Polyurethane solid microspheres with anticoagulant effect, preparation method and application thereof

Abstract

The invention discloses a polyurethane solid microsphere with an anticoagulant effect, a preparation method and application thereof, and belongs to the technical field of blood purification. The preparation method comprises the following steps: adding a polyurethane synthetic monomer and an initiator to a solvent to initiate a polymerization reaction; subsequently, adding glycerol and polyethersulfone to the reaction solution for micro-crosslinking and blending; cooling the solution after the reaction is completed, and then obtaining polyurethane anticoagulant microspheres in a coagulation bath through phase inversion; finally, removing impurities and deprotonating the microspheres; the invention also discloses an application of the microspheres obtained by the above method; the polyurethane microspheres of the invention can achieve anticoagulation by reducing the levels of blood calcium ions and coagulation factors, while not affecting the recovery of the coagulation ability in the patient's body.

Description

Technical Field

[0001] The present invention relates to the technical field of blood purification, and in particular to polyurethane solid microspheres with anticoagulant effect, and a preparation method and application thereof. Background Art

[0002] Blood purification achieves therapeutic purposes by removing a patient's blood from the body and passing it through a purification device to remove certain pathogenic substances and purify the blood. Currently, blood purification has become a primary treatment for end-stage renal disease, liver failure, and acute poisoning. However, the blood-contacting materials used in blood purification treatments are typically made of polymers, metals, ceramics, and other materials. These lack good blood compatibility, causing adverse foreign body reactions when in contact with blood, leading to functional failure. Thrombosis is the most common adverse reaction associated with blood-contact materials. When biomaterials are exposed to blood, a large number of blood proteins adsorb onto their surfaces, including proteins that trigger coagulation. Subsequently, blood cells such as platelets, endothelial cells, and white blood cells are activated to release coagulation factors, triggering plasma coagulation and forming a blood clot. Simultaneously, blood flow creates fluid friction on the implant surface, causing adsorbed fibrinogen to adhere to platelets in the blood, further activating platelets and ultimately forming a blood clot. When thrombi form on the surface of biomaterials, they can cause blockage of medical devices, forcing the suspension of purification treatment. When thrombi leave the surface of biomaterials and travel through the bloodstream, they may block small blood vessels downstream, leading to stroke or tissue death. Therefore, anticoagulation plays a fundamental role in the clinical blood purification process.

[0003] Currently, there are two main approaches to addressing coagulation during blood purification: 1. Anticoagulant therapy using anticoagulants such as heparin, argatroban, and citrate; 2. Modifying the surface of blood-contacting biomaterials to enhance their biocompatibility and prevent activation of coagulation. Various anticoagulants, such as unfractionated heparin, low-molecular-weight heparin, direct thrombin inhibitors (such as argatroban and hirudin), and heparinoids (such as fondaparinux and danapalene), are widely used for systemic anticoagulation and antithrombotic effects. However, the use of liquid anticoagulants in blood purification is a low-safety strategy, and the precise dosage of anticoagulants is difficult to determine due to interpatient variability. Overuse of anticoagulants can lead to hemorrhagic complications such as cerebral hemorrhage and severe gastrointestinal bleeding, while underuse can cause coagulation-related complications such as thrombosis in blood vessels, leading to circuit failure and premature termination of treatment. Surface modification of blood-contacting materials to prevent activation of coagulation is a viable approach. Currently, there are two main strategies for surface modification: one is to avoid activating the body's coagulation cascade by regulating or reducing protein adsorption (inert strategy); the other is to actively prevent coagulation by endothelialization, grafting coagulation pathway inhibitors or specific proteins or drug molecules (active strategy). However, neither strategy can completely replace the use of anticoagulants. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a method for preparing polyurethane solid microspheres with anticoagulant effect to solve the above problems.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for preparing polyurethane solid microspheres with anticoagulant effect, comprising the following steps:

[0006] (a) dissolving a polyurethane monomer, 4,4-diphenylmethane diisocyanate (MDI) and a monomer, 2,2-dihydroxymethylpropionic acid (DMPA), in a solvent and performing a polymerization reaction with triethylamine as a catalyst to obtain a polymer solution containing a polyurethane molecule represented by the following formula (1);

[0007]

[0008] Wherein, n=5~300;

[0009] (b) adding glycerol and polyethersulfone to the polymer solution obtained in step (a) to crosslink the linear polyurethane and simultaneously blending it with the polyethersulfone to obtain a mixed solution;

[0010] (c) after the mixed solution obtained in step (b) is cooled, electrostatic spinning is performed using an electrospinning machine to obtain microspheres;

[0011] (d) finally, washing and deprotonating the microspheres obtained in step (c) to obtain finished polyurethane solid microspheres.

[0012] At present, people often use anticoagulants such as hirudin (direct thrombin inhibitor), heparin (indirect thrombin inhibitor), citric acid (calcium chelator) for anticoagulant treatment, and groups such as carboxyl, sulfonic acid, and hydroxyl groups with anticoagulant effects have also been discovered. However, traditional anticoagulants are often accompanied by side effects such as acidosis and thrombocytopenia when used, and affect the recovery of the patient's coagulation ability. Although adding anticoagulant groups to blood-contact materials for modification can avoid side effects, it cannot completely replace anticoagulants. In order to solve the problem of anticoagulants affecting the recovery of the patient's coagulation ability, people are exploring new anticoagulant methods. For example, Zhao Changsheng's research group proposed the use of polymer microspheres combined with coagulation factors. These polymer microspheres show excellent stability and prevent blood coagulation in the extracorporeal circuit by binding to coagulation factors. Based on the above principles, the inventors of this application, through theoretical analysis and extensive experiments, synthesized a polyurethane containing a large number of carboxyl groups in its side chains using MDI and DMPA. They then used micro-crosslinking and polyethersulfone blending to improve the stability of the polyurethane in the blended microspheres. The numerous anticoagulant groups on the microspheres reduced the levels of calcium ions and endogenous coagulation factors in the blood. Furthermore, the linear polyurethane has a partial structural similarity to hirudin and can effectively bind to thrombin. By binding to multiple coagulation factors, the coagulation cascade is prevented from initiating in multiple ways, achieving the goal of replacing anticoagulant drugs for anticoagulant therapy. Furthermore, because the anticoagulant groups are fixed to the microspheres, they are prevented from entering the patient's body during treatment, preventing side effects and preventing the recovery of the patient's coagulation ability.

[0013] Compared with CN103055725A, this application has the following technical means: in addition to omitting the sulfonation step, due to the increased proportion of polyurethane, in order to stabilize the polyurethane in the microspheres, in addition to adding polyethersulfone, a glycerol cross-linking step is added in the middle, and the spinning is transformed into drop balls. Compared with membranes, the microspheres have an increased contact surface area and can better exert an anticoagulant effect. From the perspective of technical effects, this application not only prolongs the activated partial thrombin time (intrinsic coagulation pathway), but also prolongs the thrombin time (common coagulation pathway). It can inhibit both coagulation pathways and replace the coagulant.

[0014] As a preferred technical solution, in step (a), the molar ratio of monomer 4,4-diphenylmethane diisocyanate to monomer 2,2-dihydroxymethylpropionic acid is 1.0-1.2, and the total concentration of the above two monomers in the solvent is 10-20 wt.%. The amount of triethylamine added is 0.01-0.5 wt.% of the total amount of the above two monomers.

[0015] The amount of monomeric MDI should be slightly more than that of DMPA to form isocyanate-terminated linear polyurethanes to facilitate the subsequent micro-crosslinking process.

[0016] The molar ratio of monomers MDI and DMPA and the total concentration of monomers in DMAc are limited, which mainly enables the successful preparation of microspheres without gelation of the solution reaction process or the failure of electrospinning to produce microspheres.

