阅读说明：本技术 基于肝素改性壳聚糖/纤维素微球的血液灌流吸附剂、其制备方法及其应用 (Blood perfusion adsorbent based on heparin modified chitosan/cellulose microspheres, and preparation method and application thereof ) 是由 苏白海 李育霈 杨嘉庆 张斯睿 宋涛 于乔 罗欣瑶 杨钦博 耿际雯 张竹韵 赵伟 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种基于肝素改性壳聚糖/纤维素微球的血液灌流吸附剂,属于生物材料技术领域,所述血液灌流吸附剂包括交联壳聚糖/纤维素微球,所述交联壳聚糖/纤维素微球外接枝有肝素涂层,还公开了其制备方法以及应用；本发明的吸附剂具有较好的力学强度和稳定性,从而更好地满足血液灌流对血液灌流吸附剂机械强度的需要,所含壳聚糖分子中所带丰富的氨基使得吸附剂带有丰富的正电荷,可高效清除带负电荷的内毒素；吸附剂外层中的肝素涂层则可与组蛋白形成复合物,从而快速清除血液中的组蛋白；本发明的的血液灌流吸附剂可通过多吸附靶点清除脓毒症患者血液中多种致病因子,从而增强血液净化治疗对脓毒症患者的治疗效果。(The invention discloses a hemoperfusion adsorbent based on heparin modified chitosan/cellulose microspheres, which belongs to the technical field of biological materials, and comprises cross-linked chitosan/cellulose microspheres, wherein a heparin coating is grafted outside the cross-linked chitosan/cellulose microspheres; the adsorbent has better mechanical strength and stability, thereby better meeting the requirement of blood perfusion on the mechanical strength of the blood perfusion adsorbent, and the chitosan molecules have rich amino groups to ensure that the adsorbent has rich positive charges and can efficiently remove endotoxin with negative charges; the heparin coating in the outer layer of the adsorbent can form a compound with histone, so that the histone in blood can be quickly removed; the blood perfusion adsorbent can remove various pathogenic factors in blood of patients with sepsis through multiple adsorption targets, so that the treatment effect of blood purification treatment on patients with sepsis is enhanced.)

1. A hemoperfusion adsorbent based on heparin modified chitosan/cellulose microspheres is characterized in that: the hemoperfusion adsorbent comprises cross-linked chitosan/cellulose microspheres, and a heparin coating is grafted outside the cross-linked chitosan/cellulose microspheres.

2. The heparin-modified chitosan/cellulose microsphere-based hemoperfusion sorbent according to claim 1, wherein: the cross-linked chitosan/cellulose microspheres are prepared by adopting a phase inversion method.

3. The heparin-modified chitosan/cellulose microsphere-based hemoperfusion sorbent according to claim 1, wherein: the average particle size of the adsorbent is 0.5-2 mm.

4. The hemoperfusion adsorbent based on heparin modified chitosan/cellulose microspheres according to claim 1, characterized in that: the heparin coating is grafted to the surface of the chitosan/cellulose microsphere by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.

5. The method for preparing heparin-modified chitosan/cellulose microsphere-based hemoperfusion adsorbent according to any one of claims 1 to 4, characterized by comprising the following steps:

1) preparing a solvent of chitosan/cellulose, wherein the solvent comprises the following components in parts by weight:

lithium hydroxide 4.5 parts of Potassium hydroxide 7 portions of Urea 8 portions of Deionized water 80.5 portions

Weighing the components in parts by weight, adding the components into a container, and stirring at room temperature to dissolve completely to obtain a solvent of cellulose and chitosan;

2) preparing a chitosan solution from the solvent obtained in the step 1), wherein the solution comprises the following components in parts by weight:

weighing the components in parts by weight, adding the components into a container, and uniformly stirring at room temperature to obtain a suspension of chitosan. Hermetically placing the mixture in a constant-temperature refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, after freezing the ice, fully melting the ice at the room temperature, stirring the ice to uniformly dissolve the chitosan, then placing the ice in the refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, taking the ice out, fully melting the ice again at the room temperature, and stirring the ice to uniformly dissolve the chitosan to obtain a chitosan solution;

3) preparing a cellulose solution from the solvent obtained in the step 1), wherein the solution comprises the following components in parts by weight:

cellulose, process for producing the same, and process for producing the same 2-4 parts of Solvent obtained in step 1) 96-98 parts of

Weighing the components in parts by weight, adding the components into a container, and uniformly stirring at room temperature to obtain a cellulose suspension. Hermetically placing the mixture in a constant-temperature refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, after freezing, fully melting the mixture at the room temperature, then starting stirring to uniformly dissolve the cellulose, then placing the mixture in the refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, taking the mixture out, fully melting the mixture again at the room temperature, and then starting stirring to uniformly dissolve the cellulose to obtain a cellulose solution;

4) mixing the chitosan solution obtained in the step 2) with the cellulose solution obtained in the step 3) in a volume ratio of 1:1, 1:2 or 2:1 to obtain a chitosan/cellulose mixed solution;

5) adding the polymer solution obtained in the step 4) into an injector, obtaining uniform spherical liquid drops by selecting an injection needle with the diameter of 24-28G, dripping the uniform spherical liquid drops into a dilute sulfuric acid or dilute hydrochloric acid solution with the volume fraction of 5-20%, wherein the dripping speed is 30-40 drops/min, the temperature is maintained at 25-30 ℃, and standing the solution in the dilute sulfuric acid or dilute hydrochloric acid solution for 1-3 hours to obtain the solidified gel microspheres: filtering the gel microspheres through a filter screen, and washing the gel microspheres with deionized water until the pH of a washing solution is between 7 and 8 to obtain chitosan/cellulose microspheres;

6) preparing the chitosan/cellulose microspheres obtained in the step (5) into heparin modified chitosan/cellulose microspheres, wherein the reaction components comprise the following components in parts by weight:

chitosan/cellulose microspheres 4 to 8 portions of Heparin sodium 0.05 to 0.3 portion 1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride 0.76 to 1.16 portions of 2- (N-morpholine) ethanesulfonic acid 1.93-2.33 parts Deionized water 80 to 120 portions of

Weighing the components in parts by weight, stirring at normal temperature, reacting for 12-24 hours, filtering the microspheres by using a filter screen, and washing with deionized water to obtain the heparin-modified chitosan/cellulose microspheres.

