Preparation method of anticoagulant macroporous adsorption resin based on phosphorylcholine in-situ grafting

Abstract

The invention discloses a preparation method of anticoagulant macroporous adsorption resin based on phosphorylcholine in-situ grafting. The preparation method comprises the following steps: 1) constructing a resin skeleton containing reactive hydroxyl; according to the preparation method, the macroporous adsorption resin is generated by introducing the multifunctional cross-linking agent and adopting the in-situ grafting technology, the synthesis method is simple, and the generated macroporous adsorption resin is stable in structure and excellent in anticoagulation performance.

Description

Technical Field

[0001] This invention relates to the field of adsorption materials technology, specifically to an anticoagulant macroporous adsorption resin based on in-situ grafting of phosphocholine. Background Technology

[0002] Progressive kidney disease can damage nephrons, causing them to gradually lose function. Once kidney function is insufficient to maintain the body's metabolic balance, kidney failure will occur, and in severe cases, it will progress to uremia, endangering life. The annual incidence of this end-stage renal disease is 50-150 per million people worldwide. According to surveys in some provinces and cities, the incidence of end-stage renal disease in my country is approximately 100 per million people.

[0003] To treat patients on the verge of death, as early as the mid-19th century, the idea of ​​using dialysis to remove diffusible substances from the blood of patients with kidney failure was proposed, thereby alleviating the possibility of poisoning symptoms. After nearly a century of effort, in 1943, the Dutchman Kolff first applied a regenerated cellulose dialyzer clinically, ushering in a new era of using artificial devices to replace kidney function. In essence, the basic technical concept of an artificial kidney is to draw the patient's blood out of the body and, through devices made using different technical principles (such as hemodialyzers and hemofilters), complete the transfer and removal of solutes and water from the blood, before returning the purified blood to the body to achieve the therapeutic goal; that is, through the biophysical mechanism of the artificial kidney, it completes the transfer and removal of metabolic waste, toxins, pathogenic factors, as well as water and electrolytes that need to be removed from the blood, thereby achieving internal environmental balance. In recent years, due to the interdisciplinary integration, basic research on artificial kidneys has made significant progress, promoting the development of new devices and the emergence of some new technologies.

[0004] Hemoperfusion (HP) adsorbs exogenous toxic substances, creatinine, microglobulins and other medium and large molecular harmful toxins in the patient's body through physical adsorption, chemical adsorption and bioaffinity adsorption. When used in combination with hemodialysis, hemoperfusion can remove small molecule metabolites such as creatinine and uric acid, as well as medium and large molecular metabolites such as β2-microglobulin.

[0005] The core of disposable hemoperfusion device technology lies in the use of adsorbent material filled in the canister. The adsorbent material should not only be safe and non-toxic to the human body, without causing allergic reactions or pyrogens, but also have stable chemical properties, a regular appearance without breakage or detachment of microparticles, and good blood compatibility.

[0006] However, the large contact area between the adsorbent and blood in the hemoperfusion device can easily cause coagulation; how to reduce the coagulation hazards that occur during use is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a macroporous adsorption resin that is simple to synthesize, structurally stable, and exhibits excellent anticoagulant properties; the specific technical solution is as follows: A method for preparing an anticoagulant macroporous adsorption resin based on in-situ grafting of phosphocholine includes the following steps: 1) Construct a resin skeleton containing reactive hydroxyl groups; 2) In-situ chemical grafting of phosphoric acid choline; 3) Purification.

[0008] Furthermore, step 1) includes the following steps: A. Oil phase preparation: Monomer mixture: 80-85 parts by weight of styrene, 15-20 parts by weight of divinylbenzene (80% purity), 5-8 parts by weight of 2,2-bis(chloromethyl)-1,3-propanediol; Pore-forming agent: 20-40 parts by weight of toluene and 10-20 parts by weight of n-heptane; Initiator: 0.8-1.0 parts by weight of benzoyl peroxide, completely dissolved in the oil phase; B. Aqueous phase preparation: Dissolve 0.5-1.0 parts by weight of gelatin, 2-4 parts by weight of polyvinyl alcohol (PVA-1788), and 5-8 parts by weight of sodium chloride in 150-200 parts by weight of deionized water to prepare a stabilizer solution; C. Suspension polymerization reaction.