[0017] The solvent in step (a) must meet the following requirements: 1. It must be a polar solvent capable of dissolving the polyurethane and the reactive monomers; 2. It must not contain active hydrogen and must be chemically stable, i.e., it must not react with -NCO groups. 3. The solvent's purification and anhydrous treatment should be as simple as possible, ensuring a long shelf life. N,N-dimethylacetamide (i.e., DMAc) or N,N-dimethylformamide (i.e., DMF) can be selected. As a preferred technical solution, DMAc, which has lower toxicity, is selected as the solvent.

[0018] As a preferred technical solution, the reaction in step (a) is carried out in an inert gas atmosphere, such as nitrogen or argon atmosphere.

[0019] As a preferred technical solution, in step (b), the amount of glycerol added is 0.2-2% of the total amount of MDI and DMPA added in step (a), and the concentration of the added polyethersulfone in the polymer solution is 5-10 wt.%. As a preferred technical solution, in step (c), during the ball dropping process, the solution is placed in a 10 mL syringe, equipped with a stainless steel needle, and connected to a high-voltage generator. A DC high-voltage generator is used to provide a voltage between the nozzle and the grounded collector. A coagulation bath is used as a ground collector, and the solution is dropped into the syringe to obtain polyurethane solid anticoagulant microspheres.

[0020] The size of the microspheres dropped from the electrospinning machine is preferably 500 to 800 μm.

[0021] As a further preferred technical solution, the coagulation bath is a 15-30 wt.% ethanol aqueous solution or a 0.5-2.0 wt.% sodium lauryl sulfate aqueous solution.

[0022] As a preferred technical solution, in step (d), the deprotonation process is as follows: soak in a pH 9 NaOH solution for 24-72 hours, then replace with a 0.01M PBS solution every 6-12 hours until the pH of the solution no longer changes. Anticoagulant microspheres contain polyurethane, and the polyurethane functional groups will break under strong alkaline conditions. Therefore, strong bases cannot be used in the deprotonation process. After deprotonation, the microspheres must be stored in neutral saline. The saline solution should be changed every 3 days to prevent bacterial growth on the microsphere surface.

[0023] A second object of the present invention is to provide polyurethane solid microspheres with anticoagulant effect obtained by the above-mentioned preparation method.

[0024] The third object of the present invention is to provide the use of the polyurethane solid microspheres with anticoagulant effect obtained by the above preparation method in blood purification.

[0025] A fourth object of the present invention is to provide a medical device comprising the polyurethane solid microspheres prepared by the above-mentioned preparation method.

[0026] The medical device is preferably a hemoperfusion device.

[0027] Compared with the prior art, the advantages of the present invention are: (1) the anticoagulant microspheres of the present invention can reduce the levels of various coagulation factors in the blood, especially the level of thrombin in the blood, and have excellent anticoagulant effect; (2) the anticoagulant components of the anticoagulant microspheres of the present invention do not enter the patient's blood and will not affect the recovery of the patient's coagulation ability; (3) the anticoagulant polymer of the present invention is synthesized by a one-pot method, which has a simple synthesis method, and the microsphere preparation process is simple and fast, takes a short time, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 and Figure 2 Surface SEM image of polyurethane solid microspheres prepared in Example 1;

[0029] Figure 3 The molecular weight of the polyurethane of Example 1 was measured by GPC;

[0030] Figure 4 Comparison of the levels of coagulation factors VIII, IX, XI, and XII in plasma before and after incubation with pure polyethersulfone spheres and the polyurethane solid microspheres prepared in Example 4;

[0031] Figure 5 This is a photograph of the state of normal whole blood after passing through the polyurethane solid microspheres prepared in Example 5. DETAILED DESCRIPTION

[0032] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0033] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0034] Example 1

[0035] A polyurethane solid microsphere with anticoagulant effect, the preparation method of which comprises the following steps:

[0036] (a) After purging the reaction vessel with argon for 30 minutes, MDI and DMPA were dissolved in DMAc at a molar ratio of 1.06, with a total concentration of 15 wt.% of the two monomers in DMAc. Triethylamine (0.1% by weight of the total weight of the two monomers) was then added, and the solution was stirred continuously until all the monomers were completely dissolved. Polymerization was then carried out in a closed apparatus containing argon for 3 hours at a reaction temperature of 60° C. to obtain a polymer solution containing polyurethane molecules.