6. Use of the heparin-modified chitosan/cellulose microsphere-based blood perfusion adsorbent of any one of claims 1-4 in the preparation of a blood perfusion device for blood purification.

7. Use according to claim 6, wherein the blood purification is of sepsis patients.

Technical Field

The invention relates to the technical field of biological materials/blood contact materials, in particular to a blood perfusion adsorbent based on heparin modified chitosan/cellulose microspheres, and a preparation method and application thereof.

Background

Sepsis is a series of clinical syndromes in which the body's immune system is deregulated due to bacterial, viral or fungal infections, resulting in dysfunction of life-threatening organs. The sepsis has high morbidity in critical patients, is the most main cause of death of the critical patients, about 250 thousands of people are diagnosed as the sepsis in China every year, and about 70 thousands of people die of the sepsis. At present, the treatment of sepsis mainly comprises broad-spectrum antibiotic anti-infection treatment, liquid resuscitation, blood vessel pressure-boosting drug treatment and the like if necessary; however, these traditional therapies have poor therapeutic efficacy, and the mortality rate of patients with sepsis is still as high as 20%, and higher in patients with severe sepsis and septic shock.

Blood purification treatments (such as hemoperfusion, plasmapheresis, and hemodialysis rate) are important adjunctive treatments for patients with severe sepsis and septic shock. The blood purification can eliminate various pathogenic factors (such as endotoxin, inflammatory factors, pathogenic bacteria and the like) abnormally accumulated in the blood of the sepsis patient so as to improve the prognosis of the sepsis patient.

In sepsis, high endotoxin and histone concentrations in blood circulation are closely related to the severity of multi-organ failure and the fatality rate of patients.

Among them, endotoxin is the major component of the outer membrane of gram-negative bacteria and is the most studied pathogen-associated molecular pattern in sepsis. Endotoxin can specifically activate various auxiliary factors (endotoxin binding peptide, Toll-like receptor and the like) on the surface of an immune cell, thereby activating a plurality of signal paths at the downstream in the immune cell, inducing an organism to generate over-activated immune response when sepsis occurs, promoting the release of proinflammatory cytokines and inflammatory cytokines in blood circulation and causing the immune imbalance of a host. Endotoxin is a well-established trigger point for the induction of an abnormal immune response in patients with sepsis. However, in clinical trials, many drugs that neutralize endotoxin in the blood of sepsis patients do not improve the prognosis and reduce the mortality of sepsis patients. In view of this, blood purification gradually becomes the main treatment method for removing endotoxin from patients with sepsis, and the currently commercialized blood purification adsorbing material for adsorbing endotoxin mainly comprises two types, namely an oXiris membrane and a polymyxin B adsorbing column, but has poor endotoxin adsorbing effect (the maximum endotoxin adsorbing amount is low), and is not suitable for patients with severe sepsis with endotoxin concentration in blood exceeding 10 ng/mL. For example, the latest EUPHRATES randomized controlled clinical trial found that polymyxin B sorbent columns when used in the treatment of patients with sepsis did not significantly improve the 28-day survival of patients, for reasons that may be: 1) the adsorption effect of endotoxin in the multicentrin B adsorption column is poor, and the endotoxin cannot be effectively removed in the treatment process; 2) the poly-rhzomorph B adsorption column can not adsorb other pathogenic factors such as bacteria, histones and The like in The blood of Patients With sepsis, The treatment principle is single, and a single Endotoxin adsorption treatment mode can seriously influence The treatment effect on The sepsis (Dellinger RP, Bagshaw SM, Antonelli M, et al. Effect of Targeted Polymyxin B haemostasis on 28-Day Mortality in Patients With separated Shock and expressed Endotoxin Level: The EUPHRATES Rangified Clinical Trial [ J ] 2018:320(14): 1455-) 1463).

In addition, histone is another important pathogenic factor in the development and progression process of sepsis. Sepsis patients are stimulated by invading pathogens to have immune cell activation, formation of a large number of neutrophil extracellular traps and apoptosis/necrosis, so that intracellular histones are released into blood, the abnormal concentration of the histones in blood circulation is increased, and vascular endothelial cell damage, inflammatory cytokine storm, disseminated intravascular coagulation and multi-organ failure are caused. Histone is one of the most intense sepsis therapeutic targets in recent years.

Therefore, the breakthrough point for solving the difficult problem of blood purification treatment of sepsis is to develop a novel blood perfusion adsorbent which can efficiently remove endotoxin, histone and other pathogenic factors from blood at the same time. This multiple adsorption target blood purification treatment model can minimize two key pathogenic factors in the onset and progression of sepsis: endotoxin and histone, thereby avoiding the occurrence of immune imbalance and multiple organ failure of an organism, further reducing the death rate of patients with severe sepsis and septic shock on the basis of the existing treatment, relieving the sanitary economic burden caused by the sepsis, and having important clinical significance for the prevention and treatment of the sepsis.

Meanwhile, sepsis patients generally have coagulation dysfunction problems such as disseminated intravascular coagulation and platelet reduction, and the risk of bleeding of the patients is high. In the traditional blood perfusion treatment process, a patient needs to receive systemic heparin anticoagulation to prevent blood from coagulating in an extracorporeal circulation pipeline and a blood perfusion adsorbent, so that the blood purification treatment cost is increased, and the risk of severe hemorrhage of a sepsis patient after blood perfusion is increased. Previous studies by the applicant have shown that hemoperfusion adsorbents with self-anticoagulant properties affect the patient's coagulation system function to a lesser extent than conventional therapeutic modalities, and better prevent the development of (post) severe hemorrhage during blood purification (Song X, Ji HF, Li YP, et al. transient blood purification and inactivation of blood purification factors by biological hydrological microsheres [ J ]. Nature biological engineering.2021.DOI:10.1038/s 41551-020-.

