Nanometer polymer hemostatic and preparation method thereof

Abstract

The invention discloses a nano polymer hemostatic and a preparation method thereof, the hemostatic is a composite nanoparticle with a core-shell structure, and is formed by compounding a core layer, a shell layer and loaded active components through covalent crosslinking and electrostatic adsorption; the particle size (D50100nm-300nm), the porosity (60%-90%) and the active component loading capacity (0.5%-5%) of the composite nanoparticles are accurately controlled through the processes of intermittent ultrasonic treatment (working for 3s-5s, and the interval is 5s-10s), gradient freeze-drying and the like, the inter-batch variation coefficient of the product is less than 5%, and the problem of poor stability of the traditional nano preparation process is solved.

Description

Technical Field

[0001] This invention relates to the field of hemostatic agents, and in particular to a nanopolymer hemostatic agent and its preparation method. Background Technology

[0002] In medical emergencies, surgeries, and trauma treatment, rapid and effective hemostasis is crucial for reducing patient mortality and complications. Especially for life-threatening bleeding such as liver and spleen rupture or arterial injury, traditional hemostasis methods like compression and ligation often fail to quickly control the bleeding. The performance of hemostatic materials directly determines hemostatic efficiency and the success rate of treatment. With the integration of materials science and nanotechnology, micro / nano-scale hemostatic agents, due to their high specific surface area and strong bioactivity, have become a research hotspot in the field of hemostatic materials.

[0003] Existing hemostatic materials can be mainly divided into three categories: traditional natural materials, synthetic polymer materials, and composite nanomaterials. Traditional natural polymer hemostatic agents (such as chitosan and gelatin) have good biocompatibility, but their hemostatic speed is slow and their mechanical properties are poor. In high-pressure bleeding scenarios, they are easily dispersed by blood and fail to form a stable hemostatic barrier. Synthetic polymer materials (such as polylactic acid and polyethyleneimine) have high mechanical strength, but their biocompatibility is insufficient. Some materials are prone to inducing hemolytic or inflammatory reactions and lack the ability to actively regulate the coagulation process.

[0004] While nanocomposite hemostatic agents developed in recent years combine the advantages of natural and synthetic materials to some extent, they still face several technical bottlenecks: First, most materials have a simple structure, promoting platelet aggregation solely through physical adsorption, failing to form a synergistic hemostatic mechanism of "physical barrier-chemical catalysis-biostimulation," making it difficult to meet the hemostatic efficiency requirements for severe bleeding; Second, unreasonable loading methods of active ingredients often result in burst release effects leading to low bioavailability, or insufficient loading affecting hemostatic efficacy; Third, it is difficult to balance the physicochemical properties and biosafety of the materials. Some nanoparticles have uneven particle size distribution (exceeding 500 nm), easily accumulating in vivo and causing toxicity, and the residual amount of crosslinking agents is too high (often >0.1 wt%), posing a biosafety risk; Fourth, the preparation process is complex and poorly controllable, making it difficult to achieve precise control of key parameters such as particle size and porosity, resulting in insufficient batch stability of the product.

[0005] Furthermore, the clinical demand for multifunctional hemostatic materials is increasingly prominent, requiring not only rapid hemostasis but also comprehensive properties such as wound adhesion, antibacterial and anti-infection effects, and promotion of wound healing. Existing materials mostly focus on the single function of hemostasis, exhibiting weak ability to regulate the wound microenvironment, and problems such as postoperative infection and delayed healing remain unresolved. Therefore, developing a nanopolymer hemostatic agent that combines rapid and efficient hemostasis, excellent biocompatibility, and multifunctional synergistic effects has become a pressing technical challenge in the field of medical materials. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a nano-polymer hemostatic agent and its preparation method.

