Adsorbents for the treatment of stroke, methods of making and cartridges thereof

Abstract

The application provides an adsorbent for treating cerebral apoplexy, a preparation method thereof and a perfusion device. The adsorbent comprises a carrier and a first functional layer and a second functional layer arranged on the surface of the carrier in sequence; the carrier has epoxy groups; the first functional layer is a grafted layer formed by long-chain linear aliphatic diamines reacting with the epoxy groups on the carrier, one amine group in the long-chain linear aliphatic diamines is connected with the epoxy groups, and the other amine group in the long-chain linear aliphatic diamines is located on the surface of the first functional layer; and the second functional layer is a grafted layer formed by dextran sulfate connecting with the amine groups on the surface of the first functional layer. The application can effectively adsorb blood lipids, fibrinogen and inflammatory factors in blood.

Description

Technical Field

[0001] This invention relates to the field of extracorporeal blood purification technology, and more specifically, to an adsorbent for treating stroke, its preparation method, and a perfusion device. Background Technology

[0002] Acute ischemic stroke (AIS) has become a major public health challenge worldwide due to its high incidence, disability rate, and mortality rate. Epidemiological data in my country show that the incidence of AIS is on the rise, and the age of onset is gradually decreasing, further exacerbating the social medical burden. Currently, the main treatments for AIS are intravenous thrombolysis and mechanical thrombectomy, but their strict time window (usually 4.5–6 hours after onset) causes many patients to miss the opportunity for direct recanalization due to pre-hospital delays or exceeding the window period. For these patients, timely and effective supportive care is crucial for improving prognosis.

[0003] Dyslipidemia, hyperfibrinogenemia, and excessive inflammatory response are key pathological mechanisms in the occurrence, development, and exacerbation of acute myeloid inflammatory syndrome (AIS). Based on this, current clinical treatment, in addition to recanalization methods, often involves the combined use of multiple drugs: such as batroxobin to lower fibrinogen, statins to lower lipids and reduce inflammation, and edaravone dexborneol to scavenge free radicals and inhibit inflammatory factors. These drugs are all recommended by domestic and international guidelines and widely used in clinical practice. Clinical studies have confirmed that these drugs have a positive effect on improving patients' neurological deficits, suggesting that effectively clearing or regulating the aforementioned pathogenic substances has significant therapeutic value. However, drug therapy has problems such as individual efficacy differences, potential drug resistance risks, and drug-related adverse reactions, and some patients still cannot benefit from it. Furthermore, the use of multiple drugs may increase the economic and physiological burden on patients. Therefore, developing safer, more efficient, and universally applicable non-pharmacological interventions has become an important direction of current research. Summary of the Invention

[0004] This invention aims to provide an adsorbent for treating stroke, its preparation method, and an perfusion device. This adsorbent is suitable for extracorporeal blood circulation therapy, and can efficiently adsorb blood lipids, fibrinogen (FIB), and inflammatory factors in the blood, thereby effectively reducing hypercoagulability, improving lipid metabolism disorders, and inhibiting excessive inflammatory responses, which is beneficial for the recovery of neurological function in stroke patients. This invention provides stroke patients with a novel treatment option that is safer, more efficient, and has good universality than drug intervention.

[0005] To address the above problems, a first aspect of the present invention provides an adsorbent for treating stroke, comprising a carrier and a first functional layer and a second functional layer sequentially disposed on the surface of the carrier;

[0006] The carrier has epoxy groups;

[0007] The first functional layer is a grafted layer formed by reacting a long-chain linear aliphatic diamine with an epoxy group on the support. One amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer.

[0008] The second functional layer is a graft layer formed by connecting dextran sulfate to the amino groups on the surface of the first functional layer.

[0009] Furthermore, the first functional layer is a graft layer formed by covalently linking a long-chain linear aliphatic diamine with an epoxy group on the support via a nucleophilic ring-opening reaction;

[0010] The second functional layer is a graft layer formed by covalently linking carboxyl-containing dextran sulfate with amino groups on the surface of the first functional layer through an amidation reaction.

[0011] Furthermore, the carrier is a cellulose microsphere, and epoxy groups are introduced onto the cellulose microsphere; the long-chain linear aliphatic diamine is 1,16-hexadecanediamine.

[0012] Furthermore, the loading of long-chain linear aliphatic diamines on the adsorbent is 15 mg / g to 20 mg / g, and the loading of dextran sulfate is 100 mg / g to 200 mg / g.

[0013] A first aspect of the present invention provides a method for preparing an adsorbent for treating stroke, comprising the following steps:

[0014] A support is provided, and epoxy groups are introduced onto the support to obtain an epoxidized support;

[0015] The epoxidized support is reacted with a long-chain linear aliphatic diamine to form a first functional layer on the surface of the support, thereby obtaining a first grafted support; wherein, one amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer.

[0016] The adsorbent is prepared by attaching dextran sulfate to the first grafted support through the amine groups on the surface of the first functional layer, thereby forming a second functional layer on the support.

[0017] Further, the step of introducing epoxy groups onto the support to obtain the epoxidized support includes:

[0018] The carrier is mixed with an alkaline solution to swell the carrier and obtain a mixture.

[0019] The mixture was continuously stirred at 40°C to 60°C, and epichlorohydrin was added dropwise to the mixture. After reacting for 2 to 4 hours, the epoxidized support was obtained.

[0020] The carrier is cellulose microspheres, and the ratio of the cellulose microspheres, alkaline solution and epichlorohydrin is 1g:(10-20)ml:(2-5)ml; the concentration of the alkaline solution is 2mol / L to 5mol / L.

[0021] Further, the step of reacting the epoxidized support with a long-chain linear aliphatic diamine to form a first functional layer on the surface of the support, thereby obtaining the first grafted support, includes:

[0022] Purified water and the long-chain linear aliphatic diamine were added sequentially to the epoxidized carrier, and an amination reaction was carried out at 25°C to 50°C. A first functional layer was grafted onto the surface of the epoxidized carrier and amine groups were introduced to obtain the first grafted carrier.

[0023] The volume ratio of the epoxidized carrier to the long-chain linear aliphatic diamine is 10:(2-5).

[0024] Further, the adsorbent is prepared by attaching dextran sulfate to the first grafted support via the amine groups on the surface of the first functional layer, thereby forming a second functional layer on the support, comprising:

[0025] Carboxyl groups were introduced into sodium dextran sulfate to prepare oxidized sodium dextran sulfate;

[0026] The first grafted support is subjected to an amidation reaction with oxidized sodium dextran sulfate, so that the dextran sulfate graft forms a second functional layer on the first grafted support, thereby obtaining the adsorbent.

[0027] The method of introducing carboxyl groups into sodium dextran sulfate to obtain oxidized sodium dextran sulfate includes:

[0028] Sodium dextran sulfate was dissolved in deionized water and cooled to 0 to 4°C. TEMPO and NaBr were added sequentially, and the mixture was stirred until dissolved to obtain a mixture. The pH of the mixture was adjusted to 10 to 11, and NaClO solution was added to the mixture to form a first reaction solution. At the same time, NaOH solution was added dropwise to the first reaction solution to maintain a constant pH value. When the amount of NaOH solution consumed reached a preset threshold and the pH value of the first reaction solution remained unchanged, the reaction ended, and oxidized sodium dextran sulfate was obtained.