[0037] The molecular weight of the obtained polyurethane was measured by GPC. Figure 3 shown.

[0038] It should be noted that due to its very broad molecular weight distribution, it is impossible to obtain a specific degree of polymerization. The 5-300 of the present invention is a range that includes more than 95% of polyurethane after excluding molecules with too low a molecular weight. Those skilled in the art are aware that polyurethane synthesis is a polymerization reaction. During the reaction process, due to differences in the concentration of reactants around different reacting molecules, the length of the polyurethane chain varies. The peak positions of the various embodiments of the present application may vary, but the range of more than 95% is within 5-300. Since the n value is within this range, there is no significant effect on ball formation.

[0039] (b) adding glycerol and polyethersulfone, wherein the amount of glycerol is 1% of the total amount of MDI and DMPA added in step (a), and the concentration of polyethersulfone in the polymer solution is 5 wt.%; after stirring until completely dissolved, the temperature is adjusted to 55° C. for micro-crosslinking and blending for 1 hour, and if climbing occurs during this period, additional DMAc is added until the climbing phenomenon disappears;

[0040] (c) After the reaction is complete, the polymer solution is cooled and then added to a 10 mL syringe equipped with a 24-gauge needle (inner diameter approximately 0.3 mm). The solution is then dropped into a 25 wt.% ethanol aqueous solution using an electrospinning machine to produce microspheres.

[0041] (d) The obtained microspheres were then washed multiple times with deionized water to remove impurities; the microspheres were then immersed in a NaOH solution (pH = 9) for 24 hours; and finally, the microspheres were removed and replaced with a 0.01 M PBS solution every 8 hours for 3 days to obtain finished polyurethane solid microspheres.

[0042] The surface SEM of the obtained microspheres is shown in Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 It can be seen from the figure that the particle size of the microspheres is between 500 and 600 μm, and there are a large number of pores on the surface.

[0043] Example 2

[0044] A polyurethane solid microsphere with anticoagulant effect, the preparation method of which comprises the following steps:

[0045] (a) After purging the reaction vessel with argon for 30 minutes, MDI and DMPA were dissolved in DMAc at a molar ratio of 1.1, resulting in a total concentration of 10 wt.%. Triethylamine (0.1% by weight of the total weight of the two monomers) was then added, and the solution was stirred continuously until all the monomers were dissolved. Polymerization was then carried out in a closed apparatus containing argon for 3 hours at 60°C to obtain a polymer solution containing polyurethane molecules.

[0046] (b) glycerol and polyethersulfone were then added, wherein the amount of glycerol was 0.8% of the total amount of MDI and DMPA added in (a), and the concentration of polyethersulfone in the polymer solution was 7 wt.%; after stirring until completely dissolved, the temperature was adjusted to 50° C. for micro-crosslinking and blending for 2 h, and if climbing occurred during this period, DMAc was added until the climbing phenomenon disappeared;

[0047] (c) After the reaction is complete, the polymer solution is cooled and then added to a 10 mL syringe equipped with a 24-gauge needle. The solution is then dropped into a 25 wt.% ethanol aqueous solution using an electrospinning machine to obtain microspheres.

[0048] (d) The obtained microspheres were then washed multiple times with deionized water to remove impurities. The microspheres were then immersed in a NaOH solution (pH = 9) for 24 h. Finally, the microspheres were removed and replaced with a 0.01 M PBS solution every 12 h. After 5 days of replacement, the finished polyurethane solid microspheres were obtained.