[0009] Furthermore, step 2) includes the following steps: A. Activation of hydroxyl groups: 10-15 parts by weight of dry Resin-OH resin are suspended in 100 parts by weight of anhydrous dichloromethane; Under the protection of ice water bath and nitrogen, a dichloromethane solution containing 3-5 parts by mass of triethylamine and 5-8 parts by mass of 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was slowly added dropwise to the system. After the addition is complete, remove the ice bath and react at room temperature for 12-24 hours to generate the phosphoryl chloride intermediate; B. Ring-opening grafting: Without separating the phosphoryl chloride intermediate, directly add 8-10 parts by mass of a 30-40% trimethylamine ethanol solution to the reaction system.

[0010] Furthermore, step 2) can also be replaced by the following steps: A. Suspend 10-15 parts by weight of dry Resin-OH resin in 100 parts by weight of anhydrous dichloromethane; under ice-water bath and nitrogen protection, add 3-5 parts by weight of anhydrous triethylamine to the system; B. Add dropwise anhydrous dichloromethane solution containing 5-8 parts by mass of 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane; after the addition is complete, stir the reaction at room temperature for 12-24 hours. C. Filter and wash thoroughly with anhydrous dichloromethane to obtain chloroethyl phosphate intermediate; D. Suspend the chloroethyl phosphate intermediate in 100 parts by weight of anhydrous dichloromethane, add 4-6 parts by weight of ethanolamine and 3-5 parts by weight of anhydrous triethylamine; react at 40°C for 24-48 hours under nitrogen protection. E. After the reaction is complete, the resin is recovered by filtration and washed thoroughly with dichloromethane, ethanol and deionized water in sequence, and then dried under vacuum to obtain the hydroxyethyl amino phosphate intermediate. F. Suspend 10-15 parts by weight of the hydroxyethyl amino phosphate intermediate in 100 parts by weight of anhydrous dichloromethane, and add 3-5 parts by weight of anhydrous triethylamine and 3-4 parts by weight of methanesulfonyl chloride in sequence under ice-water bath and nitrogen protection, and react for 4-6 hours. G. Without separation, directly add 8-10 parts by mass of a 30-40% trimethylamine ethanol solution to the reaction system, heat to 40-50°C, and continue the reaction for 24-48 hours.

[0011] The preparation method of this invention generates macroporous adsorption resin by introducing a multifunctional crosslinking agent and in-situ grafting technology. The synthesis method is simple, and the generated macroporous adsorption resin has a stable structure and excellent anticoagulant properties. Detailed Implementation

[0012] The invention will now be described more fully by way of examples. The invention can be embodied in many different forms and should not be construed as being limited to the exemplary embodiments described herein.

[0013] The parts in Examples 1 and 2 are all by weight. Example 1

[0014] I. Synthesis of Macroporous Resin Frameworks Containing Active Functional Groups The core of this step is to utilize the synergistic effect of 2,2-bis(chloromethyl)-1,3-propanediol and DVB to construct a resin skeleton containing reactive hydroxyl groups.

[0015] 1. Oil phase preparation: Add the following components to the reaction flask: Monomer mixture: 80-85 parts styrene, 15-20 parts divinylbenzene (DVB, 80% purity), 5-8 parts 2,2-bis(chloromethyl)-1,3-propanediol.

[0016] Pore-forming agent: 30-60 parts of a mixed pore-forming agent (toluene and n-heptane mixed at a mass ratio of 2:1). The selection and proportion of the pore-forming agent can be optimized according to the desired pore size.

[0017] Initiator: 0.8~1.0 parts of benzoyl peroxide (BPO); completely dissolved in the oil phase.

[0018] 2. Aqueous phase preparation: In another container, add 150-200 parts of deionized water, dissolve 0.5-1.0 parts of gelatin, 2-4 parts of polyvinyl alcohol (PVA-1788), and 5-8 parts of sodium chloride to prepare a stabilizer solution.

[0019] 3. Suspension polymerization reaction: Add the aqueous phase to a reactor equipped with a stirrer, condenser and thermometer, and heat to 50-55℃.

[0020] While stirring, the prepared oil phase is slowly added to the aqueous phase, and the stirring speed is adjusted to control the oil droplet size, thereby controlling the final resin particle size.

[0021] After the oil phase is evenly dispersed, the temperature is increased to 75~80℃ at a rate of 1℃ / min and held for 4~6 hours.

[0022] The temperature was then increased to 85°C, and the reaction was continued for 6–10 hours to ensure complete monomer conversion. After the reaction was complete, the mixture was allowed to cool naturally to room temperature.

[0023] 4. Post-processing: The resin microspheres are collected by filtration and repeatedly washed with hot water (60-70℃) to remove porogens and stabilizers.

[0024] Extract the sample sequentially with ethanol and deionized water in a Soxhlet extractor for 24-48 hours to completely remove residual porogens and oligomers.