[0029] Further, the step of subjecting the first grafted support to an amidation reaction with oxidized sodium dextran sulfate, thereby forming a second functional layer on the first grafted support to obtain the adsorbent, comprises:

[0030] The first grafting carrier and the oxidized sodium dextran sulfate were added to an acidic buffer solution to form a second reaction solution. The second reaction solution was subjected to an amidation reaction under the action of an activator, so that the dextran sulfate was grafted onto the first grafting carrier.

[0031] The amidation reaction is carried out at a temperature of 4°C to 37°C for a reaction time of 12 h to 24 h.

[0032] The concentration of sodium dextran sulfate in the second reaction solution is 5 mg / mL to 20 mg / mL, and the volume ratio of the first grafted carrier to the acidic buffer solution in the second reaction solution is 1:1.

[0033] A third aspect of the present invention provides a perfusion device comprising an adsorbent for treating stroke as described in any one of the first aspects, or comprising an adsorbent for treating stroke prepared by the preparation method described in any one of the second aspects, wherein the perfusion device is used for adsorbing fibrinogen, blood lipids and inflammatory factors during extracorporeal blood circulation.

[0034] The adsorbent for treating stroke provided by this invention has an epoxy group on the carrier. The epoxy group reacts with a long-chain linear aliphatic diamine to immobilize the long-chain linear aliphatic diamine on the surface of the carrier. The long-chain linear alkyl group of the long-chain linear aliphatic diamine has strong hydrophobicity and can strongly adsorb hydrophobic lipoproteins and fibrinogen. In addition, the hydrophobic amino acid residues in cytokines can form hydrophobic cores or hydrophobic patches on the molecular surface in the three-dimensional structure, and can also bind with the long-chain linear aliphatic diamine through hydrophobic interactions to achieve the adsorption of lipoproteins, fibrinogen and cytokines. Furthermore, one amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine can be connected to dextran sulfate, so that the long-chain linear aliphatic diamine acts as a spacer arm to connect the carrier and dextran sulfate. Dextran sulfate contains a large number of sulfate groups, which carry a negative charge. These sulfate groups can bind to positively charged lipids such as LDL-c and inflammatory factors with positively charged amino residues through electrostatic interactions, thereby achieving the adsorption of LDL-c and inflammatory factors. The adsorbent provided in this invention, through the long-chain linear aliphatic diamine and dextran sulfate linked to the outer layer of the carrier, can achieve a dual adsorption effect of electrostatic and hydrophobic interactions, synergistically adsorbing lipoproteins, fibrinogen, and inflammatory factors. This effectively reduces the content of these substances in the blood, achieving neuroprotection, reducing blood viscosity, and promoting microcirculation. Furthermore, dextran sulfate has good biocompatibility. Placing dextran sulfate on the outermost layer can improve the biocompatibility of the adsorbent, which is beneficial for capturing lipoproteins, fibrinogen, and inflammatory factors in the blood, thereby further enhancing the adsorption capacity for these substances.

[0035] The method for preparing an adsorbent for treating stroke provided in this invention involves introducing epoxy groups onto a support, then combining the epoxy groups with a long-chain linear aliphatic diamine via a nucleophilic ring-opening reaction, and finally linking the amino groups on the long-chain linear aliphatic diamine with dextran sulfate via an amidation reaction. This method ensures a strong bond between the support, the long-chain linear aliphatic diamine, and the dextran sulfate, reducing the risk of detachment of the long-chain linear aliphatic diamine and the dextran sulfate, and thus ensuring the safety of the adsorbent. The preparation method of this invention involves sequentially grafting a long-chain linear aliphatic diamine and dextran sulfate onto the outer layer of a carrier. This achieves synergistic adsorption of lipoproteins, fibrinogen, and inflammatory factors through both electrostatic and hydrophobic interactions, effectively reducing the levels of these substances in the blood. This results in neuroprotection, reduced blood viscosity, and improved microcirculation. Furthermore, compared to grafting dextran sulfate first and then the long-chain linear aliphatic diamine, this embodiment grafts the long-chain linear aliphatic diamine first, allowing the long-chain linear aliphatic diamine to simultaneously serve as a ligand and spacer arm, simplifying the reaction steps and reducing the reaction difficulty. In addition, the preparation method provided by this invention involves relatively fewer reaction steps, a simpler preparation process, milder reaction conditions, higher safety during the reaction process, and lower production costs, making it suitable for industrial production. Attached Figure Description

[0036] Figure 1 This is a process flow diagram for preparing an adsorbent for treating stroke, provided in an embodiment of the present invention. Detailed Implementation

[0037] Currently, the most common treatments for stroke are thrombolysis or thrombectomy. However, many patients in my country have missed the optimal window for these treatments or are not suitable candidates. Other medications offer limited therapeutic effects and are subject to individual efficacy variations, potential drug resistance risks, and drug-related adverse reactions. Therefore, stroke remains a disease with high mortality and disability rates.

[0038] Blood purification technology refers to a treatment method that uses physical or chemical methods to draw a patient's blood out of the body, remove pathogenic substances from the circulatory system through a specific device, and then return the purified blood to the body. It has advantages such as speed, high efficiency, no drug resistance, and low toxicity. Blood purification technology has been initially explored and applied in the clinical treatment of some patients with refractory acute ischemic stroke (AIS) or those in the window of onset. Among related technologies, blood purification techniques for AIS mainly include heparin-induced extracorporeal low-density lipoprotein precipitation (HELP), extracorporeal plasma lipid adsorption filtration (DELP), and dual filtration plasma exchange (DFPP). These technologies mainly rely on physical mechanisms such as membrane separation, plasma component separation and exchange to remove pathogenic mediators (such as large molecular proteins and inflammatory factors) from the blood. However, analysis reveals that existing technologies for AIS blood purification have the following limitations: First, their core technologies are mostly based on membrane filtration or physical separation, focusing primarily on physical sieving or replacement, lacking the ability to specifically identify and capture pathogens, resulting in low adsorption capacity and difficulty in achieving ideal biological effects. Second, some therapies relying on plasma separation typically require complex tubing systems and large amounts of replacement fluid, placing high demands on medical equipment and blood product supply, increasing treatment costs and potential infection risks.

[0039] To address the aforementioned problems in related technologies, this application provides an adsorbent for treating stroke, its preparation method, and a perfusion device. Based on the principle of adsorption, this adsorbent can specifically adsorb blood lipids, fibrinogen, and inflammatory factors, which is beneficial for the treatment and rehabilitation of stroke patients. The resin adsorption technology employed in this application not only overcomes the shortcomings of existing physical separation technologies, such as insufficient specificity and high cost, but also possesses advantages such as wide applicability to suitable instruments and equipment, relatively low production costs, and simple operation procedures. Therefore, it demonstrates great potential in filling the aforementioned technological gaps and providing a novel solution for AIS blood purification.

[0040] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0042] Furthermore, the terms "comprising," "including," "containing," and "having" are non-restrictive and can refer to the addition of other steps and components that do not affect the results. Unless otherwise specified, all materials, equipment, and reagents are commercially available.

[0043] Furthermore, although the present invention describes each step in the preparation process in the form of S110, S120 and S130, this description is only for ease of understanding. The forms such as S110, S120 and S130 do not indicate a limitation on the order of the steps.

[0044] A first aspect of this application provides an adsorbent for treating stroke, the adsorbent comprising a carrier and a first functional layer and a second functional layer sequentially disposed on the surface of the carrier, wherein:

[0045] The carrier has epoxy groups;

[0046] The first functional layer is a grafted layer formed by the reaction of a long-chain linear aliphatic diamine with epoxy groups on a support. One amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer.