[0007] One of the objectives of this invention is achieved through the following technical solution: A nanopolymer hemostatic agent, wherein the hemostatic agent is a core-shell structured composite nanoparticle, which is composed of a core layer, a shell layer and a loaded active ingredient through covalent cross-linking and electrostatic adsorption. The core layer is a modified natural polymer material selected from one or more of carboxymethyl chitosan, oxidized dextran, gallic acid-grafted chitosan, and acylated starch. The modified natural polymer material is modified by carboxylation, acylation, or grafting reaction, and has a molecular weight of 10,000 Da to 100,000 Da. The shell is a functional synthetic polymer material selected from one or more of polyethylene glycol-modified polylactic acid-glycolic acid copolymer (PEG-PLGA, PEG segment molecular weight 2000Da~5000Da), branched polyethyleneimine (generation 2.0~4.0G), and poly(L-lysine) hydrobromide, with a molecular weight of 5000Da~50000Da. The active ingredient is a hemostatic, synergistic, and repairing compound, including one or more of thrombin, ε-polylysine, and bovine basic fibroblast growth factor (bFGF), with a loading of 0.5% to 5% of the total mass of the nanopolymer hemostatic agent; The composite nanoparticles have a particle size of 50 nm to 500 nm (median particle size D50 of 100 nm to 300 nm), a three-dimensional network porous structure with a pore size of 20 μm to 40 μm, a porosity of 60% to 90%, and a specific surface area of ​​15 m². 2 / g~40m 2 / g, bulk density is 0.025g / cm³ 3 ~0.05g / cm 3 ; The hemostatic agent can change from a dispersed state to a gel state within 10s to 30s under physiological conditions, with a water absorption rate ≥1800% (determined by distillation water method), an adhesion strength ≥0.5MPa, and can withstand a liquid pressure ≥600mmHg. The hemostatic time on rat abdominal aortic wounds is ≤60s.

[0008] As a further improvement to the above technical solution: In the core-layer modified natural polymer material: The degree of substitution of carboxymethyl chitosan is 0.6–0.8, and the degree of deacetylation is ≥85%. The degree of oxidation of oxidized dextran is 20%–40%; The grafting rate of gallic acid onto chitosan is 15%–30%.

[0009] In the aforementioned shell-functional synthetic polymer material, the molar ratio of lactic acid to glycolic acid in PEG-PLGA is 50:50 to 75:25, and the amine value of branched polyethyleneimine is 400 mg KOH / g to 800 mg KOH / g.

[0010] The active ingredient includes: Specific activity of thrombin ≥200 U / mg; The purity of ε-polylysine is ≥95%, and the molecular weight is 3000 Da~4000 Da; Bovine basic fibroblast growth factor bioactivity ≥1×10 5 IU / mg.

[0011] It also includes crosslinking agents and stabilizers, among which: The crosslinking agent is selected from one or more of carbodiimide (EDC), N-hydroxysuccinimide (NHS), and genipin, with a residual amount ≤0.1wt%; The stabilizer is selected from Tween 80 and polysorbate 60, and its content is 0.1% to 1% of the total mass of the hemostatic agent.

[0012] The biocompatibility and safety indicators of the hemostatic agent meet the following requirements: Hemolysis rate ≤5% (determined according to GB / T16886.4 standard); The cytotoxicity level is Grade 1 (cell viability ≥70% as determined by MTT assay). Antibacterial rate against Staphylococcus aureus and Escherichia coli ≥80%; Endotoxin content <0.5 EU / cm³ 2 The residual amount of ethylene oxide is ≤4μg / g (if ethylene oxide sterilization is used).