[0025] The resin was dried to constant weight in a vacuum oven at 60°C to obtain a white, opaque macroporous resin skeleton containing active hydroxyl groups (denoted as Resin-OH).

[0026] In this step, the introduction of 2,2-bis(chloromethyl)-1,3-propanediol is crucial. It participates in the polymerization as a crosslinking agent, but its diol structure is retained in the backbone, which is fundamentally different from conventional resins containing only DVB, laying the foundation for subsequent differentiated grafting.

[0027] II. In-situ chemical grafting of phosphocholine This step utilizes the hydroxyl groups in the Resin-OH skeleton to covalently attach phosphocholine to the resin through an efficient activation-grafting reaction.

[0028] 1. Activation of hydroxyl groups: 10-15 parts of dry Resin-OH resin were suspended in 100 parts of anhydrous dichloromethane.

[0029] Under the protection of ice water bath and nitrogen, a solution of dichloromethane containing 3-5 parts triethylamine and 5-8 parts 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane (this compound is the key phosphorylation reagent) is slowly added dropwise to the system.

[0030] After the addition is complete, remove the ice bath and allow the reaction to proceed at room temperature for 12–24 hours. During this time, the hydroxyl groups on the resin react with phosphoryl chloride to generate a highly reactive phosphoryl chloride intermediate (Resin-P-Cl).

[0031] Chemical reaction formula:

[0032] 2. Open-loop grafting: There is no need to separate Resin-P-Cl; simply add 8-10 parts of trimethylamine ethanol solution (concentration 30-40%) directly to the reaction system.

[0033] The temperature is raised to 40-50℃, and the reaction continues for 24-48 hours. During this process, the phosphoryl chloride intermediate undergoes a ring-opening quaternization reaction with trimethylamine to form the final phosphorocholine structure.

[0034] Chemical reaction formula:

[0035] 3. Purification: After the reaction is complete, the resin is recovered by filtration.

[0036] The resin was washed repeatedly with dichloromethane, ethanol, and deionized water in sequence until the washing solution was neutral and no chloride ions were detected (test with silver nitrate solution).

[0037] Finally, the resin was washed with acetone and dried under vacuum at 40°C to obtain the final product—phosphocholine-grafted anticoagulant macroporous adsorption resin (denoted as Resin-PC).

[0038] This "in-situ activation-ring-opening grafting" route is completely different from the lengthy path of amination and grafting via chloromethyl resin in existing patents. We utilize the hydroxyl groups reserved in a novel crosslinking agent to directly construct phosphocholine through a concise phosphorochemistry process. The reaction pathway is unique, with fewer steps and higher efficiency.

[0039] Summary of the advantages of this synthetic route: Route novelty: 2,2-bis(chloromethyl)-1,3-propanediol is used as a functional crosslinking agent for the synthesis of anticoagulant resin for the first time, and a corresponding in-situ phosphocholine grafting technique is developed. This combination has not been reported in the published patent literature.

[0040] Simplified process: The "one-pot two-step" method avoids the post-chloromethylation treatment of highly toxic chloromethyl ether and the complex purification process caused by multiple post-modification steps, making production safer and more environmentally friendly.

[0041] Controllable structure and superior performance: Phosphocholine groups are directly attached to the polymer backbone via stable covalent bonds, making them difficult to detach and ensuring long-lasting anticoagulant properties. Because the grafting sites originate from within the backbone, the distribution of phosphocholine may be more uniform, contributing to improved blood compatibility. This avoids the damage to the resin pore structure caused by harsh post-processing, thus preserving its adsorption performance. Example 2

[0042] The difference between this embodiment and Embodiment 2 lies in the different in-situ chemical grafting methods for phosphocholine.

[0043] Step 1: Ring-opening reaction – Production of chloroethyl phosphate intermediate This step utilizes the reaction of cyclic phosphoryl chloride with the hydroxyl groups of the resin to introduce a highly reactive chloroethyl terminus into the backbone.

[0044] Reaction system preparation: Suspend 10-15 parts of dry Resin-OH resin in 100 parts of anhydrous dichloromethane. Under ice-water bath (0-5°C) and nitrogen protection, add 3-5 parts of anhydrous triethylamine (as acid absorbent) to the system.

[0045] Ring-opening reaction: Slowly add anhydrous dichloromethane solution containing 5-8 parts of 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane. After the addition is complete, remove the ice bath and stir the reaction at room temperature for 12-24 hours. During this process, the cyclic phosphoryl chloride undergoes ring-opening, forming a phosphate ester bond with the resin hydroxyl group, and generating an intermediate with a chloroethyl terminus.