Chitosan is the only positively charged polysaccharide existing in nature, has good biocompatibility and hydrophilicity, and has been studied as a base material for the development of blood purification materials. The applicant previously disclosed that a blood perfusion adsorbent based on genipin cross-linked chitosan microspheres can effectively remove endotoxin and bacteria in blood, but cannot adsorb histone (chitosan-based blood perfusion adsorbent and application thereof in preparing a blood perfusion device for sepsis blood purification, patent No. ZL 202010095433.5).

Heparin is an anticoagulant substance extracted from the mucous membrane of the small intestine of animals, and is mucopolysaccharide sulfate consisting of D-glucosamine, L-iduronic acid and D-glucuronic acid alternately, has high negative charge density, can form a complex with histone through electrostatic action so as to neutralize the pathophysiological action of histone and play an immunoregulatory role, but cannot directly eliminate histone from the plasma of patients with sepsis (Alhamdi Y, Abrams ST, Lane S, et al. histone-associated thrombocytenophora in tissues of tissue of the heart are critical ill [ J.JAMA, 2016,315(8): 817-) 819.).

Disclosure of Invention

One of the objectives of the present invention is to provide a hemoperfusion adsorbent based on heparin modified chitosan/cellulose microspheres to solve the above problems.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the blood perfusion adsorbent based on the heparin modified chitosan/cellulose microspheres comprises cross-linked chitosan/cellulose microspheres, and a heparin coating is grafted outside the cross-linked chitosan/cellulose microspheres.

As a preferred technical scheme: the cross-linked chitosan/cellulose microspheres are prepared by adopting a phase inversion method.

The microsphere of the invention preferably uses a mixed solution of chitosan and cellulose to prepare the chitosan/cellulose microsphere by one step through a phase inversion method, and introduces cellulose molecules into the microsphere to increase the mechanical strength and stability of the microsphere through physical crosslinking with the chitosan molecules, so that other chemical crosslinking agents (such as glutaraldehyde, genipin and the like) with cytotoxic effect are not required to be added in the preparation process of the microsphere.

As a preferred technical scheme: the average particle size of the adsorbent is 0.5-2 mm.

The appropriate particle size is convenient for filling the disposable hemoperfusion apparatus by using the adsorbent material. If the size of the adsorbent is too small, the adsorbent is difficult to recover in the using process, and gaps stacked in the perfusion column are too small, so that great resistance is generated to the blood passing, and even the blood passing can be blocked, and the use of the blood perfusion device is influenced; if the size of the adsorbent is too large, the adsorption material filled in the perfusion column is too little, the space utilization rate of the perfusion column is greatly reduced, and the toxin removing capacity is further influenced.

As a preferred technical scheme: the heparin coating is grafted to the surface of the chitosan/cellulose microsphere by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.

The second purpose of the present invention is to provide a method for preparing the hemoperfusion adsorbent based on heparin modified chitosan/cellulose microspheres, which adopts the technical scheme that the method comprises the following steps:

1) preparing a solvent of chitosan/cellulose, wherein the solvent comprises the following components in parts by weight:

lithium hydroxide	4.5 parts of
		Potassium hydroxide	7 portions of
Urea	8 portions of
		Deionized water	80.5 portions

Weighing the components in parts by weight, adding the components into a container, and stirring at room temperature to dissolve completely to obtain a solvent of cellulose and chitosan;

2) preparing a chitosan solution from the solvent obtained in the step 1), wherein the solution comprises the following components in parts by weight:

chitosan	2-4 parts of
		Solvent obtained in step 1)	96-98 parts of

Weighing the components in parts by weight, adding the components into a container, and uniformly stirring at room temperature to obtain a suspension of chitosan. Hermetically placing the mixture in a constant-temperature refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, after freezing the ice, fully melting the ice at the room temperature, stirring the ice to uniformly dissolve the chitosan, then placing the ice in the refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, taking the ice out, fully melting the ice again at the room temperature, and uniformly stirring the ice to obtain a chitosan solution;

3) preparing a cellulose solution from the solvent obtained in the step 1), wherein the solution comprises the following components in parts by weight:

cellulose, process for producing the same, and process for producing the same	2-4 parts of
		Solvent obtained in step 1)	96-98 parts of

Weighing the components in parts by weight, adding the components into a container, and uniformly stirring at room temperature to obtain a cellulose suspension. Hermetically placing the mixture in a constant-temperature refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, after freezing the ice, fully melting the ice at the room temperature, stirring the ice to uniformly dissolve the chitosan, then placing the ice in the refrigerator at the temperature of between 40 ℃ below zero and 20 ℃ below zero for 2 to 6 hours, taking the ice out, fully melting the ice again at the room temperature, and uniformly stirring the ice to obtain a cellulose solution;

4) mixing the chitosan solution obtained in the step 2) with the cellulose solution obtained in the step 3) in a volume ratio of 1:1, 1:2 or 2:1 to obtain a chitosan/cellulose mixed solution;

5) adding the polymer solution obtained in the step 4) into an injector, obtaining uniform spherical liquid drops by selecting an injection needle with the diameter of 24-28G, dripping the uniform spherical liquid drops into a dilute sulfuric acid or dilute hydrochloric acid solution with the volume fraction of 5-20%, wherein the dripping speed is 30-40 drops/min, the temperature is maintained at 25-30 ℃, and standing the solution in the dilute sulfuric acid or dilute hydrochloric acid solution for 1-3 hours to obtain the solidified gel microspheres: filtering the gel microspheres through a filter screen, and washing the gel microspheres with deionized water until the pH of a washing solution is between 7 and 8 to obtain chitosan/cellulose microspheres;

6) preparing the chitosan/cellulose microspheres obtained in the step (5) into heparin modified chitosan/cellulose microspheres, wherein the reaction components comprise the following components in parts by weight:

weighing the components in parts by weight, stirring at normal temperature, reacting for 12-24 hours, filtering the microspheres by using a filter screen, and washing with deionized water to obtain the heparin-modified chitosan/cellulose microspheres.

In order to achieve the required performance, the preparation method of the invention is different from the prior method, and firstly forms the crosslinked chitosan/cellulose microspheres, and then forms the heparin coating on the surfaces of the composite microspheres.