[0013] A method for preparing the nanopolymer hemostatic agent as described in any one of claims 1-6, comprising the following steps: Core layer solution preparation: The modified natural polymer material is dissolved in a buffer solution with a pH of 5.0–7.0 and stirred at 25℃–37℃ for 1–2 hours until completely dissolved to obtain a core layer solution with a concentration of 5 mg / mL–50 mg / mL; the buffer solution is selected from phosphate buffer (PBS) and acetate-sodium acetate buffer, and the buffer concentration is 0.01 mol / L–0.05 mol / L; Preparation of shell prepolymer: The functional synthetic polymer material is dissolved in a water-soluble organic solvent, a crosslinking agent and a surfactant are added, and the mixture is ultrasonically dispersed for 10 min to 30 min (power 100W to 200W) to obtain a shell prepolymer with a concentration of 10 mg / mL to 100 mg / mL; the water-soluble organic solvent is selected from one or more of acetone, acetonitrile, and dimethyl sulfoxide (DMSO), and the surfactant is selected from one of glycerol stearate and polyoxyethylene sorbitan fatty acid ester, with the addition amount being 5% to 15% of the mass of the synthetic polymer material; the mass ratio of the crosslinking agent to the synthetic polymer material is 0.1 to 1:1; Emulsification and compounding: Under the conditions of ultrasonic power of 200W to 500W and intermittent ultrasound (working for 3s to 5s, with an interval of 5s to 10s), the shell prepolymer solution is slowly added dropwise to the core solution at a rate of 0.5mL / min to 2mL / min, with an oil-water phase volume ratio of 1:3 to 1:10. After the addition is completed, emulsification is continued for 20min to 60min to obtain a primary emulsion with uniform particle size. Active ingredient loading: Dissolve the active ingredient in deionized water (concentration 1 mg / mL to 10 mg / mL), and slowly add it to the primary emulsion at a stirring speed of 1000 rpm to 3000 rpm and a temperature of 30℃ to 50℃. The reaction is carried out at a constant temperature for 1 to 3 hours. If the active ingredient is bFGF, the pH of the reaction system is controlled at 7.2 to 7.4, and the reaction time after addition does not exceed 2 hours. Curing and molding: The reaction solution is placed in a dialysis bag with a molecular weight cutoff of 3500 Da to 10000 Da and dialyzed in deionized water for 24 h to 48 h. The deionized water is replaced every 4 h to 6 h to remove organic solvents and unreacted small molecules. After dialysis, the liquid preparation is placed in a freeze dryer and pre-frozen at -40℃ to -60℃ for 4 h to 8 h. Then it is dried under a vacuum of 1 Pa to 10 Pa for 24 h to 48 h to obtain a powdered nano-polymer hemostatic agent. Sterilization treatment: Sterilization is carried out by gamma ray irradiation (dose 25kGy±2kGy) or ethylene oxide sterilization. After sterilization, sterility inspection and endotoxin testing are performed.

[0014] As a further improvement to the above technical solution: The surfactant mentioned in step 2 is polyoxyethylene sorbitan fatty acid ester (Tween 80). When the synthesized polymer material is PEG-PLGA, the amount of surfactant added is 10% to 15% of the mass of PEG-PLGA, which can reduce the interfacial tension of the emulsion to below 25 mN / m.

[0015] In step 3, the emulsification process uses an ice-water bath to control the temperature, keeping the system temperature between 0°C and 10°C, to avoid high temperatures causing degradation of the synthesized polymer materials or deactivation of the active ingredients (if the active ingredients are added in advance).

[0016] The freezing-drying temperature rise procedure in step 5 is as follows: after pre-freezing, raise the temperature to -20℃ at a rate of 1℃ / min and hold for 8 hours; then raise the temperature to 0℃ at a rate of 0.5℃ / min and hold for 4 hours; finally raise the temperature to 25℃ and hold for 8 hours to ensure that the product moisture content is ≤5%; if ethylene oxide sterilization is used in step 6, the sterilization conditions are: ethylene oxide concentration 400mg / L~600mg / L, temperature 37℃~55℃, relative humidity 40%~80%, sterilization time 2h~4h, and ventilation and desorption for at least 12 hours after sterilization.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (i) Establish a synergistic hemostasis mechanism to achieve rapid control of severe bleeding. Rapid formation of physical barrier: The core-shell structured composite nanoparticles have a particle size precisely controlled between 50nm and 500nm and a three-dimensional network porous structure with pore sizes of 20μm to 40μm and a porosity of 60% to 90%. They can rapidly absorb water from the blood within 10s to 30s (water absorption rate ≥1800%), concentrate clotting factors and platelets, and at the same time, the nanoparticles can penetrate deep bleeding points to form a dense physical hemostatic barrier. The hemostasis time for rat abdominal aortic wounds is ≤60s, which is significantly better than existing materials (usually >90s).

[0018] Synergistic activation of chemical and biological signals: Functional synthetic polymers in the shell layer (such as PEG-PLGA and branched polyethyleneimine) adsorb platelets through surface charge effect, activating the PI3K / Akt signaling pathway and promoting platelet aggregation; Highly active components loaded in the core layer (thrombin specific activity ≥200U / mg, ε-polylysine purity ≥95%) can directly catalyze the coagulation cascade reaction, forming a triple synergistic mechanism of "physical adsorption-chemical catalysis-biostimulation", which improves hemostasis efficiency by more than 40% compared with single-component materials.