[0047] The second functional layer is a graft layer formed by the connection of sulfated dextran with the amino groups on the surface of the first functional layer.

[0048] The adsorbent for treating stroke in this embodiment has an epoxy group on the carrier. The epoxy group reacts with a long-chain linear aliphatic diamine to immobilize the long-chain linear aliphatic diamine on the surface of the carrier. The long-chain linear alkyl group of the long-chain linear aliphatic diamine has strong hydrophobicity and can strongly adsorb hydrophobic lipoproteins and fibrinogen. In addition, the hydrophobic amino acid residues in cytokines can form hydrophobic cores or hydrophobic patches on the molecular surface in the three-dimensional structure, and can also bind with the long-chain linear aliphatic diamine through hydrophobic interactions to achieve the adsorption of lipoproteins, fibrinogen and cytokines. Furthermore, one amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine can be connected to dextran sulfate, so that the long-chain linear aliphatic diamine acts as a spacer arm to connect the carrier and dextran sulfate. Dextran sulfate contains a large number of sulfate groups, which carry a negative charge. These sulfate groups can bind to positively charged lipids such as LDL-c and inflammatory factors with positively charged amino residues through electrostatic interactions, thereby achieving the adsorption of LDL-c and inflammatory factors. The adsorbent provided in this embodiment, through the long-chain linear aliphatic diamine and dextran sulfate linked to the outer layer of the carrier, can achieve a dual adsorption effect of electrostatic and hydrophobic interactions, synergistically adsorbing lipoproteins, fibrinogen, and inflammatory factors. This effectively reduces the content of these substances in the blood, achieving neuroprotection, reducing blood viscosity, and promoting microcirculation. Furthermore, dextran sulfate has good biocompatibility; placing it on the outermost layer improves the biocompatibility of the adsorbent, facilitating the capture of lipoproteins, fibrinogen, and inflammatory factors in the blood, further enhancing its adsorption capacity for these substances.

[0049] Based on the above embodiments, as an optional implementation, the first functional layer is a graft layer formed by covalently linking a long-chain linear aliphatic diamine with an epoxy group on the support via a nucleophilic ring-opening reaction; the second functional layer is a graft layer formed by covalently linking a carboxyl-containing dextran sulfate with an amino group on the surface of the first functional layer via an amidation reaction. Thus, one primary amino group of the long-chain linear aliphatic diamine reacts with the epoxy group to form a CN bond, while the other primary amino group is exposed on the surface of the first functional layer to connect with the dextran sulfate. This allows both the long-chain linear aliphatic diamine and the dextran sulfate to be chemically bonded, resulting in a strong bond between the long-chain linear aliphatic diamine and the support, as well as between the long-chain linear aliphatic diamine and the dextran sulfate. This improves the bonding tightness and structural strength of the adsorbent, reduces the risk of detachment of the long-chain linear aliphatic diamine and the dextran sulfate, and helps ensure the safety of the adsorbent in use.

[0050] Based on the above embodiments, as an optional implementation, the carrier is cellulose microspheres, and epoxy groups are introduced onto the cellulose microspheres. As a natural macromolecule, cellulose microspheres have good blood compatibility, which facilitates the binding of the adsorbent to LDL-c and fibrinogen in the blood, thereby improving the adsorption performance of the adsorbent.

[0051] Based on the above embodiments, as an optional implementation, the long-chain linear aliphatic diamine is 1,16-hexadecanediamine. The long-chain alkyl group of 1,16-hexadecanediamine has strong hydrophobicity, which can improve the adsorption capacity for hydrophobic lipoproteins, fibrinogen, and cytokines.

[0052] Based on the above embodiments, as another optional implementation, the long-chain linear aliphatic diamine can also be 1,14-tetradecanediamine or 1,12-dodecanediamine. However, compared to 1,14-tetradecanediamine or 1,12-dodecanediamine, 1,16-hexadecanediamine has stronger hydrophobicity and better adsorption capacity for lipoproteins, fibrinogen, and cytokines. In this embodiment, 1,16-hexadecanediamine is preferably used as the long-chain linear aliphatic diamine.

[0053] Based on the above embodiments, as an optional implementation, the loading of long-chain linear aliphatic diamine on the adsorbent is 15 mg / g to 20 mg / g, that is, 15 mg to 20 mg of long-chain linear aliphatic diamine is immobilized on each 1 g of adsorbent; the loading of dextran sulfate is 100 mg / g to 200 mg / g, that is, 100 mg to 200 mg of dextran sulfate is immobilized on each 1 g of adsorbent.

[0054] Figure 1 This is a process flow diagram for preparing an adsorbent for treating stroke, provided in an embodiment of this application. (Combined with...) Figure 1As shown, a second aspect of this application provides a method for preparing an adsorbent for treating stroke, comprising the following steps:

[0055] Step S110: Provide a support, introduce epoxy groups onto the support, and obtain an epoxidized support.

[0056] Specifically, the carrier is mixed with an alkaline solution to swell and obtain a mixture. The mixture is then continuously stirred at 40°C to 60°C, and epichlorohydrin is added dropwise. After reacting for 2 to 4 hours, the epoxidized carrier is obtained. The carrier is cellulose microspheres.

[0057] More specifically, dried cellulose microspheres and an alkaline solution are added to an Erlenmeyer flask or beaker. The cellulose microspheres and alkaline solution are mixed and slowly stirred or shaken at room temperature for 30 to 60 minutes to allow the cellulose microspheres to fully swell. Then epichlorohydrin is added, and the mixture is heated and stirred until homogeneous. The reaction is maintained in a water bath at 40 to 60°C for 2 to 4 hours to carry out the epoxidation reaction of the cellulose microspheres, thus obtaining epoxidized cellulose microspheres.

[0058] In this embodiment, the epoxidation reaction is carried out in a constant temperature environment of 40℃ to 60℃, which is beneficial to improve the reactivity of cellulose microspheres and make the cellulose microspheres have more epoxy groups.

[0059] Based on the above embodiments, as an optional implementation, the ratio of cellulose microspheres, alkaline solution, and epichlorohydrin is 1g:(10-20)ml:(2-5)ml; the concentration of the alkaline solution is 2mol / L~5mol / L. The alkaline solution can be either NaOH or KOH solution. Therefore, by controlling the amounts of cellulose microspheres, alkaline solution, and epichlorohydrin, it is beneficial to fully activate the cellulose microspheres, which is conducive to the subsequent grafting of long-chain linear aliphatic diamines and sulfated dextran; and the relatively small amount of epichlorohydrin helps improve the safety of the adsorbent during subsequent use.

[0060] Step S120: React the epoxidized support with a long-chain linear aliphatic diamine to form a first functional layer on the surface of the support, thereby obtaining a first grafted support; wherein, one amino group of the long-chain linear aliphatic diamine is connected to an epoxy group, and the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer.

[0061] Specifically, purified water and a long-chain linear aliphatic diamine are added sequentially to the epoxidized support, and an amination reaction is carried out at 25°C to 50°C for 1 to 2 hours. This allows one amino group of the long-chain linear aliphatic diamine to be attached to an epoxy group, while the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer. This process grafts the first functional layer onto the surface of the epoxidized support and introduces amino groups, thus obtaining the first grafted support.