[0046] Intermediate separation: After the reaction was complete, the mixture was filtered and thoroughly washed with anhydrous dichloromethane to remove the byproduct triethylamine hydrochloride and unreacted reagents. The chloroethyl phosphate intermediate (denoted as Resin-P-ClEt) was obtained and can be used directly in the next reaction step.

[0047] Chemical reaction formula:

[0048] Second step: Aminolysis reaction – introduction of hydroxyethylamine This step utilizes the amino group of ethanolamine to replace the active chlorine atom, introducing a hydroxyl group in preparation for quaternization.

[0049] Amine hydrolysis: The Resin-P-ClEt intermediate obtained in the previous step was resuspended in 100 parts of anhydrous dichloromethane. 4-6 parts of ethanolamine and 3-5 parts of anhydrous triethylamine were added.

[0050] Reaction process: Under nitrogen protection, the reaction is carried out at 40°C for 24-48 hours. The amino group of ethanolamine nucleophilically attacks the chlorine atom at the end of the chloroethyl group, forming a stable amine bond and introducing a crucial terminal primary hydroxyl group.

[0051] Purification: After the reaction was completed, the resin was recovered by filtration and washed thoroughly with dichloromethane, ethanol and deionized water in sequence, and then dried under vacuum to obtain the hydroxyethyl aminophosphate intermediate (denoted as Resin-P-NHEtOH). Chemical reaction formula:

[0052]

[0053] After reacting with ethanolamine, we obtained an intermediate terminated with a primary hydroxyl group. In organic synthesis, it can be selectively and efficiently converted into other functional groups.

[0054] (1) High selectivity: We can specifically activate primary hydroxyl groups to methanesulfonyl chloride (MsCl) without affecting the amide bond (-NH-) or phosphate bond already present in the molecule.

[0055] (2) High efficiency: Methanesulfonate is an excellent leaving group, and its subsequent quaternization reaction with trimethylamine is highly efficient and complete. Step 3: Quaternization reaction – constructing the phosphoric choline terminus This step involves activating the terminal hydroxyl group and reacting it with trimethylamine to complete the construction of the zwitterionic structure of phosphocholine.

[0056] Hydroxyl activation: 10-15 parts of Resin-P-NHEtOH intermediate were suspended in 100 parts of anhydrous dichloromethane. Under ice-water bath and nitrogen protection, 3-5 parts of anhydrous triethylamine and 3-4 parts of methanesulfonyl chloride (MsCl) were added sequentially, and the reaction was carried out for 4-6 hours to activate the primary hydroxyl groups at the resin end to highly reactive methanesulfonate esters (-OMs).

[0057] Quaternization: No separation is required. Directly add 8-10 parts of trimethylamine ethanol solution (concentration 30-40%) to the reaction system. Heat to 40-50°C and continue the reaction for 24-48 hours. Trimethylamine nucleophilically attacks the methanesulfonate group to form the quaternary ammonium salt.

[0058] Final purification: After the reaction, the resin was recovered by filtration. It was washed successively with dichloromethane, ethanol, and large amounts of deionized water until the washings were neutral. Finally, it was washed with acetone and dried under vacuum at 40°C to constant weight to obtain the final product—phosphocholine-grafted anticoagulant macroporous adsorption resin (denoted as Resin-PC).

[0059] Chemical reaction formula: 1. Activation:

[0060] 2. Quaternization:

[0061] Ultimately, a phosphocholine structure was formed on the resin:

[0063] The phosphorocholine structure formed in Example 2 allows for the formation of a flexible 4-carbon chain between the phosphate and quaternary ammonium salts. This longer, flexible chain better mimics the natural molecule, making its spatial conformation closer to the natural phosphorocholine structure. This allows the terminal phosphorocholine groups to more easily swing and orient on the resin surface, thus enabling more effective interaction with the biological environment and exerting its anticoagulant function.

[0064] Verification Experiment 1. Coagulation test (PTT test) Based on the national standard GB / T16886.4-2022 "Biological evaluation of medical devices - Part 4: Selection of blood interaction tests", the partial thromboplastin time (PTT) test method was used to conduct in vitro blood compatibility tests to evaluate the effect of the sample on human plasma clotting time.

[0065]

[0066] Negative control: high-density polyethylene; Blank control: human plasma; Positive control: glass beads Examples 1 and 2 showed significant anticoagulant effects compared to ungrafted resin.

[0067] 2. Platelet test Based on the national standard GB / T16886.4-2022 "Biological evaluation of medical devices - Part 4: Selection of blood interaction tests", the platelet counting method was used to conduct in vitro blood compatibility tests to evaluate the effect of the sample on the platelet count in human blood.