[0049] Example 3

[0050] A polyurethane solid microsphere with anticoagulant effect, the preparation method of which comprises the following steps:

[0051] (a) After purging the reaction vessel with argon for 30 minutes, MDI and DMPA were dissolved in DMAc at a molar ratio of 1.07 and a total concentration of 10 wt.% of the two monomers in DMAc. Triethylamine (0.05% by weight of the total weight of the two monomers) was then added, and the solution was stirred continuously until all the monomers were completely dissolved. Polymerization was then carried out in a closed apparatus containing argon for 2.5 hours at a reaction temperature of 70° C. to obtain a polymer solution containing polyurethane molecules.

[0052] (b) glycerol and polyethersulfone were then added, wherein the amount of glycerol was 0.5% of the total amount of MDI and DMPA added in (a), and the concentration of polyethersulfone in the polymer solution was 8 wt.%; after stirring until completely dissolved, the temperature was adjusted to 55° C. for micro-crosslinking and blending for 2 h, and if climbing occurred during this period, DMAc was added until the climbing phenomenon disappeared;

[0053] (c) After the reaction is complete, the polymer solution is cooled and then added to a 10 mL syringe equipped with a 24-gauge needle. The solution is then dropped into a coagulation bath of a 0.5 wt.% aqueous sodium lauryl sulfate solution using an electrospinning machine to produce microspheres.

[0054] (d) The obtained microspheres were then washed multiple times with deionized water to remove impurities; the microspheres were then immersed in a NaOH solution (pH = 9) for 24 hours. Finally, the microspheres were removed and replaced with a 0.01 M PBS solution every 8 hours. After 4 days of replacement, the finished polyurethane solid microspheres were obtained.

[0055] Example 4

[0056] This embodiment is a preferred embodiment of the present invention.

[0057] A polyurethane solid microsphere with anticoagulant effect, the preparation method of which comprises the following steps:

[0058] (a) After purging the reaction vessel with argon for 30 minutes, MDI and DMPA were dissolved in DMAc at a molar ratio of 1.06, resulting in a total concentration of 10 wt.% of the two monomers in DMAc. Triethylamine (0.2% by weight of the total monomer weight) was then added, and the solution was stirred continuously until all the monomers were completely dissolved. Polymerization was then carried out in a closed apparatus containing argon for 3 hours at a reaction temperature of 65°C to obtain a polymer solution containing polyurethane molecules.

[0059] (b) glycerol and polyethersulfone were then added, wherein the amount of glycerol was 0.8% of the total amount of MDI and DMPA added in (a), and the concentration of polyethersulfone in the polymer solution was 5 wt.%; after stirring until completely dissolved, the temperature was adjusted to 55° C. for micro-crosslinking and blending for 2 h, and if climbing occurred during this period, DMAc was added until the climbing phenomenon disappeared;

[0060] (c) After the reaction is complete, the polymer solution is cooled and then added to a 10 mL syringe equipped with a 24-gauge needle. The solution is then dropped into a coagulation bath of 25 wt.% ethanol in water to produce microspheres using an electrospinning machine.

[0061] (d) The obtained microspheres were then washed multiple times with deionized water to remove impurities. The microspheres were then immersed in a NaOH solution (pH = 9) for three days. Finally, the microspheres were removed and replaced with a 0.01 M PBS solution every 12 hours for four days to obtain finished polyurethane solid microspheres.

[0062] Example 5

[0063] A polyurethane solid microsphere with anticoagulant effect, the preparation method of which comprises the following steps:

[0064] (a) After purging the reaction vessel with argon for 30 minutes, MDI and DMPA were dissolved in DMAc at a molar ratio of 1.2, resulting in a total monomer concentration of 20 wt.%. Triethylamine (0.05% by weight of the total monomer weight) was then added, and the solution was stirred continuously until all the monomers were completely dissolved. Polymerization was then carried out in a closed apparatus containing argon for 2.5 hours at a reaction temperature of 70°C to obtain a polymer solution containing polyurethane molecules.