In the preparation method, the dilute sulfuric acid or dilute hydrochloric acid selected in the preparation process of the microsphere is used as a coagulating bath, so that amino groups in chitosan molecules can be fully protonated, and the positive charges carried by the inner layer of the microsphere are increased, thereby enhancing the adsorption effect of the microsphere on endotoxin.

In step (6), the group is activated by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and heparin is grafted by an amide reaction.

The invention provides a heparin modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent, which comprises chitosan/cellulose microspheres formed by physically crosslinking cellulose and chitosan and heparin modified chitosan/cellulose microspheres (namely, the self-anticoagulation blood perfusion adsorbent) subjected to heparin surface modification.

The chitosan is the only polysaccharide with positive charge existing in nature, has good biocompatibility and hydrophilicity, can be used as a substrate (also a ligand) for adsorbing endotoxin, the mechanical strength of the chitosan/cellulose microsphere formed by cellulose crosslinking is obviously improved compared with that before the chitosan/cellulose microsphere is not crosslinked, the mechanical strength of the chitosan/cellulose microsphere is improved by more than 50% compared with that of the original chitosan microsphere, and the requirement of blood perfusion on the mechanical strength of a blood perfusion device in extracorporeal circulation is met.

Heparin is an anticoagulant drug extracted from animal internal organs, and has the function of specifically adsorbing histone in addition to the anticoagulant function. Introducing heparin into the surface of the chitosan/cellulose microsphere formed by crosslinking to prepare the heparin modified chitosan/cellulose microsphere, wherein the heparin modified chitosan/cellulose microsphere has the characteristics of toxin adsorption (endotoxin and histone) and anticoagulation; when the adsorbent is used as an adsorbent and filled into a blood perfusion device, the in-vivo injection of the traditional exogenous anticoagulant heparin in the blood perfusion treatment process can be reduced to a great extent, and the treatment cost and the bleeding risk caused by the exogenous heparin are greatly reduced.

Although heparin is reported in the prior art to neutralize toxins such as histone in blood, the "neutralization" is different from the "adsorption" in the present application, and after the "neutralization", a complex formed by histone and heparin still exists in blood, the concentration of histone is not changed, and the body may be damaged; in the application, after heparin is grafted on the adsorbent, the adsorbent can directly adsorb histone, so that the concentration of the histone in blood can be effectively and substantially reduced; that is, the molecule heparin itself is not capable of "adsorbing" histone, it is invisible in blood, it can only neutralize histone, it is not capable of removing histone from blood in this application, it must rely on the chitosan microsphere adsorbent in this application to produce the effect that heparin in this application is a ligand (providing functional group) for adsorbing histone. In addition, from the anticoagulation point of view, compared with the prior anticoagulation with carrageenan by the applicant, in the present application, the adsorbent is self-anticoagulated, because heparin is grafted on the surface of the adsorbent, and can only interact with blood directly contacted with the adsorbent in extracorporeal circulation, namely a local anticoagulation strategy, which is obviously different from the clinically existing hemoperfusion (purification) treatment: clinical heparin is directly injected into veins, belongs to a systemic anticoagulation strategy, and has higher bleeding risk (also proved by clinical data).

Abundant amino groups carried in chitosan molecules in the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent are fully protonated in dilute hydrochloric acid or dilute sulfuric acid solution, so that an inner-layer chitosan base material of the adsorbent is provided with abundant positive charges, and endotoxin with negative charges can be efficiently eliminated; the heparin coating in the outer layer of the adsorbent can form a complex with histone, thereby rapidly removing histone from blood. Therefore, the heparin modified chitosan/cellulose microspheres have higher adsorption capacity for endotoxin and histone.

The heparin modified chitosan/cellulose microsphere based self-anticoagulation blood perfusion adsorbent is substantially gel microspheres. The loose and porous network structure of the gel microsphere can be in more sufficient contact with endotoxin and histone, so that the gel microsphere has good capability of clearing the endotoxin and the histone.

The third object of the present invention is to provide an application of the blood perfusion adsorbent based on heparin-modified chitosan/cellulose microspheres in the preparation of a blood perfusion device for blood purification.

Preferably, the blood purification is blood purification of patients suffering from sepsis.

The heparin modified chitosan/cellulose microspheres can be applied to blood purification (perfusion) treatment of patients with sepsis through a unique multi-target adsorption mode, and the principle is as follows:

1. in the heparin modified chitosan/cellulose microspheres, protonated amino groups in chitosan molecules can act on endotoxin, so that the endotoxin can be efficiently adsorbed;

2. in the heparin-modified chitosan/cellulose microspheres, outer layer heparin molecules can form a compound with histone, so that the histone in blood is removed;

3. the surface modification of the heparin can endow the chitosan/cellulose microspheres with unique anticoagulation performance, and the anticoagulation performance can avoid the bleeding risk caused by additional injection of the anticoagulant and use of an exogenous anticoagulant in the blood purification process;

4. therefore, the heparin modified chitosan/cellulose microspheres can better protect patients with sepsis and realize blood purification from more targets.

At present, no heparin modified chitosan/cellulose microspheres prepared by the method of the invention are used for hemoperfusion treatment; in addition, the heparin modified chitosan/cellulose microspheres are particularly suitable for blood purification treatment of patients with sepsis, and compared with the traditional blood perfusion application fields, such as acute drug and toxicant poisoning, acute and chronic renal failure, hepatic encephalopathy and the like, blood purification of patients with sepsis has special treatment requirements, and the existing common blood perfusion materials cannot meet the requirements. The development of a special blood perfusion device for special diseases is the trend of blood purification (perfusion) treatment at present and in future

The application discloses a preparation method of a blood perfusion adsorbent based on chitosan/cellulose microspheres, which has better mechanical strength and stability, thereby better meeting the requirement of blood perfusion on the mechanical strength of the blood perfusion adsorbent. Heparin is grafted on the surface of the obtained chitosan/cellulose microspheres through a chemical reaction to prepare heparin modified chitosan/cellulose microspheres, the heparin modified chitosan/cellulose microspheres are used for simultaneously adsorbing endotoxin and histone in blood of patients with sepsis, and various pathogenic factors in the blood of patients with sepsis are eliminated through multiple adsorption targets, so that the treatment effect of blood purification treatment on patients with sepsis is improved.