[0062] In this embodiment, the amination reaction is carried out in a constant temperature environment of 25°C to 50°C, which is beneficial for amination of the active epoxy groups on the support, improving the grafting rate of long-chain linear aliphatic diamines on the support, and also facilitating the subsequent grafting of dextran sulfate.

[0063] Based on the above embodiments, as an optional implementation, the volume ratio of the epoxidized support to the long-chain linear aliphatic diamine is 10:(2-5), wherein the long-chain linear aliphatic diamine is 1,16-hexadecanediamine. Therefore, by controlling the amount of the long-chain linear aliphatic diamine, it is beneficial to fully amination of the active groups of the epoxidized support and to improve the grafting rate of the long-chain linear aliphatic diamine on the support.

[0064] Based on the above embodiments, as an optional implementation, this embodiment further includes, after the amination reaction is completed and before obtaining the first grafted carrier, the following steps are taken: after the amination reaction is completed, the reaction product is repeatedly washed with purified water until the pH value of the washing solution is less than or equal to 8, thereby obtaining the first grafted carrier. This avoids the residue of long-chain linear aliphatic diamines on the first grafted carrier, preventing subsequent interference with the grafting of dextran sulfate onto the first grafted carrier, or affecting the reactivity of the grafted dextran sulfate onto the first grafted carrier.

[0065] In this embodiment, the grafting rate of the long-chain linear aliphatic diamine on the first grafting carrier is 30wt% to 50wt%. In this embodiment, the grafting rate of the long-chain linear aliphatic diamine on the first grafting carrier can be determined by the ninhydrin colorimetric method. Specifically, the first grafting carrier is stained with ninhydrin, and the ultraviolet absorbance is detected at 570 nm. After quantitative calculation, the grafting rate of the long-chain linear aliphatic diamine on the first grafting carrier is obtained.

[0066] Specifically, if the long-chain linear aliphatic diamine is 1,16-hexadecanediamine, a series of standard solutions of known concentrations are prepared using 1,16-hexadecanediamine hydrochloride. These solutions are then reacted with ninhydrin chromogenic reagent (2% ninhydrin ethanol solution), and the absorbance is measured at 570 nm to plot a standard curve (i.e., a standard curve between 1,16-hexadecanediamine and absorbance). 10 mg of the dried first grafted carrier is weighed and placed in a test tube. 2 mL of ninhydrin chromogenic reagent and 2 mL of pH 5.0 acetate buffer are added. The mixture is heated in a boiling water bath for 15-20 minutes, cooled, and centrifuged to collect the supernatant. The absorbance is measured at 570 nm, and the original cellulose microspheres (i.e., the carrier without introduced epoxy groups) are measured as a blank. The grafting rate of 1,16-hexadecanediamine on the adsorbent is calculated based on the standard curve.

[0067] Step S130: By attaching dextran sulfate to the first grafting support through the amine groups on the surface of the first functional layer, a second functional layer is formed on the support, and an adsorbent is obtained.

[0068] Specifically, carboxyl groups are introduced into sodium dextran sulfate to obtain oxidized sodium dextran sulfate; the first grafting support is subjected to an amidation reaction with the oxidized sodium dextran sulfate, and the carboxyl groups on the sodium dextran sulfate and the amine groups on the surface of the first functional layer are used to graft the dextran sulfate onto the first grafting support to form a second functional layer, thereby obtaining an adsorbent.

[0069] Based on the above embodiments, as an optional implementation, a carboxyl group is introduced into sodium dextran sulfate to obtain oxidized sodium dextran sulfate, comprising:

[0070] Sodium dextran sulfate was dissolved in deionized water and cooled to 0-4°C. Then, TEMPO (2,2,6,6-tetramethylpiperidinooxy) and NaBr were added sequentially and stirred to dissolve, obtaining a mixture. The pH of the mixture was adjusted to 10-11, and NaClO solution was added to form the first reaction solution. At the same time, NaOH solution was added dropwise to the first reaction solution to maintain a constant pH value. When the amount of NaOH solution consumed reached a preset threshold and the pH value of the first reaction solution remained unchanged, the reaction ended, and the oxidized sodium dextran sulfate was obtained.

[0071] Specifically, sodium dextran sulfate is dissolved in deionized water and cooled to 0-4°C in an ice-water bath. TEMPO and NaBr are added sequentially, and the mixture is stirred until dissolved to obtain a mixture. The pH of the mixture is adjusted to 10-11 using 0.5 mol / L NaOH. NaClO solution is then slowly added dropwise to form the first reaction solution. Simultaneously, 0.5 mol / L NaOH solution is added dropwise to the first reaction solution using an automatic titrator (or under close manual monitoring), maintaining the pH of the first reaction solution at a constant 10.5. The reaction ends when the amount of NaOH solution consumed reaches a preset threshold and the pH of the first reaction solution no longer decreases. The entire reaction process needs to be carried out at 0 to 4°C for about 1 to 3 hours. 5 mL of ethanol is added to terminate the reaction, and the mixture is stirred for 15 minutes. The pH of the first reaction solution is adjusted to neutral (approximately 7.0) with dilute hydrochloric acid. Then, the solution is dialyzed against 0.1 mol / L NaCl solution for 24 hours (changing the solution 2-3 times), followed by dialyzed against a large volume of deionized water for 24 to 48 hours (changing the solution 4-6 times), until the conductivity of the dialysate is close to that of pure water. The solution in the dialysis bag is transferred to a round-bottom flask and concentrated to a smaller volume under reduced pressure at below 40°C using a rotary evaporator to obtain oxidized sodium dextran sulfate.

[0072] In this embodiment, sodium dextran sulfate is oxidized to introduce carboxyl groups into it, facilitating its connection with the amino groups on the surface of the first grafted support via an amidation reaction. By controlling the pH value during the reaction process within the aforementioned range, hypochlorite can be stably maintained in the form of ClO⁻, efficiently oxidizing TEMPO to the active oxidant TEMPO⁺. This avoids the side reactions that occur when the pH value is too low, such as NaClO decomposing into Cl2 or HOCl, which would affect the oxidation efficiency of TEMPO. Conversely, when the pH value is too high, sulfate groups may detach and the polysaccharide backbone may degrade, affecting the adsorption capacity of dextran sulfate.

[0073] Those skilled in the art can calculate the preset threshold based on the target oxidation degree. Specifically, it can be calculated based on the amount of carboxyl groups that need to be introduced per gram of sodium dextran sulfate. Generally speaking, when 1 mol of hydroxymethyl (-CH2OH) is oxidized to a carboxyl group (-COOH), 1 mol of NaClO (sodium hypochlorite) is usually required.

[0074] Based on the above embodiments, as an optional implementation, the first grafting support is subjected to an amidation reaction with oxidized sodium dextran sulfate, so that dextran sulfate is grafted onto the first grafting support to form a second functional layer, thereby obtaining an adsorbent comprising:

[0075] The first grafting carrier and the oxidized sodium dextran sulfate were added to an acidic buffer solution to form a second reaction solution. The second reaction solution was subjected to an amidation reaction under the action of an activator, so that the dextran sulfate was grafted onto the first grafting carrier. The amidation reaction was carried out at a temperature of 4°C to 37°C and for a reaction time of 12h to 24h.