[0065] (b) glycerol and polyethersulfone were then added, wherein the amount of glycerol was 2% of the total amount of MDI and DMPA added in (a), and the concentration of polyethersulfone in the polymer solution was 6 wt.%; after stirring until completely dissolved, the temperature was adjusted to 50° C. for micro-crosslinking and blending for 2 h, and if climbing occurred during this period, DMAc was added until the climbing phenomenon disappeared;

[0066] (c) After the reaction is complete, the polymer solution is cooled and then added to a 10 mL syringe equipped with a 24-gauge needle. The solution is then dropped into a coagulation bath of 15 wt.% ethanol in water to produce microspheres using an electrospinning machine.

[0067] (d) The obtained microspheres were then washed multiple times with deionized water to remove impurities; the microspheres were then immersed in a NaOH solution (pH = 9) for 24 hours. Finally, the microspheres were removed and replaced with a 0.01 M PBS solution every 8 hours. After 4 days of replacement, the finished polyurethane solid anticoagulant microspheres were obtained.

[0068] Example 6

[0069] A polyurethane solid microsphere with anticoagulant effect, the preparation method of which comprises the following steps:

[0070] (a) After purging the reaction vessel with argon for 30 minutes, MDI and DMPA monomers were dissolved in anhydrous grade DMAc at a ratio of 1.1, for a total monomer concentration of 14 wt.%. Triethylamine (0.1% by weight of the total monomer weight) was then added, and the solution was stirred continuously until all monomers were dissolved. Polymerization was carried out in a closed apparatus containing argon for 2 hours at 70°C to obtain a polymer solution containing polyurethane molecules.

[0071] (b) glycerol and polyethersulfone were then added, wherein the amount of glycerol was 0.8% of the total amount of MDI and DMPA added in (a), and the concentration of polyethersulfone in the polymer solution was 5 wt.%; after stirring until completely dissolved, the temperature was adjusted to 50° C. for micro-crosslinking and blending for 2 h, and if climbing occurred during this period, anhydrous DMAc was added until the climbing phenomenon disappeared;

[0072] (c) After the reaction is complete, the polymer solution is cooled and then added to a 10 mL syringe equipped with a 24-gauge needle. The solution is then dropped into a coagulation bath of a 0.5 wt.% aqueous sodium lauryl sulfate solution using an electrospinning machine to produce microspheres.

[0073] (d) The obtained microspheres were then washed multiple times with deionized water to remove impurities. The microspheres were then immersed in a NaOH solution (pH = 9) for three days. Finally, the microspheres were removed and replaced with a 0.01 M PBS solution every 12 hours for five days to obtain finished polyurethane solid microspheres.

[0074] Performance Test Example 1: Determination of Coagulation Time

[0075] To evaluate the anticoagulant activity of the microspheres, activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured using an automated coagulation analyzer. Fresh blood was collected from healthy volunteers using vacuum tubes containing sodium citrate as an anticoagulant. After collection, platelet-poor plasma (PPP) was obtained by centrifugation for clotting time determination.

[0076] In addition, polyethersulfone was dissolved in dimethylacetamide to obtain a 16 wt.% polyethersulfone solution, and then pure polyethersulfone microspheres were obtained as Comparative Example 1 by the same steps (c) and (d) as in Example 1;

[0077] The linear polyurethane obtained in step (a) of Example 1 was not subjected to step (b), but directly subjected to steps (c) and (d) to obtain pure linear polyurethane balls, as Comparative Example 2;

[0078] No microspheres were added as blank control;

[0079] The polyurethane solid microspheres prepared in Examples 1 to 6 and Comparative Examples 1 to 2 were soaked in PBS overnight, and then the PBS was removed. Fresh PPP (concentration: 10 mg microspheres / 100 μL PPP) was introduced, and then incubated at 37°C for 30 min. After the incubation was completed, the microspheres were removed, and the APTT, PT, and TT of PPP before and after incubation were measured using an automatic coagulation analyzer. The results are shown in Table 1.