Compared with the prior art, the invention has the advantages that:

1. the heparin modified chitosan/cellulose microsphere based self-anticoagulation blood perfusion adsorbent provided by the invention is a microsphere adsorbent which is formed by physically crosslinking chitosan microspheres with a network structure through cellulose and then modifying the surfaces of the chitosan microspheres through heparin; the adsorbent is prepared based on natural polysaccharide chitosan, cellulose and heparin, has excellent blood compatibility, has an anticoagulation group of heparin, and shows excellent anticoagulation performance;

2. according to the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent, abundant amino groups (from chitosan) enable the adsorbent to have abundant positive charges, so that the adsorbent has good endotoxin removing capacity; according to the invention, ligands for adsorbing endotoxin in the traditional blood perfusion adsorbent design and corresponding base materials (chitosan in the invention) are organically combined together, and the ligands for adsorbing endotoxin are grafted to the surface of the base materials without additional chemical reaction, so that the preparation process is simplified;

3. according to the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent, heparin modification can enable heparin to be coated on the surface of the chitosan/cellulose microsphere, so that specific adsorption of a heparin coating on histone is realized, and therefore the adsorbent has good cleaning capacity on the histone;

4. the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent provided by the invention has a loose and porous network structure, and can be in more sufficient contact with endotoxin and histone, so that the adsorbent has a high-efficiency adsorption function on the endotoxin and the histone;

5. according to the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent, the chitosan is crosslinked by cellulose, so that the mechanical property of the microsphere is improved, the microsphere can be effectively prevented from being broken in the using process, the requirement of clinical blood perfusion treatment is met, and the size stability of the microsphere can be kept in use;

6. according to the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent, heparin is grafted on the surface of a chitosan microsphere through an amide reaction between carboxyl in heparin molecules and amino in the chitosan molecules, so that the coating of the heparin on the surface of the chitosan/cellulose microsphere is realized, the blood compatibility of the chitosan/cellulose microsphere is improved while the adsorbent obtains a histone adsorption function, the self-anticoagulation characteristic is endowed to the chitosan/cellulose microsphere, the preparation process is simple, the conditions are mild, the whole reaction is carried out in a solution, an organic solvent is not required to be used as a reaction medium, the adsorbent is green and environment-friendly, the harm of the organic solvent to the environment and a human body is avoided, and meanwhile, the complicated treatment process caused by the recovery of the organic solvent is reduced;

7. the preparation method of the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent provided by the invention has the advantages that the chitosan/cellulose microsphere is prepared by a phase inversion method, and the operation is simple and convenient.

8. According to the preparation method of the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent, chitosan and cellulose are natural polysaccharides and are common chemical raw materials, heparin is widely present in animal viscera, resources are rich, cost is low, and industrial production is facilitated, so that the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent is easy to popularize and apply in the field of biological medicines;

9. the heparin modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent provided by the invention has excellent adsorption performance, good biocompatibility and mechanical performance, and excellent anticoagulation performance, and is a biological material (blood contact material) which has application prospect and is especially specially used for blood purification treatment of sepsis patients.

Drawings

FIG. 1 is a schematic diagram of the chemical reaction of grafting heparin when preparing the heparin-modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent according to the method of the present invention;

FIG. 2 is a Fourier infrared spectrum of chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1 and heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75, respectively) obtained in step (6) of example 1 and step (6) of example 2;

FIG. 3 shows the deformation amount of the chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1, the heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75, respectively) obtained in step (6) of example 1 and step (6) of example 2, and the chitosan microspheres (CS) obtained in step (3) of example 5 under the stress of 66.7 kPa;

FIG. 4 is a graph showing endotoxin adsorption capacities (results are shown in bar charts) of chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1, heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75, respectively) obtained in step (6) of example 1, chitosan/cellulose microspheres (CSCE (HCl)) obtained in step (5) of example 3, and chitosan/cellulose microspheres (CSCE (HAc)) obtained in step (5) of example 4, for adsorbing endotoxin in blood according to the present invention;

FIG. 5 is a graph showing adsorption capacities of chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1 of the present invention, and heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75, respectively) obtained in step (6) of example 1 and example 2, for adsorbing histone in blood (including adsorption capacities of experimental histone and bovine serum albumin of control group, the results are shown in black and gray bars, respectively);

fig. 6 is a blood coagulation time chart (including partial prothrombin time, thrombin time and prothrombin time, results are shown in gray, black and white bar charts, respectively) of chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1 of the present invention and heparin-modified chitosan/cellulose microspheres (cschep 150 and cschep 75, respectively) obtained in step (6) of example 1 and example 2.

The specific implementation mode is as follows:

the technical solutions of the present invention will be described in detail and fully below with reference to the following examples, and it should be understood that the described examples are only a part of the examples of the present invention, and not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example prepares the self-anticoagulation blood perfusion adsorbent based on heparin modified chitosan/cellulose microspheres by the following steps:

(1) quantitatively weighing 4.5 parts of lithium hydroxide, 7 parts of potassium hydroxide, 8 parts of urea and 80.5 parts of deionized water according to parts by weight of the raw materials, adding the raw materials into a container, and stirring at room temperature to completely dissolve the raw materials to obtain a solvent of cellulose and chitosan;

(2) quantitatively weighing 4 parts of cellulose and 96 parts of the cellulose solvent obtained in the step 1 by weight of raw materials, adding the raw materials into a container, and uniformly stirring at room temperature to obtain a cellulose suspension; sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing ice, starting stirring to dissolve cellulose uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a cellulose solution;

(3) quantitatively weighing 4 parts of chitosan and 96 parts of the chitosan solvent obtained in the step 1 according to parts by weight of the raw materials, adding the raw materials into a container, and uniformly stirring at room temperature to obtain a chitosan suspension. Sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing the ice, starting stirring to dissolve the chitosan uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a chitosan solution;

(4) mixing the chitosan solution and the cellulose solution in equal volume to obtain a chitosan/cellulose solution;

(5) adding a chitosan/cellulose solution into an injector, obtaining uniform spherical liquid drops through a 28G injection needle, sequentially dripping the uniform spherical liquid drops into dilute sulfuric acid with the volume fraction of 10%, wherein the dripping speed is 30-40 drops/min, and the temperature is maintained at 25-30 ℃ to obtain solidified gel microspheres; filtering the gel microspheres through a filter screen and washing the gel microspheres with deionized water until the pH of a washing solution is between 7 and 8 to obtain CSCE microspheres;

(6) quantitatively weighing 6g of chitosan/cellulose microspheres, 150mg of heparin sodium, 0.96g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 2.13g of 2- (N-morpholine) ethanesulfonic acid and 100g of deionized water, stirring at normal temperature for reaction for 24 hours, filtering the microspheres, and washing with the deionized water for 3-4 times to obtain CSCEHEP150 microspheres, wherein the grafting reaction principle is shown in figure 1.