[0019] (ii) Optimize material composition and structure, taking into account both biosafety and reliability in use. Significantly improved biocompatibility: The core layer uses high-purity modified natural polymers (carboxymethyl chitosan deacetylation degree ≥85%, oxidized dextran oxidation degree 20%~40%), while the shell layer uses PEG-modified biodegradable materials. Combined with strictly controlled residual cross-linking agents (≤0.1wt%), the material achieves a hemolysis rate ≤5%, a cytotoxicity level of 1 (cell viability ≥70%), and an endotoxin content <0.5EU / cm³. 2 It fully complies with the GB / T16886.4 biocompatibility standard.

[0020] Mechanical properties and adhesion meet clinical needs: Through core-shell covalent cross-linking and porous structure design, the material has an adhesion strength of ≥0.5MPa and can withstand a liquid pressure of ≥600mmHg. It will not be washed away in arterial hypertension bleeding scenarios, solving the problem of easy displacement of traditional nanomaterials; at the same time, the freeze-dried powder dosage form is easy to store and transport, and it quickly gels upon contact with blood, adapting to the need for adhesion of irregular wounds.

[0021] (III) Integrating multifunctional features to promote wound healing and infection control Broad-spectrum antibacterial effect: The ε-polylysine in the active ingredient works synergistically with the shell polymer to achieve an antibacterial rate of ≥80% against Staphylococcus aureus and Escherichia coli, which can effectively reduce the risk of postoperative wound infection and solve the shortcomings of existing hemostatic agents that lack antibacterial function.

[0022] Synergistic effect of wound repair: Loaded bovine basic fibroblast growth factor (bioactivity ≥1×10⁻⁶) 5 IU / mg can be continuously released after hemostasis, stimulating wound tissue regeneration, shortening the healing cycle, and achieving integrated treatment of "hemostasis-antibacterial-repair".

[0023] (iv) The preparation process is highly controllable, ensuring product stability and industrialization potential. Precise control of key parameters: Through intermittent ultrasound (working for 3s to 5s, with intervals of 5s to 10s) and gradient freeze drying, the particle size (D50 100nm to 300nm), porosity (60% to 90%) and active ingredient loading (0.5% to 5%) of composite nanoparticles are precisely controlled, with batch-to-batch variation coefficients of <5%, solving the problem of poor stability in traditional nanoparticle preparation processes.

[0024] Green, efficient, and cost-controllable: The preparation process uses water-soluble organic solvents (acetone, acetonitrile, etc.), which can be completely removed by dialysis (residual amount <0.01%), without the need for complex purification equipment; the raw materials are all commercially mass-produced materials, and the production cost is reduced by more than 60% compared with precious metal nano-hemostatic agents, which has the prospect of large-scale industrial application.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the formation process of the core-shell structured primary emulsion in this embodiment. Figure 2 This is a flowchart illustrating the active ingredient loading and curing process in this embodiment; Figure 3 This is a flowchart of the sterilization and finished product testing process in this embodiment. Detailed Implementation

[0027] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0028] It should be noted that when a component is described as "fixed to" another component, it can be directly on the other component or may have a component in between. When a component is considered "connected to" another component, it can be directly connected to the other component or may have a component in between. When a component is considered "set on" another component, it can be directly set on the other component or may have a component in between. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. I. Specific Implementation Methods Example 1: Carboxymethyl chitosan-PEG-PLGA core-shell nano-hemostatic agent (thrombin-loaded) 1. Raw material preparation Core material: Carboxymethyl chitosan (degree of substitution 0.7, degree of deacetylation 88%, molecular weight 50,000 Da) Shell material: PEG-PLGA (PEG segment molecular weight 3000 Da, PLGA lactic acid: glycolic acid = 65:35, total molecular weight 20000 Da) Active ingredient: Thrombin (specific activity 250 U / mg) Crosslinking agent: EDC and NHS (mass ratio 1:1) Buffer solution: 0.02 mol / L PBS (pH 6.5) Water-soluble organic solvent: acetone 2. Preparation steps (1) Preparation of core layer solution: Weigh 2.5g of carboxymethyl chitosan, dissolve it in 500mL of PBS buffer, stir at 30℃ for 1.5h to obtain a core layer solution with a concentration of 5mg / mL.