[0080] Table 1. Coagulation time of microspheres in various examples and comparative examples

[0081]

[0082]

[0083] Note: The unit of the above adsorption amount is "s".

[0084] As shown in Table 1, pure linear polyurethane microspheres significantly prolonged the APTT, PT, and TT. The significant prolongation of PT, in particular, suggests the presence of anticoagulant substances in the plasma, likely due to partial dissolution of the linear polyurethane in the plasma. After repeated incubations of the same batch of microspheres with plasma, the pure linear polyurethane microspheres gradually softened and eventually dissolved in the plasma, while the morphology of the pure polyethersulfone microspheres and the anticoagulant microspheres remained unchanged, indicating that the glycerol crosslinking and polyethersulfone blend effectively immobilized the polyurethane.

[0085] Compared to the blank control and plasma incubated with pure polyethersulfone microspheres, plasma incubated with the anticoagulant microspheres of Examples 1 to 6 showed significant prolongation of both APTT and TT, while PT remained unchanged. The longest APTT prolongation was 54.4 seconds, and the longest TT prolongation was 40.1 seconds. This demonstrates that the polyurethane solid anticoagulant microspheres have a strong inhibitory effect on both intrinsic and common coagulation pathways, demonstrating their excellent anticoagulant capacity.

[0086] Performance Test Example 2: Plasma levels of coagulation factors VIII, IX, XI, and XII

[0087] In order to detect the activity of coagulation factors VIII, IX, XI and XII in plasma after incubation with microspheres, standard plasma and corresponding coagulation factor-deficient plasma (coagulation factor VIII-deficient plasma, coagulation factor IX-deficient plasma, coagulation factor XI-deficient plasma, coagulation factor XII-deficient plasma) were used, and the APTT of each coagulation factor was measured using a fully automatic coagulation analyzer to obtain a calibration curve of each coagulation factor level. Pure polyethersulfone microspheres and the polyurethane anticoagulant microspheres prepared in Example 4 were soaked in PBS overnight, and then the PBS was removed. Fresh PPP (concentration 10 mg microspheres / 100 μL PPP) was introduced respectively, and then incubated at 37°C for 30 minutes. After that, the APTT of PPP before and after incubation was measured using a fully automatic coagulation analyzer. The activity of the coagulation factor to be tested was obtained according to the calibration curve. The results are as follows: Figure 4 shown.

[0088] Depend on Figure 4 It can be seen that the levels of endogenous coagulation factors VIII, IX, XI and XII in the plasma incubated with the anticoagulant microspheres of Example 4 were reduced by about 30-70%, indicating that the polyurethane solid anticoagulant microspheres of the present invention can achieve the purpose of anticoagulation by inhibiting multiple endogenous coagulation factors.

[0089] Performance Test Example 3: Changes in Thrombin Levels Before and After Incubation with Anticoagulant Microspheres

[0090] Three prepared polyurethane anticoagulant microspheres were added to the wells of the well plate, and the wells without microspheres served as blank controls. T-TE buffer (preparation method, see Table 2) and thrombin, a special solvent for the enzymatic reaction, were added to two wells, respectively, and the cells were incubated at 37°C for 15 minutes. Then, liquid was collected from each well, and the thrombin chromogenic substrate S-2238 was added. The kinetics of OD-405 nm were read on a microplate reader for 10 minutes. The thrombin level was determined by absorbance, with the blank control as 100%. The results are shown in Table 3.

[0091] Table 2. Preparation of T-TE buffer

[0092]

[0093] Note: At the end of the preparation, adjust the pH to 7.6 with concentrated hydrochloric acid.