Example 2

This example prepares the self-anticoagulation blood perfusion adsorbent based on heparin modified chitosan/cellulose microspheres by the following steps:

(1) quantitatively weighing 4.5 parts of lithium hydroxide, 7 parts of potassium hydroxide, 8 parts of urea and 80.5 parts of deionized water according to parts by weight of the raw materials, adding the raw materials into a container, and stirring at room temperature to completely dissolve the raw materials to obtain a solvent of cellulose and chitosan;

(2) quantitatively weighing 4 parts of cellulose and 96 parts of the cellulose solvent obtained in the step 1 by weight of raw materials, adding the raw materials into a container, and uniformly stirring at room temperature to obtain a cellulose suspension; sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing ice, starting stirring to dissolve cellulose uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a cellulose solution;

(3) quantitatively weighing 4 parts of chitosan and 96 parts of the chitosan solvent obtained in the step 1 according to parts by weight of the raw materials, adding the chitosan solvent into a container, and uniformly stirring at room temperature to obtain a chitosan suspension; sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing the ice, starting stirring to dissolve the chitosan uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a chitosan solution;

(4) mixing the chitosan solution and the cellulose solution in equal volume to obtain a chitosan/cellulose solution;

(5) adding a chitosan/cellulose solution into an injector, obtaining uniform spherical liquid drops through a 28G injection needle, sequentially dripping the uniform spherical liquid drops into dilute sulfuric acid with the volume fraction of 10%, wherein the dripping speed is 30-40 drops/min, and the temperature is maintained at 25-30 ℃ to obtain solidified gel microspheres; filtering the gel microspheres through a filter screen, and washing the gel microspheres with deionized water until the pH value of a washing solution is between 7 and 8 to obtain the chitosan/cellulose microspheres.

(6) 6g of chitosan/cellulose microspheres, 75mg of heparin sodium (92.5USP), 0.96g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 2.13g of 2- (N-morpholine) ethanesulfonic acid and 100g of deionized water are weighed quantitatively, stirred and reacted for 24 hours at normal temperature, and the microspheres are filtered and washed for 3-4 times by using the deionized water to obtain CSCEHEP75 microspheres.

Example 3

This example prepares the self-anticoagulation blood perfusion adsorbent based on heparin modified chitosan/cellulose microspheres by the following steps:

(1) quantitatively weighing 4.5 parts of lithium hydroxide, 7 parts of potassium hydroxide, 8 parts of urea and 80.5 parts of deionized water according to parts by weight of the raw materials, adding the raw materials into a container, and stirring at room temperature to completely dissolve the raw materials to obtain a solvent of cellulose and chitosan;

(2) quantitatively weighing 4 parts of cellulose and 96 parts of the cellulose solvent obtained in the step 1 by weight, adding the weighed materials into a container, and uniformly stirring at room temperature to obtain a cellulose suspension. Sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing ice, starting stirring to dissolve cellulose uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a cellulose solution;

(3) quantitatively weighing 4 parts of chitosan and 96 parts of the chitosan solvent obtained in the step 1 according to parts by weight of the raw materials, adding the raw materials into a container, and uniformly stirring at room temperature to obtain a chitosan suspension. Sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing the ice, starting stirring to dissolve the chitosan uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a chitosan solution;

(4) and mixing the chitosan solution and the cellulose solution in equal volume to obtain the chitosan/cellulose solution.

(5) Adding a chitosan/cellulose solution into an injector, obtaining uniform spherical liquid drops through a 28G injection needle, sequentially dripping the uniform spherical liquid drops into dilute hydrochloric acid with the volume fraction of 10%, wherein the dripping speed is 30-40 drops/min, and the temperature is maintained at 25-30 ℃ to obtain solidified gel microspheres; filtering the gel microspheres through a filter screen and washing the gel microspheres with deionized water until the pH of a washing solution is between 7 and 8 to obtain CSCE (HCl) microspheres.

Example 4

The preparation process of chitosan/cellulose microsphere blood perfusion adsorbent in this example is as follows:

(1) quantitatively weighing 4.5 parts of lithium hydroxide, 7 parts of potassium hydroxide, 8 parts of urea and 80.5 parts of deionized water according to parts by weight of the raw materials, adding the raw materials into a container, and stirring at room temperature to completely dissolve the raw materials to obtain a solvent of cellulose and chitosan;

(2) quantitatively weighing 4 parts of cellulose and 96 parts of the cellulose solvent obtained in the step 1 by weight of raw materials, adding the raw materials into a container, and uniformly stirring at room temperature to obtain a cellulose suspension; sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing ice, starting stirring to dissolve cellulose uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a cellulose solution;

(3) quantitatively weighing 4 parts of chitosan and 96 parts of the chitosan solvent obtained in the step 1 according to parts by weight of the raw materials, adding the chitosan solvent into a container, and uniformly stirring at room temperature to obtain a chitosan suspension; sealing and placing in a constant temperature refrigerator at minus 30 ℃ for 4 hours, after freezing the ice, starting stirring to dissolve the chitosan uniformly after the ice is fully melted at the room temperature, then placing in the refrigerator at minus 30 ℃ for 4 hours, taking out the ice and fully melting at the room temperature again, and then stirring uniformly to obtain a chitosan solution;

(4) mixing the chitosan solution and the cellulose solution in equal volume to obtain a chitosan/cellulose solution;

(5) adding a chitosan/cellulose solution into an injector, obtaining uniform spherical liquid drops through a 28G injection needle, sequentially dripping the uniform spherical liquid drops into dilute acetic acid with the volume fraction of 10%, wherein the dripping speed is 30-40 drops/min, and the temperature is maintained at 25-30 ℃ to obtain solidified gel microspheres; filtering the gel microspheres through a filter screen and washing the gel microspheres by deionized water until the pH of a washing solution is between 7 and 8 to obtain CSCE (HAc) microspheres.