[0031] (2) Preparation of shell prepolymer: Weigh 5g of PEG-PLGA and dissolve it in 50mL of acetone. Add 0.5g of EDC-NHS mixed crosslinking agent (accounting for 10% of the mass of PLGA) and 0.5g of Tween 80 (accounting for 10% of the mass of PLGA). Disperse the mixture by ultrasonication at 150W for 20min to obtain a shell prepolymer of 100mg / mL.

[0032] (3) Emulsification and composite: Under the conditions of 300W intermittent ultrasound (working for 4s, with an interval of 8s) and ice-water bath temperature control (5℃), the shell prepolymer solution was added to the core solution at a rate of 1mL / min (oil-water phase volume ratio 1:5). After the addition was completed, emulsification was continued for 40min to obtain the primary emulsion.

[0033] (4) Loading of active ingredients: Weigh 0.15g of thrombin and dissolve it in 10mL of deionized water. Stir at 2000rpm and add the primary emulsion at 40℃. React at a constant temperature for 2h.

[0034] (5) Curing and molding: The reaction solution was transferred into a dialysis bag with a molecular weight cutoff of 5000 Da, and dialyzed with deionized water for 36 hours (with water changed every 5 hours). Then it was freeze-dried (pre-frozen at -50℃ for 6 hours, and dried at 1 Pa vacuum for 36 hours according to the program) to obtain a powdered hemostatic agent.

[0035] (6) Sterilization treatment: γ-ray irradiation sterilization (dose 25kGy).

[0036] Example 2: Gallic acid grafted chitosan-branched PEI core-shell nano-hemostatic agent (loaded with ε-polylysine) 1. Raw material preparation Core material: Gallic acid grafted chitosan (grafting rate 25%, molecular weight 80,000 Da) Shell material: 3.0G branched polyethyleneimine (amine value 600mgKOH / g, molecular weight 10000Da) Active ingredient: ε-polylysine (98% purity, molecular weight 3500 Da) Crosslinking agent: Genipin Buffer solution: 0.03 mol / L acetate-sodium acetate buffer (pH 5.5) Water-soluble organic solvent: acetonitrile 2. Preparation steps (1) Preparation of core layer solution: Weigh 5g gallic acid-grafted chitosan, dissolve it in 100mL buffer solution, stir at 37℃ for 2h to obtain a core layer solution of 50mg / mL.

[0037] (2) Preparation of shell prepolymer: Weigh 2g of branched PEI and dissolve it in 20mL of acetonitrile. Add 0.4g of genipin (accounting for 20% of the mass of PEI) and 0.2g of polysorbate 60. Disperse the mixture by ultrasonication at 100W for 30min to obtain a shell prepolymer of 100mg / mL.

[0038] (3) Emulsification and composite: Under the conditions of 200W intermittent ultrasound (working for 3s, with a 10s interval) and ice-water bath (8℃), the shell prepolymer solution was added dropwise to the core solution at a rate of 0.5mL / min (oil-water phase volume ratio 1:8), and emulsified for 30min to obtain the primary emulsion.

[0039] (4) Loading of active ingredients: Weigh 0.1g of ε-polylysine and dissolve it in 5mL of deionized water. Stir at 1500rpm and add the primary emulsion at 35℃. React for 1.5h.

[0040] (5) Curing and molding: Dialysis bag (molecular weight cutoff 8000Da) was dialyzed for 48 hours and then freeze-dried (pre-frozen at -40℃ for 8 hours and dried under 5Pa vacuum for 40 hours).

[0041] (6) Sterilization treatment: Ethylene oxide sterilization (concentration 500mg / L, 45℃, humidity 60%, sterilization for 3h, desorption for 18h).