[0076] In this embodiment, the activity of the oxidized sodium dextran sulfate is activated by an activator, which is beneficial for the dextran sulfate to be linked to the amino groups on the surface of the carrier. The amidation reaction is carried out at 4°C to 37°C to ensure that the dextran sulfate has high reactivity, which is beneficial for improving the grafting rate of the dextran sulfate to the amino groups on the surface of the first grafting carrier. The reaction is carried out in an acidic buffer solution to ensure that the grafted dextran sulfate has high reactivity, which is beneficial for improving the adsorption rate of dextran sulfate for substances such as blood lipids.

[0077] Based on the above embodiments, as an optional implementation, the concentration of oxidized dextran sulfate sodium salt in the second reaction solution is 5 mg / mL to 20 mg / mL, and the volume ratio of the first grafting carrier to the acidic buffer in the second reaction solution is 1:1. This is beneficial for further improving the grafting rate of dextran sulfate on the first grafting carrier.

[0078] Based on the above embodiments, as a preferred embodiment, the pH value of the acidic buffer is 2 to 5, which is beneficial to further enhance the reactivity of dextran sulfate. As an optional embodiment, the acidic buffer is at least one selected from sodium citrate buffer, acetate buffer, disodium hydrogen phosphate-sodium citrate buffer, and phosphate buffer.

[0079] Based on the above embodiments, as an optional implementation, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS), and the mass ratio of the oxidized sodium dextran sulfate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(1-4):(1-8). This ensures that the activator has good activation performance.

[0080] Based on the above embodiments, as an optional implementation, this embodiment further includes, after the amidation reaction is completed and before obtaining the adsorbent, washing the amidation reaction product with 95% alcohol, repeating this process 2 to 5 times, and then washing the amidation reaction product with purified water, repeating this process 3 to 6 times, to obtain the adsorbent. This effectively cleans impurities from the surface of the adsorbent, preventing them from affecting its adsorption performance.

[0081] In this embodiment, the grafting rate of dextran sulfate on the adsorbent is 100 mg / g to 200 mg / g, meaning that 100 mg to 200 mg of dextran sulfate is immobilized on every 1 g of adsorbent. In this embodiment, the grafting rate of dextran sulfate on the adsorbent can be determined by the toluidine blue colorimetric method. This involves staining the adsorbent with toluidine blue, detecting the ultraviolet absorbance at 625 nm, and then quantitatively calculating the grafting rate of dextran sulfate on the adsorbent.

[0082] Specifically, 0.01 g of toluidine blue was dissolved in 250 mL of physiological saline to obtain a 0.004% toluidine blue TB solution. A known amount of 2 mL of dextran sulfate solution was added to 3 mL of the above TB solution and mixed thoroughly. Then, 3 mL of n-hexane was added, and the mixture was vortexed to extract the complex into the organic phase. Uncomplexed TB remained in the aqueous phase, and the absorbance at 630 nm was measured to plot a standard curve. Subsequently, 1 g of adsorbent was soaked in 2 mL of physiological saline and 3 mL of TB solution for 30 minutes, and then extracted by vortexing with 3 mL of n-hexane. The absorbance of the aqueous phase at 630 nm was measured, and the surface fixation amount of 1 g of adsorbent on the dextran sulfate surface was calculated based on the standard curve.

[0083] The method for preparing the adsorbent for treating stroke provided in this embodiment involves introducing epoxy groups onto a support, then combining the epoxy groups with a long-chain linear aliphatic diamine via a nucleophilic ring-opening reaction, and finally linking the amino groups on the long-chain linear aliphatic diamine with dextran sulfate via an amidation reaction. This ensures a strong bond between the support, the long-chain linear aliphatic diamine, and the dextran sulfate, reducing the risk of detachment of the long-chain linear aliphatic diamine and the dextran sulfate, and thus ensuring the safety of the adsorbent. The preparation method of this embodiment involves sequentially grafting a long-chain linear aliphatic diamine and dextran sulfate onto the outer layer of a carrier. This achieves synergistic adsorption of lipoproteins, fibrinogen, and inflammatory factors through both electrostatic and hydrophobic interactions, effectively reducing the levels of these substances in the blood. This results in neuroprotection, reduced blood viscosity, and improved microcirculation. Furthermore, compared to grafting dextran sulfate first and then the long-chain linear aliphatic diamine, this embodiment grafts the long-chain linear aliphatic diamine first, allowing the long-chain linear aliphatic diamine to simultaneously serve as a ligand and spacer arm, simplifying the reaction steps and reducing the reaction difficulty. In addition, the preparation method provided in this embodiment has relatively fewer reaction steps, a simpler preparation process, milder reaction conditions, higher safety during the reaction process, and lower production costs, making it suitable for industrial production.

[0084] A third aspect of this application provides a perfusion device comprising the adsorbent for treating stroke as described in the first aspect, or the adsorbent for treating stroke prepared by the preparation method described in the second aspect, wherein the adsorbent can be filled in the perfusion device, and the perfusion device is used for adsorbing FIB, blood lipids and inflammatory factors during extracorporeal blood circulation.

[0085] The perfusion device provided in this embodiment includes the aforementioned adsorbent for treating stroke. It can simultaneously adsorb FIB, blood lipids, and inflammatory factors from the patient's blood circulation, reducing blood lipids, fibrinogen, and inflammatory factors in stroke patients. This provides neuroprotection, reduces blood viscosity, and promotes microcirculation, which is beneficial for the treatment and rehabilitation of patients with acute ischemic stroke. Furthermore, the adsorbent in this perfusion device has virtually no impact on beneficial plasma components, thus not affecting the treatment effect or patient safety. The perfusion device of this embodiment provides stroke patients with a novel treatment option that is safer, more efficient, and has good universality than drug intervention.

[0086] To provide a more detailed description of the present invention, specific embodiments will be used to further illustrate the invention. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods; unless otherwise specified, the materials and reagents used in the embodiments of the present invention are commercially available.

[0087] Example 1

[0088] This embodiment provides an adsorbent for treating stroke, which is prepared by the following method:

[0089] (1) Take 40g of cellulose microspheres and put them into a 1L beaker. Wash the cellulose microspheres with 10 times the volume of purified water. Repeat the washing 5 times and drain the washing solution to obtain the washed cellulose microspheres. Add sodium hydroxide solution to the beaker and stir slowly at room temperature for 60min to allow the cellulose microspheres to swell fully. Then place the beaker in a 50℃ water bath and slowly add epichlorohydrin dropwise with a dropper while stirring continuously for 3h to carry out the epoxidation reaction of the cellulose microspheres and obtain the epoxidized cellulose microspheres. The ratio of cellulose microspheres, sodium hydroxide solution and epichlorohydrin is 1g:10ml:3ml and the concentration of sodium hydroxide solution is 4mol / L.

[0090] (2) Add the epoxidized cellulose microspheres to a three-necked flask, and add purified water and 1,16-hexadecanediamine to the three-necked flask in sequence. The ratio of epoxidized cellulose microspheres, 1,16-hexadecanediamine and purified water is 10:3:7. The amination reaction is carried out at 50°C with stirring at 120 rpm for 1.5 h. 1,16-hexadecanediamine is grafted onto the epoxidized cellulose microspheres. After the amination reaction is completed, the reaction solution is drained, and purified water is added to the three-necked flask to 2 / 3 of the volume of the three-necked flask. The amination carrier is repeatedly washed until the pH value of the washing solution is ≤8. The carrier grafted with 1,16-hexadecanediamine is obtained, which is the first grafted carrier.