Example 5

The preparation process of the chitosan microsphere blood perfusion adsorbent in this example is as follows:

(1) quantitatively weighing 4.5 parts of lithium hydroxide, 7 parts of potassium hydroxide, 8 parts of urea and 80.5 parts of deionized water according to parts by weight of the raw materials, adding the raw materials into a container, and stirring at room temperature to completely dissolve the raw materials to obtain a chitosan solvent;

(2) quantitatively weighing 4 parts of chitosan and 96 parts of the chitosan solvent obtained in the step 1 according to parts by weight of the raw materials, adding the chitosan solvent into a container, and uniformly stirring at room temperature to obtain a chitosan suspension; sealing and placing in a constant temperature refrigerator at-30 deg.C for 4 hr, after freezing, melting at room temperature, stirring to dissolve chitosan uniformly, placing in a refrigerator at-30 deg.C for 4 hr, taking out, melting at room temperature again, and stirring uniformly to obtain chitosan solution;

(3) adding a chitosan solution into an injector, obtaining uniform spherical liquid drops through a 28G injection needle, sequentially dripping the uniform spherical liquid drops into dilute sulfuric acid with the volume fraction of 10%, wherein the dripping speed is 30-40 drops/min, and the temperature is maintained at 25-30 ℃ to obtain the solidified gel microspheres; and filtering the gel microspheres through a filter screen and washing the gel microspheres with deionized water until the pH value of a washing solution is between 7 and 8 to obtain the CS microspheres.

Example 6

The components, structures, mechanical strengths, adsorption properties and anti-coagulation properties of the heparin-modified chitosan/cellulose microsphere-based self-anti-coagulation blood perfusion adsorbents prepared in examples 1 and 2, the chitosan/cellulose microsphere blood perfusion adsorbents prepared in examples 3 and 4, and the chitosan microsphere blood perfusion adsorbent prepared in example 5 were measured:

1. component and structure detection

Fourier transform infrared spectroscopy was performed on the chitosan/cellulose microspheres (CSCE) prepared in the step (5) of example 1 and the heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75, respectively) obtained in the step (6) of example 1 and the step (6) of example 2, respectively, and the results are shown in fig. 2; from FIG. 2, it can be found that the Fourier infrared spectra of CSCEHEP150 and CSCEHEP75 are at a wavelength of 1217cm with respect to CSCE^-1And 941cm^-1Characteristic absorption peaks of sulfonic acid groups appear at the positions respectively, and the heparin is proved to be successfully introduced into the surface of the crosslinked chitosan microsphere, namely the heparin modified chitosan/cellulose microsphere-based self-anticoagulation blood perfusion adsorbent is successfully prepared.

2. Mechanical Strength detection

The chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1, the heparin-modified chitosan/cellulose microspheres (cscehp 150 and cscehp 75, respectively) obtained in step (6) of example 1 and step (6) of example 2, and the chitosan microspheres (CS) obtained in step (3) of example 5 were subjected to a compression test using a universal material testing machine, and a graph of the deformation rate thereof as a function of pressure was obtained. As shown in fig. 3, under the maximum theoretical pressure condition (66.7kPa) of the hemoperfusion adsorbent, the deformation of the CSCE microspheres added with cellulose is significantly lower than that of the CS microspheres without fibers, and the mechanical strength is better, that is, the mechanical strength of the hemoperfusion adsorbent in the present invention is caused by physical cross-linking of chitosan and cellulose; after heparin modification, the deformation of heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75) is slightly increased compared with that of CSCE microspheres, which is related to that carboxyl groups in heparin molecules and amino groups in the chitosan molecules undergo amidation reaction to partially sacrifice the skeleton of the chitosan/cellulose microspheres in the modification process, but the mechanical strength of the chitosan/cellulose microspheres still meets the requirement on the mechanical strength of an adsorbent in blood perfusion treatment.

3. Detection of adsorption Properties

Endotoxin and histone in blood were removed by using chitosan/cellulose microspheres (CSCE) prepared in step (5) of example 1 and heparin-modified chitosan/cellulose microspheres (cscehp 150 and cscehp 75, respectively) obtained in step (6) of example 1 and step (6) of example 2, respectively.

The adsorption amount is calculated as follows:

q＝(C₀-C_t)*V/m

wherein q in the formula represents the adsorption amount of the microspheres, C₀And C_tRepresents the concentration of endotoxin or histone at the start and at t hours of adsorption, respectively, v (ml) represents the volume of endotoxin, and m (g) represents the dry mass of the chitosan/cellulose microspheres or heparin-modified chitosan/cellulose microspheres employed.

3.1 taking 1g (wet weight) of each of the chitosan/cellulose microspheres (CSCE) prepared in the step (5) of example 1, the heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75 respectively) obtained in the step (6) of example 1 and the heparin-modified chitosan/cellulose microspheres (CSCEHEP75) obtained in the step (6) of example 2, the chitosan/cellulose microspheres (CSCE (HCl)) obtained in the step (5) of example 3 and the chitosan/cellulose microspheres (CSCE (HAc)) obtained in the step (5) of example 4, putting the mixture into 2mL of endotoxin phosphate buffer solution with initial concentration of 84.5EU/mL, carrying out constant temperature oscillation adsorption at 37 ℃ for 3 hours, and carrying out Endosafee Portable Test System (PTS) to obtain chitosan/cellulose microspheres^TM) The instrument (from Charles River Laboratories International, inc. us) measures endotoxin changes in phosphate buffer before and after adsorption at limulus reagent test kit (sensitivity 0.05-5.0EU/mL), calculates the amount of autoadhesion blood perfusion adsorbent, other chitosan/cellulose microspheres, and adsorption amount to endotoxin (EU/g) of heparin-modified chitosan/cellulose microspheres, and the results are shown in fig. 4:

example 1 the chitosan/cellulose microspheres (CSCE) prepared in step (5) had an adsorption amount of endotoxin in phosphate buffer of 577.7 EU/g;

example 1 the adsorption amount of heparin-modified chitosan/cellulose microspheres (CSCEHEP150) obtained in step (6) in phosphate buffer solution to endotoxin is 250.8 EU/g;

example 2 the adsorption amount of heparin-modified chitosan/cellulose microspheres (CSCEHEP75) obtained in step (6) in phosphate buffer solution to endotoxin was 475.9 EU/g.