[0042] Example 3: Oxidized dextran-poly(L-lysine) core-shell nano-hemostatic agent (loaded with bFGF) 1. Raw material preparation Core material: Oxidized dextran (oxidation degree 30%, molecular weight 30000 Da) Shell material: Poly(L-lysine) hydrobromide (molecular weight 30,000 Da) Active ingredient: bFGF (bioactivity 1.5×10⁻⁶) 5 IU / mg Crosslinking agent: EDC Buffer solution: 0.01 mol / L PBS (pH 7.4) Water-soluble organic solvent: DMSO 2. Preparation steps (1) Preparation of core layer solution: Weigh 1g of oxidized dextran and dissolve it in 200mL PBS. Stir at 25℃ for 1h to obtain a core layer solution of 5mg / mL.

[0043] (2) Preparation of shell prepolymer: Weigh 3g of poly(L-lysine) and dissolve it in 30mL of DMSO. Add 0.3g of EDC and 0.3g of Tween 80, and sonicate at 200W for 15min to obtain a shell prepolymer of 100mg / mL.

[0044] (3) Emulsification and composite: 400W intermittent ultrasound (5s working, 5s interval), ice water bath (0℃), shell prepolymer solution is added dropwise to core solution at 2mL / min (volume ratio 1:10), emulsification for 60min.

[0045] (4) Loading of active ingredients: Weigh 0.05gbFGF and dissolve it in 5mLPBS (pH7.4), stir at 3000rpm, add the primary emulsion at 30℃, and react for 1h (control pH7.3).

[0046] (5) Curing and molding: Dialysis bag (molecular weight cutoff 10000Da) is dialyzed for 24 hours and then freeze-dried (pre-frozen at -60℃ for 4 hours and dried under 10Pa vacuum for 24 hours).

[0047] (6) Sterilization treatment: γ-ray irradiation sterilization (dose 23kGy).

[0048] II. Experimental Procedure 1. Physicochemical property testing (1) Particle size and morphology characterization Test method: Particle size distribution was measured using a Malvern laser particle size analyzer, and morphology was observed using a scanning electron microscope (SEM).

[0049] result:

[0050] (2) Water absorption rate and gelation time Test method: Weigh 0.1g of sample, add 20mL of distilled water, let stand at 37℃ for 30min, and calculate the water absorption rate; mix with simulated blood (volume ratio 1:10) at 37℃ and record the gelation time.

[0051] result:

[0052] (3) Loading and release of active ingredients Test methods: High performance liquid chromatography (HPLC) was used to determine the loading; dynamic dialysis in simulated body fluids was used to determine the release curve.

[0053] result:

[0054] 2. Hemostatic performance test (1) Rat liver wound hemostasis model Experimental method: SD rats (250-300g) were anesthetized and their livers were exposed. A wound with a diameter of 5mm and a depth of 2mm was prepared, and 0.1g of hemostatic agent was applied immediately. The hemostasis time and the amount of bleeding were recorded.

[0055] Control group: Commercially available chitosan hemostatic powder.

[0056] result:

[0057] (2) Rat abdominal aortic hemostasis model Experimental method: The abdominal aorta of SD rats was exposed, a 0.5 mm wound was created by puncture, 0.2 g of hemostatic agent was applied, and the hemostasis time and hemostasis success rate were recorded (hemostasis within 300 s was considered successful).

[0058] result:

[0059] 3. Biosafety Testing (1) Hemolysis test Test method: According to GB / T16886.4 standard, the material was incubated with rabbit blood red blood cell suspension and the hemolysis rate was measured.

[0060] Results: The hemolysis rates in Examples 1-3 were 2.1%, 2.8%, and 1.9%, respectively (all ≤5%).

[0061] (2) Cytotoxicity assay (MTT method) Test method: L929 cells were co-cultured with the material extract for 24 hours, and cell viability was measured.

[0062] Results: The cell survival rates in Examples 1-3 were 85%, 82%, and 88%, respectively (all ≥70%, toxicity grade 1).

[0063] (3) Antibacterial test Test method: Plate count method was used to determine the antibacterial rate against Staphylococcus aureus (ATCC25923) and Escherichia coli (ATCC25922).

[0064] result:

[0065] 4. Stability Test (1) Acceleration stability Test method: Store at 40℃ and RH75% for 3 months, and measure changes in particle size and hemostasis time.

[0066] Results: Particle size change rate <5%, hemostasis time extension <10%, and performance was stable.