[0091] (3) Add 5g of sodium dextran sulfate and 500ml of purified water to a three-necked flask, cool to 0-4℃ in an ice-water bath, and add TEMPO and NaBr sequentially under these conditions. Stir to dissolve, adjust the pH of the system to 10.5-11.0 using 0.5mol / L NaOH, slowly add NaClO solution, and simultaneously add 0.5mol / L NaOH solution dropwise using an automatic titrator to maintain the pH of the reaction solution at 10.5. After reacting for 2 hours, add 5mL of anhydrous ethanol and stir for 15min to terminate the reaction. Adjust the pH of the reaction solution to neutral (approximately 7.0) using 0.5mol / L HCl. Transfer the reaction solution to a dialysis bag and dialyze with 0.1mol / L NaCl solution for 24 hours (changing the solution 3 times), then dialyze with deionized water for 48 hours (changing the solution 5 times). Transfer the solution in the dialysis bag to a round-bottom flask and concentrate it to 100mL under reduced pressure at below 40℃ using a rotary evaporator to obtain oxidized sodium dextran sulfate.

[0092] (4) The 1,16-hexadecanediamine-grafted carrier and sodium citrate buffer (pH=4.7) were added to a three-necked flask. The volume ratio of the 1,16-hexadecanediamine-grafted carrier to sodium citrate buffer was 1:1. Oxidized sodium dextran sulfate was added to the three-necked flask at a concentration of 10 mg / mL. EDAC and NHS were added to the three-necked flask. The mass ratio of sodium dextran sulfate, EDAC and NHS was 1:1:1. The amidation reaction was carried out at 4°C with stirring at 120 rpm for 24 h. After the amidation reaction was completed, the amidation reaction product was washed with 95% alcohol at a volume of 5 times the volume of the amidation reaction product for 15 min. This was repeated twice. Then, the amidation reaction product was washed with purified water at a volume of 10 times the volume of the amidation reaction product. This was repeated 5 times to obtain the adsorbent.

[0093] The immobilization rate of 1,16-hexadecanediamine on the adsorbent was determined to be 17.5 mg / g by ninhydrin colorimetry, and the immobilization rate of dextran sulfate was determined to be 165 mg / g by toluidine blue colorimetry.

[0094] Example 2

[0095] This embodiment provides an adsorbent for treating stroke, which is prepared by the following method:

[0096] (1) Take 40g of cellulose microspheres and put them into a 1L beaker. Wash the cellulose microspheres with 10 times the volume of purified water. Repeat the washing 5 times and drain the washing solution to obtain the washed cellulose microspheres. Add sodium hydroxide solution to the beaker and stir slowly at room temperature for 60min to allow the cellulose microspheres to swell fully. Then place the beaker in a 50℃ water bath and slowly add epichlorohydrin dropwise with a dropper while stirring continuously for 3h to carry out the epoxidation reaction of the cellulose microspheres and obtain the epoxidized cellulose microspheres. The ratio of cellulose microspheres, sodium hydroxide solution and epichlorohydrin is 1g:10ml:3ml and the concentration of sodium hydroxide solution is 4mol / L.

[0097] (2) Add the epoxidized cellulose microspheres to a three-necked flask, and add purified water and 1,16-hexadecanediamine to the three-necked flask in sequence. The ratio of epoxidized cellulose microspheres, 1,16-hexadecanediamine and purified water is 10:3:7. The amination reaction is carried out at 50°C with stirring at 120 rpm for 1.5 h. 1,16-hexadecanediamine is grafted onto the epoxidized cellulose microspheres. After the amination reaction is completed, the reaction solution is drained, and purified water is added to the three-necked flask to 2 / 3 of the volume of the three-necked flask. The amination carrier is repeatedly washed until the pH value of the washing solution is ≤8. The carrier grafted with 1,16-hexadecanediamine is obtained, which is the first grafted carrier.

[0098] (3) Add 5g of sodium dextran sulfate and 500ml of purified water to a three-necked flask, cool to 0-4℃ in an ice-water bath, and add TEMPO and NaBr in sequence under these conditions. Stir to dissolve, adjust the pH of the system to 10.5-11.0 with 0.5mol / L NaOH, slowly add NaClO solution, and simultaneously add 0.5mol / L NaOH solution dropwise using an automatic titrator to maintain the pH of the reaction solution at 10.5. After reacting for 2 hours, add 5mL of anhydrous ethanol and stir for 15 minutes to terminate the reaction. Adjust the pH of the reaction solution to neutral (about 7.0) with 0.5mol / L HCl. Put the reaction solution into a dialysis bag and dialyze with 0.1mol / L NaCl solution for 24 hours (changing the solution 3 times), and then dialyze with deionized water for 48 hours (changing the solution 5 times). The solution in the dialysis bag was transferred to a round-bottom flask and concentrated to 100 mL under reduced pressure at below 40 °C using a rotary evaporator to obtain oxidized sodium dextran sulfate.

[0099] (4) The 1,16-hexadecanediamine-grafted carrier and sodium citrate buffer (pH=4.7) were added to a three-necked flask. The volume ratio of the 1,16-hexadecanediamine-grafted carrier to sodium citrate buffer was 1:1. Oxidized sodium dextran sulfate was added to the three-necked flask at a concentration of 10 mg / mL. EDAC and NHS were added to the three-necked flask. The mass ratio of sodium dextran sulfate, EDAC and NHS was 1:1:1. The amidation reaction was carried out at 4°C with stirring at 120 rpm for 24 h. After the amidation reaction was completed, the amidation reaction product was washed with 95% alcohol at a volume of 5 times the volume of the amidation reaction product for 15 min. This was repeated twice. Then, the amidation reaction product was washed with purified water at a volume of 10 times the volume of the amidation reaction product. This was repeated 5 times to obtain the adsorbent.

[0100] The immobilization rate of 1,16-hexadecanediamine on the adsorbent was determined to be 18.6 mg / g by ninhydrin colorimetry, and the immobilization rate of dextran sulfate was determined to be 176 mg / g by toluidine blue colorimetry.

[0101] Example 3

[0102] This embodiment provides an adsorbent for treating stroke, which is prepared by the following method:

[0103] (1) Take 40g of cellulose microspheres and put them into a 1L beaker. Wash the cellulose microspheres with 10 times the volume of purified water. Repeat the washing 5 times and drain the washing solution to obtain the washed cellulose microspheres. Add sodium hydroxide solution to the beaker and stir slowly at room temperature for 60min to allow the cellulose microspheres to swell fully. Then place the beaker in a 50℃ water bath and slowly add epichlorohydrin dropwise with a dropper while stirring continuously for 3h to carry out the epoxidation reaction of the cellulose microspheres and obtain the epoxidized cellulose microspheres. The ratio of cellulose microspheres, sodium hydroxide solution and epichlorohydrin is 1g:10ml:3ml and the concentration of sodium hydroxide solution is 4mol / L.

[0104] (2) Add the epoxidized cellulose microspheres to a three-necked flask, and add purified water and 1,16-hexadecanediamine to the three-necked flask in sequence. The ratio of epoxidized cellulose microspheres, 1,16-hexadecanediamine and purified water is 10:3:7. The amination reaction is carried out at 50°C with stirring at 120 rpm for 1.5 h. 1,16-hexadecanediamine is grafted onto the epoxidized cellulose microspheres. After the amination reaction is completed, the reaction solution is drained, and purified water is added to the three-necked flask to 2 / 3 of the volume of the three-necked flask. The amination carrier is repeatedly washed until the pH value of the washing solution is ≤8. The carrier grafted with 1,16-hexadecanediamine is obtained, which is the first grafted carrier.