Example 3 the chitosan/cellulose microspheres (CSCE (HCl)) obtained in step (5) adsorbed endotoxin at 498.3EU/g in phosphate buffer.

Example 4 the chitosan/cellulose microspheres (CSCE (HAc)) obtained in step (5) adsorbed endotoxin at 205.4EU/g in phosphate buffer.

From the above results, it can be seen that different types of chitosan/cellulose microspheres formed by dropping chitosan/cellulose solutions into different types of dilute acid solutions have different endotoxin adsorption properties: the highest endotoxin adsorption capacity of chitosan/cellulose microspheres (CSCE) prepared from dilute sulfuric acid solution, and the second lowest endotoxin adsorption capacity of chitosan/cellulose microspheres (CSCE (HCl)) prepared from dilute hydrochloric acid solution; the chitosan/cellulose microspheres (csce (hac)) prepared from dilute acetic acid solution had the lowest endotoxin adsorption capacity. The difference is caused by that in the process of forming the microspheres, compared with hydrochloric acid and acetic acid which are used as coagulating baths, the chitosan/cellulose microspheres can receive more protons in sulfuric acid, so that the microspheres have more positive charges; therefore, the subsequent heparin modified chitosan/cellulose microspheres are prepared on the basis of CSCE. In addition, the above results show that the adsorption performance of endotoxin is reduced after the heparin coating is grafted on the surface, which accords with the theory that: the negative charge of heparin reduces the adsorption of endotoxin. In the invention, heparin is mainly used for adsorbing histone, can bring self-anticoagulation performance and improve biocompatibility, has a balance of heparin content and chitosan content in microspheres, and the more chitosan, the better endotoxin adsorption performance, but the poor anticoagulation performance and the lower histone adsorption capacity.

Therefore, the focus of the invention is how to balance and choose the amount of heparin, and finally, the amount of heparin selected for adsorbing endotoxin in blood is optimized, so that a certain endotoxin adsorption performance is kept, and good histone adsorption performance, anticoagulant performance and biocompatibility are also achieved.

3.2 the chitosan/cellulose microspheres (CSCE) prepared in the step (5) of example 1, and 1g (wet weight) of the heparin-modified chitosan/cellulose microspheres (CSCEHEP150 and CSCEHEP75, respectively) obtained in the step (6) of example 1 and the step (6) of example 2 were put into 2mL of a histone or bovine serum albumin phosphate buffer solution with an initial concentration of 100. mu.g/mL, and adsorbed at a constant temperature of 37 ℃ for 3 hours under shaking, the change in the concentration of histone or bovine serum albumin in the phosphate buffer solution before and after adsorption was detected, and the adsorption amount (μ g/g) of the self-anticoagulant blood perfusion adsorbent based on the heparin-modified chitosan/cellulose microspheres was calculated, and the results are shown in FIG. 5:

example 1 the chitosan/cellulose microspheres (CSCE) obtained in step (5) had an adsorption amount of 39.9. mu.g/g for histone and 105.7. mu.g/g for bovine serum albumin in a phosphate buffer;

example 1 the heparin-modified chitosan/cellulose microspheres (CSCEHEP150) obtained in step (6) adsorbed the histone in the phosphate buffer solution at 314.3. mu.g/g; the adsorption capacity to bovine serum albumin was 38.9. mu.g/g;

example 2 the resulting heparin-modified chitosan/cellulose microspheres (cschep 75) obtained in step (6) had an adsorption amount of histone of 222.9 μ g/g in a phosphate buffer; the adsorption capacity to bovine serum albumin was 55.3. mu.g/g;

from the above analysis, it can be seen that the present invention provides a heparin-modified chitosan/cellulose microsphere-based self-anticoagulant blood perfusion adsorbent, which can efficiently remove histone in a phosphate buffer solution, and the adsorption amount of histone increases with the increase of heparin content on the surface of the microsphere, and the adsorption amount of bovine serum albumin decreases with the increase of heparin content on the surface of the microsphere, because the negatively charged heparin coating can reduce the adsorption of negatively charged bovine serum albumin by the microspheres through electrostatic repulsion, i.e. the heparin-modified chitosan/cellulose microspheres have selectivity on the adsorption of the histone, and do not affect the normal albumin concentration in the body of the patient, and can be safely and effectively used for the blood purification treatment of the patient with sepsis.

4. Anticoagulant performance testing

The chitosan/cellulose microspheres (CSCE) prepared in the step (5) of example 1 and 1g (wet weight) of the heparin-modified chitosan/cellulose microspheres (cschep 150 and cschep 75, respectively) obtained in the step (6) of example 1 and the step (6) of example 2 were co-incubated with 5mL of platelet poor plasma for 30min, respectively, after which the plasma coagulation time was measured using an automated coagulation analyzer, and the results are shown in fig. 6:

the heparin modified chitosan/cellulose microspheres can prolong the partial thromboplastin time and the thrombin time, but do not influence the prothrombin time, show excellent anticoagulation capacity, particularly obviously improve the anticoagulation capacity along with the increase of the heparin content, and can prolong the activated partial thromboplastin time from 32.8 seconds to 100.5 seconds. Therefore, the heparin modified chitosan/cellulose microsphere self-anticoagulation blood perfusion adsorbent provided by the invention has excellent anticoagulation performance, can directly prevent blood coagulation during blood purification, and thus reduces the application of exogenous heparin in blood purification; the method not only reduces the treatment cost, but also can prevent patients from generating the risk of serious hemorrhage and other side effects caused by systemic anticoagulation generated by exogenous heparin injection.