[0067] (2) Sterilization residue detection Test methods: Gas chromatography was used to determine ethylene oxide residues, and the Limulus Amebocyte Lysate (LAL) method was used to determine endotoxins.

[0068] Results: Ethylene oxide residue ≤2 μg / g (Example 2), endotoxin <0.2 EU / cm 2 (All embodiments).

[0069] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A nanopolymer hemostatic agent, characterized by, The hemostatic agent is a composite nanoparticle with core-shell structure, which is composed of a core layer, a shell layer and a loaded active ingredient by covalent cross-linking and electrostatic adsorption; The core layer is a modified natural polymer material selected from one or more of carboxymethyl chitosan, oxidized dextran, gallic acid grafted chitosan and acylated starch, which is modified by carboxylation, acylation or grafting reaction, and has a molecular weight of 10000 Da to 100000 Da; The shell layer is a functional synthetic polymer material selected from one or more of polyethylene glycol modified polylactic acid-glycolic acid copolymer (PEG-PLGA, PEG segment molecular weight 2000 Da to 5000 Da), dendritic polyethyleneimine (generation number 2.0 to 4.0 G) and poly(L-lysine) hydrobromide, and has a molecular weight of 5000 Da to 50000 Da; The active ingredient is a hemostatic synergistic and repair composite ingredient, including one or more of thrombin, ε-polylysine and bovine basic fibroblast growth factor (bFGF), and has a loading amount of 0.5% to 5% of the total mass of the nanoparticle hemostatic agent; The composite nanoparticles have a particle size of 50 nm to 500 nm (median particle size D50 of 100 nm to 300 nm), a three-dimensional network porous structure with a pore size of 20 μm to 40 μm, a porosity of 60% to 90%, and a specific surface area of ​​15 m². 2 / g~40m 2 / g, bulk density is 0.025g / cm³ 3 ~0.05g / cm 3 ; The hemostatic agent can be converted from a dispersed state to a gelled state within 10s to 30s under physiological conditions, has a water absorption rate of ≥1800% (determined by the distilled water method), an adhesion strength of ≥0.5 MPa, can withstand a liquid pressure of ≥600 mmHg, and has a hemostatic time of ≤60s for a rat abdominal aortic wound.

2. The nanopolymer hemostatic agent of claim 1, wherein, In the modified natural polymer material of the core layer: The degree of substitution of carboxymethyl chitosan is 0.6 to 0.8, and the degree of deacetylation is ≥85%; The oxidation degree of oxidized dextran is 20% to 40%; The grafting rate of gallic acid grafted chitosan is 15% to 30%.

3. The nanopolymer hemostatic agent of claim 1, wherein, In the functional synthetic polymer material of the shell layer, the molar ratio of lactic acid to glycolic acid of PEG-PLGA is 50:50 to 75:25, and the amine value of dendritic polyethyleneimine is 400 mgKOH / g to 800 mgKOH / g.

4. The nanopolymer hemostatic agent of claim 1, wherein, In the active ingredient: The specific activity of thrombin is ≥200 U / mg; The purity of ε-polylysine is ≥95%, and the molecular weight is 3000 Da to 4000 Da; The biological activity of bovine basic fibroblast growth factor is ≥ 1 x 10 5 IU / mg.

5. The nanopolymer hemostatic agent of claim 1, wherein, It also includes a cross-linking agent and a stabilizer, wherein: The cross-linking agent is selected from one or more of carbodiimide (EDC), N-hydroxysuccinimide (NHS) and genipin, and the residual amount is ≤0.1 wt%; The stabilizer is selected from one of Tween 80 and polysorbate 60, and the content is 0.1% to 1% of the total mass of the hemostatic agent.

6. The nanopolymer hemostatic agent of claim 1, wherein, The biocompatibility and safety indicators of the hemostatic agent meet the following requirements: Hemolysis rate ≤5% (determined according to GB / T16886.4 standard); Cell toxicity grade is 1 (MTT method detects cell survival rate ≥70%); The antibacterial rate against Staphylococcus aureus and Escherichia coli is ≥80%; Endotoxin content <0.5 EU / cm 2 Ethylene oxide residual amount <4 μg / g (if ethylene oxide sterilization is used).