[0068]

[0069] Negative control: High-density polyethylene; Blank control: Human blood; Positive control: Black rubber It can be seen that the platelet adsorption capacity in Examples 1 and 2 is significantly lower than that in the ungrafted resin.

[0070] 3. In vitro thrombosis test The test was conducted in accordance with the national standard GB / T16886.4-2022 "Biological evaluation of medical devices - Part 4: Selection of blood interaction tests", and the in vitro thrombosis test method was used to evaluate whether the sample could induce thrombus production in vitro.

[0071]

[0072] Experimental blank medium: rabbit blood partially anticoagulated with sodium citrate (30 mL rabbit blood, with 1.2 mL of 3.8% sodium citrate solution added for partial anticoagulation). In Examples 1 and 2, the content of residual fibrinogen was higher than that of ungrafted resin, and the amount of fibrinogen adsorbed by the examples was reduced.

[0073] The above examples are only for illustrating the present invention. In addition, there are many other different implementations, which can be conceived by those skilled in the art after understanding the concept of the present invention. Therefore, they will not be listed one by one here.

Claims

1. A method for preparing an anticoagulant macroporous adsorbent resin grafted with phosphorylcholine in situ, characterized in that, Comprising the following steps: 1) Constructing a resin skeleton containing reactive hydroxyl groups; 2) In-situ chemical grafting of phosphocholine; 3) Purification.

2. The production method according to claim 1, wherein Step 1) comprises the following steps: A, oil phase preparation, monomer mixture: 80~85 mass parts of styrene, 15~20 mass parts of divinyl benzene with a purity of 80%, 5~8 mass parts of 2,2-bis(chloromethyl)-1,3-propanediol; pore forming agent: 20~40 mass parts of toluene and 10~20 mass parts of n-heptane; initiator: 0.8~1.0 mass parts of benzoyl peroxide, completely dissolved in the oil phase; B, water phase preparation: 150~200 mass parts of deionized water, 0.5~1.0 mass parts of gelatin, 2~4 mass parts of polyvinyl alcohol (PVA-1788) and 5~8 mass parts of sodium chloride are dissolved to prepare a stabilizer solution; C, suspension polymerization reaction.

3. The production method according to claim 1, wherein Step 2) comprises the following steps: A, activation of hydroxyl groups: 10~15 mass parts of dry Resin-OH resin is suspended in 100 mass parts of anhydrous dichloromethane; Under ice water bath and nitrogen protection, 3~5 mass parts of triethylamine and 5~8 mass parts of 2-chloro-2-oxo-1,3,2-dioxaphospholane in dichloromethane solution are slowly added to the system; After the addition is completed, the ice bath is removed and the reaction is carried out at room temperature for 12~24 hours to generate a phosphoryl chloride intermediate; B, ring-opening grafting: without separation of the phosphoryl chloride intermediate, 8~10 mass parts of 30~40% trimethylamine ethanol solution is directly added to the reaction system.

4. The production method according to claim 1, wherein Step 2) comprises the following steps: A, 10~15 mass parts of dry Resin-OH resin is suspended in 100 mass parts of anhydrous dichloromethane; under ice water bath and nitrogen protection, 3~5 mass parts of anhydrous triethylamine is added to the system; B, 5~8 mass parts of 2-chloro-2-oxo-1,3,2-dioxaphospholane in anhydrous dichloromethane solution is added dropwise; after the addition is completed, the reaction is carried out at room temperature for 12~24 hours under stirring; C, filtration, and washing with anhydrous dichloromethane to obtain a chloroethyl phosphate intermediate; D, the chloroethyl phosphate intermediate is suspended in 100 mass parts of anhydrous dichloromethane, 4~6 mass parts of ethanolamine and 3~5 mass parts of anhydrous triethylamine are added; under nitrogen protection, the reaction is carried out at 40°C for 24~48 hours; E, after the reaction is completed, the resin is recovered by filtration, and is sequentially washed with dichloromethane, ethanol and deionized water, and is vacuum dried to obtain a hydroxyethyl amine phosphate intermediate; F, 10~15 mass parts of the hydroxyethyl amine phosphate intermediate is suspended in 100 mass parts of anhydrous dichloromethane, under ice water bath and nitrogen protection, 3~5 mass parts of anhydrous triethylamine and 3~4 mass parts of methane sulfonyl chloride are sequentially added, and the reaction is carried out for 4~6 hours; G, without separation, 8~10 mass parts of 30~40% trimethylamine ethanol solution is directly added to the reaction system, and the temperature is raised to 40~50°C, and the reaction is continued for 24~48 hours.

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