorption columns, blood purification products, histone antagonists, or human umbilical vein endothelial cell apoptosis antagonists. Preferably, the blood-purifying product is selected from a blood purifier or a perfusion adsorption column.

10. A medicament for treating at least one of the following diseases, characterized in that, The disease is selected from at least one of the following: histone-mediated critical illness, thrombosis and coagulation dysfunction, autoimmune disease, and nervous system disease; Preferably, the histone-mediated critical illnesses are selected from sepsis, septic shock, severe trauma and traumatic coagulopathy, acute respiratory distress syndrome, severe acute pancreatitis, severe burns or ischemia-reperfusion injury.

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