7. A method for preparing the nanopolymer hemostatic agent according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: Nuclear layer solution preparation: the modified natural polymer material is dissolved in a buffer solution with pH 5.0-7.0, stirred at 25-37℃ for 1-2h until completely dissolved, to obtain a nuclear layer solution with a concentration of 5-50mg / mL; the buffer solution is selected from one of phosphate buffered saline (PBS), acetic acid-sodium acetate buffer, and the buffer solution has a concentration of 0.01-0.05mol / L; Shell layer pre-polymer solution preparation: the functional synthetic polymer material is dissolved in a water-soluble organic solvent, a crosslinking agent and a surfactant are added, and ultrasonic dispersion is performed for 10-30min (power 100-200W) to obtain a shell layer pre-polymer solution with a concentration of 10-100mg / mL; the water-soluble organic solvent is selected from one or more of acetone, acetonitrile, and dimethyl sulfoxide (DMSO), the surfactant is selected from one of glycerol stearate and polyoxyethylene sorbitan fatty acid ester, and the addition amount of the surfactant is 5-15% of the mass of the synthetic polymer material; the mass ratio of the crosslinking agent to the synthetic polymer material is 0.1-1:1; Emulsification and compounding: under the conditions of ultrasonic power 200-500W and intermittent ultrasonic waves (working for 3-5s, interval 5-10s), the shell layer pre-polymer solution is slowly added to the nuclear layer solution at a rate of 0.5-2mL / min, the volume ratio of oil to water is 1:3-1:10, and after the addition is completed, emulsification is continued for 20-60min to obtain a primary emulsion with uniform particle size; Active ingredient loading: the active ingredient is dissolved in deionized water (concentration 1-10mg / mL), slowly added to the primary emulsion under the conditions of stirring speed 1000-3000rpm and temperature 30-50℃, and constant-temperature reaction is performed for 1-3h; if the active ingredient is bFGF, the pH of the reaction system is controlled to be 7.2-7.4, and the reaction time after the addition is not more than 2h; Solidification and molding: the reaction liquid is placed in a dialysis bag with a molecular weight cut-off of 3500-10000Da, dialyzed in deionized water for 24-48h, and the deionized water is replaced every 4-6h to remove organic solvents and unreacted small molecules; after the dialysis is completed, the liquid preparation is placed in a freeze-drying machine, pre-frozen at -40--60℃ for 4-8h, and then dried under a vacuum degree of 1-10Pa for 24-48h to obtain a powdered nanometer high-molecular hemostatic agent; Sterilization treatment: γ-ray irradiation sterilization (dose 25kGy±2kGy) or ethylene oxide sterilization is adopted, and after the sterilization, sterile examination and endotoxin detection are performed.

8. The preparation method according to claim 7, characterized in that, The surfactant in step 2 is polyoxyethylene sorbitan fatty acid ester (Tween 80), and when the synthetic polymer material is PEG-PLGA, the addition amount of the surfactant is 10-15% of the mass of PEG-PLGA, which can reduce the interfacial tension of the emulsion to below 25mN / m.

9. The preparation method according to claim 7, characterized in that, In step 3, the emulsification process adopts an ice-water bath for temperature control, so that the system temperature is maintained at 0-10℃, to avoid high temperature leading to degradation of the synthetic polymer material or inactivation of the active ingredient (if the active ingredient is added in advance).

10. The preparation method according to claim 7, characterized in that, The temperature rising procedure for freeze-drying in step 5 is as follows: after pre-freezing, the temperature is raised to -20℃ at a rate of 1℃ / min, and the temperature is kept for 8h; then the temperature is raised to 0℃ at a rate of 0.5℃ / min, and the temperature is kept for 4h; finally, the temperature is raised to 25℃, and the temperature is kept for 8h, so as to ensure that the water content of the product is less than or equal to 5%; if ethylene oxide is used for sterilization in step 6, the sterilization conditions are as follows: the concentration of ethylene oxide is 400mg / L-600mg / L, the temperature is 37℃-55℃, the relative humidity is 40%-80%, the sterilization time is 2h-4h, and the ventilation resolution after sterilization is at least 12h.

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