[0094] Table 3. Thrombin levels of microspheres in various examples and blank controls

[0095]

[0096]

[0097] As shown in Table 3, compared with the blank control, the thrombin level in the solution incubated with anticoagulant microspheres was reduced by more than 70%, among which the lowest thrombin level dropped to only 13.6% of the original level, proving that the polyurethane solid anticoagulant microspheres have a strong inhibitory effect on thrombin, which is also one of the sources of their excellent anticoagulant ability.

[0098] Performance Test Example 4: Polyurethane Microsphere Whole Blood Anticoagulation Test

[0099] A 2 mL syringe was filled with 0.5 mL of the polyurethane anticoagulant microspheres prepared in Example 5. Whole blood without any anticoagulant was then collected and passed directly through the syringe. Digital photographs of normal whole blood and whole blood passing through the syringe containing anticoagulant microspheres were taken, and the whole blood clotting time was recorded.

[0100] Depend on Figure 5 As can be seen, normal whole blood clots within minutes. However, whole blood passing through a syringe filled with anticoagulant microspheres showed no clots and remained unclotted for over two hours. This demonstrates that polyurethane solid anticoagulant microspheres possess excellent anticoagulant properties, enabling adequate anticoagulation in the extracorporeal blood purification circuit without the need for any anticoagulant drugs.

[0101] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing polyurethane solid microspheres with anticoagulant effect, characterized in that: The steps include: (a) dissolving a polyurethane monomer, 4,4-diphenylmethane diisocyanate, and a monomer, 2,2-dihydroxymethylpropionic acid, in a solvent, and performing a polymerization reaction with triethylamine as a catalyst to obtain a polymer solution containing polyurethane molecules represented by the following formula (1); wherein n = 5 to 300; The molar ratio of the monomer 4,4-diphenylmethane diisocyanate to the monomer 2,2-dihydroxymethylpropionic acid is 1.0-1.2, the total concentration of the two monomers in the solvent is 10-20 wt.%, and the amount of triethylamine added is 0.01-0.5 wt.% of the total amount of the two monomers; The solvent is N, N-dimethylacetamide or N, N-dimethylformamide; (b) after the reaction in step (a) is completed, glycerol and polyethersulfone are added to the obtained polymer solution to crosslink the polyurethane therein, and the mixture is blended with the polyethersulfone to obtain a mixed solution, wherein the amount of glycerol added is 0.2 to 2% of the total amount of 4,4-diphenylmethane diisocyanate and 2,2-dimethylolpropionic acid added in step (a), and the concentration of the added polyethersulfone in the polymer solution is 5 to 10 wt.%; (c) after the mixed solution obtained in step (b) is cooled, electrostatic spinning is performed using an electrospinning machine to obtain microspheres; (d) finally, washing and deprotonating the microspheres obtained in step (c) to obtain finished polyurethane solid microspheres.

2. The method according to claim 1, characterized in that The reaction in step (a) is carried out in an inert gas atmosphere.

3. The method according to claim 1, characterized in that In step (c), during the ball dropping process, the solution is placed in a 10 mL syringe, equipped with a stainless steel needle, connected to a high voltage generator, a DC high voltage generator is used to provide voltage between the nozzle and the ground collector, and a coagulation bath is used as a ground collector, and the solution is dropped into it to obtain polyurethane solid anticoagulant microspheres.

4. The method according to claim 3, characterized in that The coagulation bath is a 15-30 wt. % ethanol aqueous solution or a 0.5-2.0 wt. % sodium lauryl sulfate aqueous solution.

5. The method according to claim 1, wherein In step (d), the deprotonation process is as follows: soaking in a NaOH solution with a pH of 9 for 24 to 72 hours, and then replacing it with a PBS solution every 6 to 12 hours until the pH of the solution no longer changes.

6. Polyurethane solid microspheres with anticoagulant effect obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the polyurethane solid microspheres with anticoagulant effect obtained by the preparation method according to any one of claims 1 to 5 in the preparation of blood purification materials.

8. A medical device, characterized in that The medical device comprises polyurethane solid microspheres prepared by the preparation method according to any one of claims 1 to 5.

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