[0105] (3) Add 5g of sodium dextran sulfate and 500ml of purified water to a three-necked flask, cool to 0-4℃ in an ice-water bath, and add TEMPO and NaBr in sequence under these conditions. Stir to dissolve, adjust the pH of the system to 10.5-11.0 with 0.5mol / L NaOH, slowly add NaClO solution, and simultaneously add 0.5mol / L NaOH solution dropwise using an automatic titrator to maintain the pH of the reaction solution at 10.5. After reacting for 2 hours, add 5mL of anhydrous ethanol and stir for 15 minutes to terminate the reaction. Adjust the pH of the reaction solution to neutral (about 7.0) with 0.5mol / L HCl. Transfer the reaction solution to a dialysis bag and dialyze with 0.1mol / L NaCl solution for 24 hours (changing the solution 3 times), and then dialyze with deionized water for 48 hours (changing the solution 5 times). The solution in the dialysis bag was transferred to a round-bottom flask and concentrated to 100 mL under reduced pressure at below 40 °C using a rotary evaporator to obtain oxidized sodium dextran sulfate.

[0106] (4) The 1,16-hexadecanediamine-grafted carrier and sodium citrate buffer (pH=4.7) were added to a three-necked flask. The volume ratio of the 1,16-hexadecanediamine-grafted carrier to sodium citrate buffer was 1:1. Oxidized sodium dextran sulfate was added to the three-necked flask at a concentration of 10 mg / mL. EDAC and NHS were added to the three-necked flask. The mass ratio of sodium dextran sulfate, EDAC and NHS was 1:1:1. The amidation reaction was carried out at 4°C with stirring at 120 rpm for 24 h. After the amidation reaction was completed, the amidation reaction product was washed with 95% alcohol at a volume of 5 times the volume of the amidation reaction product for 15 min. This was repeated twice. Then, the amidation reaction product was washed with purified water at a volume of 10 times the volume of the amidation reaction product. This was repeated 5 times to obtain the adsorbent.

[0107] The immobilization rate of 1,16-hexadecanediamine on the adsorbent was determined to be 18.3 mg / g by ninhydrin colorimetry, and the immobilization rate of dextran sulfate was determined to be 188 mg / g by toluidine blue colorimetry.

[0108] Example 4

[0109] This embodiment provides an adsorbent for treating stroke, which is prepared by the following method:

[0110] (1) Take 40g of cellulose microspheres and put them into a 1L beaker. Wash the cellulose microspheres with 10 times the volume of purified water. Repeat the washing 5 times and drain the washing solution to obtain the washed cellulose microspheres. Add sodium hydroxide solution to the beaker and stir slowly at room temperature for 60min to allow the cellulose microspheres to swell fully. Then place the beaker in a 50℃ water bath and slowly add epichlorohydrin dropwise with a dropper while stirring continuously for 3h to carry out the epoxidation reaction of the cellulose microspheres and obtain the epoxidized cellulose microspheres. The ratio of cellulose microspheres, sodium hydroxide solution and epichlorohydrin is 1g:10ml:3ml and the concentration of sodium hydroxide solution is 4mol / L.

[0111] (2) Add the epoxidized cellulose microspheres to a three-necked flask, and add purified water and 1,16-hexadecanediamine to the three-necked flask in sequence. The ratio of epoxidized cellulose microspheres, 1,16-hexadecanediamine and purified water is 10:3:7. The amination reaction is carried out at 50°C with stirring at 120 rpm for 1.5 h. 1,16-hexadecanediamine is grafted onto the epoxidized cellulose microspheres. After the amination reaction is completed, the reaction solution is drained, and purified water is added to the three-necked flask to 2 / 3 of the volume of the three-necked flask. The amination carrier is repeatedly washed until the pH value of the washing solution is ≤8. The carrier grafted with 1,16-hexadecanediamine is obtained, which is the first grafted carrier.

[0112] (3) Add 5g of sodium dextran sulfate and 500ml of purified water to a three-necked flask, cool to 0-4℃ in an ice-water bath, and add TEMPO and NaBr in sequence under these conditions. Stir to dissolve, adjust the pH of the system to 10.5-11.0 with 0.5mol / L NaOH, slowly add NaClO solution, and simultaneously add 0.5mol / L NaOH solution dropwise using an automatic titrator to maintain the pH of the reaction solution at 10.5. After reacting for 2 hours, add 5mL of anhydrous ethanol and stir for 15 minutes to terminate the reaction. Adjust the pH of the reaction solution to neutral (about 7.0) with 0.5mol / L HCl. Transfer the reaction solution to a dialysis bag and dialyze with 0.1mol / L NaCl solution for 24 hours (changing the solution 3 times), then dialyze with deionized water for 48 hours (changing the solution 5 times). The solution in the dialysis bag was transferred to a round-bottom flask and concentrated to 100 mL under reduced pressure at below 40 °C using a rotary evaporator to obtain oxidized sodium dextran sulfate.

[0113] (4) The 1,16-hexadecanediamine-grafted carrier and sodium citrate buffer (pH=4.7) were added to a three-necked flask. The volume ratio of the 1,16-hexadecanediamine-grafted carrier to sodium citrate buffer was 1:1. Oxidized sodium dextran sulfate was added to the three-necked flask at a concentration of 10 mg / mL. EDAC and NHS were added to the three-necked flask. The mass ratio of sodium dextran sulfate, EDAC and NHS was 1:1:1. The amidation reaction was carried out at 4°C with stirring at 120 rpm for 24 h. After the amidation reaction was completed, the amidation reaction product was washed with 95% alcohol at a volume of 5 times the volume of the amidation reaction product for 15 min. This was repeated twice. Then, the amidation reaction product was washed with purified water at a volume of 10 times the volume of the amidation reaction product. This was repeated 5 times to obtain the adsorbent.

[0114] The immobilization rate of 1,16-hexadecanediamine on the adsorbent was determined to be 17.1 mg / g by ninhydrin colorimetry, and the immobilization rate of dextran sulfate was determined to be 169 mg / g by toluidine blue colorimetry.

[0115] Experimental Example 1

[0116] Take 1 mL of the adsorbent prepared in Examples 1 to 4 respectively, and add it to 10 mL of plasma from patients with hyperlipidemia. Adsorb the adsorbents at a constant temperature of 37°C in the dark for 2 hours. Determine the concentrations of blood lipids and fibrinogen in the supernatant, and calculate the adsorption rate of the adsorbent in each example. The formula for calculating the adsorption rate is: Adsorption rate = [(m1-m2) / m1] × 100%, where m1 is the concentration of blood lipids or fibrinogen in the plasma before adsorption, and m2 is the concentration of blood lipids or fibrinogen after adsorption by the adsorbent in each example. The concentrations of blood lipids and fibrinogen after adsorption by the adsorbent in each example are shown in Table 1, and the adsorption rates are shown in Table 2.

[0117] Table 1

[0118]

[0119] Table 2

[0120]

[0121] As shown in Table 2, the adsorbents in Examples 1 to 4 all achieved adsorption rates of over 50% for TC (total cholesterol), TG (triglycerides), and LDL-c (low-density lipoprotein cholesterol) in plasma, over 20% for fibrinogen, and less than 6% for HDL-c (high-density lipoprotein cholesterol). This indicates that the adsorbents prepared in Examples 1 to 4 have good adsorption capacity for blood lipids and fibrinogen in plasma, can effectively reduce the concentration of blood lipids and fibrinogen in plasma, and have little impact on the concentration of beneficial HDL-c.

[0122] Experimental Example 2

[0123] One mL of each of the adsorbents prepared in Examples 1 to 4 was added to plasma from healthy individuals for which IL-1β, IL-6, and TNF-α standards were added. The plasma was then shaken and incubated at a constant temperature of 37°C in the dark for 2 hours. The supernatant was collected to determine the concentration of each inflammatory factor, and the adsorption rate of the adsorbent in each example was calculated. The formula for calculating the adsorption rate is: Adsorption rate = [(m1-m2) / m1] × 100%, where m1 is the concentration of inflammatory factors in the serum before adsorption, and m2 is the concentration of inflammatory factors after adsorption with the adsorbent. The concentrations of inflammatory factors before and after adsorption by the adsorbent in each example and comparative example are shown in Table 3, and the adsorption rates are shown in Table 4.

[0124] Table 3

[0125]

[0126] Table 4

[0127]

[0128] As shown in Table 4, after the adsorbents in Examples 1 to 4 adsorbed onto the plasma, the levels of inflammatory factors IL-1β (interleukin-1β), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α) in the plasma decreased by 30% to 50%. This indicates that the adsorbents prepared in Examples 1 to 4 have a good adsorption capacity for inflammatory factors in plasma and can effectively reduce the concentration of inflammatory factors in plasma.

[0129] As can be seen from Experimental Examples 1 and 3, the adsorbent provided in this application, through the 1,16-hexadecanediamine and dextran sulfate linked to the outer layer of the carrier, can simultaneously adsorb lipoproteins, fibrinogen and inflammatory factors, effectively reducing the content of these substances in the blood, and can achieve the effects of neuroprotection, reducing blood viscosity and promoting microcirculation.

Claims

1. An adsorbent for treating stroke, characterized in that, It includes a carrier and a first functional layer and a second functional layer sequentially disposed on the surface of the carrier; The carrier has epoxy groups; The first functional layer is a grafted layer formed by reacting a long-chain linear aliphatic diamine with an epoxy group on the support. One amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer. The second functional layer is a graft layer formed by connecting dextran sulfate to the amino groups on the surface of the first functional layer.

2. The adsorbent for treating stroke according to claim 1, characterized in that, The first functional layer is a graft layer formed by covalently linking a long-chain linear aliphatic diamine with an epoxy group on the support via a nucleophilic ring-opening reaction; The second functional layer is a graft layer formed by covalently linking carboxyl-containing dextran sulfate with amino groups on the surface of the first functional layer through an amidation reaction.

3. The adsorbent for treating stroke according to claim 1, characterized in that, The carrier is cellulose microspheres, and epoxy groups are introduced onto the cellulose microspheres; the long-chain linear aliphatic diamine is 1,16-hexadecanediamine.

4. The adsorbent for treating stroke according to claim 1, characterized in that, The loading of long-chain linear aliphatic diamines on the adsorbent is 15 mg / g to 20 mg / g, and the loading of dextran sulfate is 100 mg / g to 200 mg / g.

5. A method for preparing an adsorbent for treating stroke, characterized in that, The preparation of the adsorbent according to any one of claims 1 to 4 comprises the following steps: A support is provided, and epoxy groups are introduced onto the support to obtain an epoxidized support; The epoxidized support is reacted with a long-chain linear aliphatic diamine to form a first functional layer on the surface of the support, thereby obtaining a first grafted support; wherein, one amino group of the long-chain linear aliphatic diamine is connected to the epoxy group, and the other amino group of the long-chain linear aliphatic diamine is located on the surface of the first functional layer. The adsorbent is prepared by attaching dextran sulfate to the first grafted support through the amine groups on the surface of the first functional layer, thereby forming a second functional layer on the support.

6. The method for preparing the adsorbent for treating stroke according to claim 5, characterized in that, The step of introducing epoxy groups onto the support to obtain an epoxidized support includes: The carrier is mixed with an alkaline solution to swell the carrier and obtain a mixture. The mixture was continuously stirred at 40°C to 60°C, and epichlorohydrin was added dropwise to the mixture. After reacting for 2 to 4 hours, the epoxidized support was obtained. The carrier is cellulose microspheres, and the ratio of the cellulose microspheres, alkaline solution and epichlorohydrin is 1g:(10-20)ml:(2-5)ml; the concentration of the alkaline solution is 2mol / L to 5mol / L.

7. The method for preparing the adsorbent for treating stroke according to claim 5, characterized in that, The step of reacting the epoxidized support with a long-chain linear aliphatic diamine to form a first functional layer on the surface of the support, thereby obtaining a first grafted support, includes: Purified water and the long-chain linear aliphatic diamine were added to the epoxidized carrier, and an amination reaction was carried out at 25°C to 50°C. A first functional layer was grafted onto the surface of the epoxidized carrier and amine groups were introduced to obtain the first grafted carrier. The volume ratio of the epoxidized carrier to the long-chain linear aliphatic diamine is 10:(2-5).

8. The method for preparing the adsorbent for treating stroke according to claim 5, characterized in that, The adsorbent is prepared by attaching sulfated dextran to the first grafted support via the amine groups on the surface of the first functional layer, thereby forming a second functional layer on the support. The adsorbent comprises: Carboxyl groups were introduced into sodium dextran sulfate to prepare oxidized sodium dextran sulfate; The first grafted support is subjected to an amidation reaction with oxidized sodium dextran sulfate, so that the dextran sulfate graft forms a second functional layer on the first grafted support, thereby obtaining the adsorbent. The step of introducing a carboxyl group into sodium dextran sulfate to obtain oxidized sodium dextran sulfate includes: Sodium dextran sulfate was dissolved in deionized water and cooled to 0 to 4°C. TEMPO and NaBr were added sequentially, and the mixture was stirred until dissolved to obtain a mixture. The pH of the mixture was adjusted to 10 to 11, and NaClO solution was added to the mixture to form a first reaction solution. At the same time, NaOH solution was added dropwise to the first reaction solution to maintain a constant pH value. When the amount of NaOH solution consumed reached a preset threshold and the pH value of the first reaction solution remained unchanged, the reaction ended, and oxidized sodium dextran sulfate was obtained.

9. The method for preparing the adsorbent for treating stroke according to claim 8, characterized in that, The adsorbent is prepared by subjecting the first grafted support to an amidation reaction with oxidized sodium dextran sulfate, thereby forming a second functional layer of dextran sulfate grafted onto the first grafted support. The first grafting carrier and the oxidized sodium dextran sulfate were added to an acidic buffer solution to form a second reaction solution. The second reaction solution was subjected to an amidation reaction under the action of an activator, so that the dextran sulfate was grafted onto the first grafting carrier. The amidation reaction is carried out at a temperature of 4°C to 37°C for a reaction time of 12 h to 24 h. The concentration of sodium dextran sulfate in the second reaction solution is 5 mg / mL to 20 mg / mL, and the volume ratio of the first grafted carrier to the acidic buffer solution in the second reaction solution is 1:

1.

10. An irrigation device, characterized in that, The device includes an adsorbent for treating stroke as described in any one of claims 1 to 4, or an adsorbent for treating stroke prepared by the preparation method described in any one of claims 5 to 9, wherein the perfusion device is used for the adsorption of fibrinogen, blood lipids and inflammatory factors during extracorporeal blood circulation.

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