WO2021027006A1 - 新型可降解的止血材料及其制备方法 - Google Patents

新型可降解的止血材料及其制备方法 Download PDF

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WO2021027006A1
WO2021027006A1 PCT/CN2019/106172 CN2019106172W WO2021027006A1 WO 2021027006 A1 WO2021027006 A1 WO 2021027006A1 CN 2019106172 W CN2019106172 W CN 2019106172W WO 2021027006 A1 WO2021027006 A1 WO 2021027006A1
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cellulose
oxidation
reaction
oxidation system
solution
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PCT/CN2019/106172
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English (en)
French (fr)
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朱翠兰
石凌锋
黄建国
陶秀梅
陈鹏
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北京诺康达医药科技股份有限公司
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Publication of WO2021027006A1 publication Critical patent/WO2021027006A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0042—Materials resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/08—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/02—Oxycellulose; Hydrocellulose; Cellulosehydrate, e.g. microcrystalline cellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • the invention belongs to the field of medical dressings, and specifically relates to a novel degradable hemostatic material and a preparation method thereof.
  • Oxidized cellulose is a gauze-like or cloth-like fibrous material in which cellulose is oxidized to cellulose acid. It looks like cotton yarn, soft and thin, and has good biocompatibility, degradability and safety. .
  • oxidized cellulose has been successfully used in neurosurgery, otolaryngology, hepatobiliary surgery and other operations to stop bleeding, and the intraoperative operation is simple and easy.
  • Surgicel is widely used clinically, which is a kind of woven yarn block of oxidized regenerated fiber. It belongs to the carboxymethyl cellulose hemostatic material. It is a water-soluble, fully-absorbable neutral cellulose with very good properties. It is water-soluble and has a strong affinity for water and saline. When it is dissolved in water, it forms a hydrocolloid with a certain viscosity, which expands and fills the gaps in the wound or compresses blood vessels to achieve hemostasis.
  • the hemostatic yarn cannot play a role in the parts where the gauze cannot be filled, which limits its use.
  • research has been carried out on oxidized cellulose hemostatic powder, which broadens the scope of clinical application of oxidized cellulose hemostatic materials.
  • the oxidized cellulose hemostatic materials prepared by the prior art all have the disadvantages of low oxidation degree, high cost, large particle size and unevenness, and they need to be improved urgently.
  • the purpose of the present invention is to provide a novel degradable hemostatic material and a preparation method thereof.
  • the concept of the present invention is as follows:
  • the present invention improves the degree of oxidation of cellulose by performing pre-oxidation crushing treatment, pre-oxidation swelling treatment and oxidation process on cellulose, so that the carboxyl content of oxidized cellulose can reach more than 22% , In line with the carboxyl content requirements of oxidized cellulose hemostatic materials; the present invention selects NO 2 oxidation system, hypochlorite oxidation system, TEMPO oxidation system, HNO 3 /H 3 PO 4 ⁇ NaNO 2 oxidation system for C 6 selection of cellulose Oxidation improves the oxidation efficiency, has fewer side reactions, and can effectively control the degree of oxidation; the present invention effectively improves the coagulation effect of the hemostatic material by complexing coagulation factors, and significantly shortens the hemostasis time compared with the positive control; the present invention uses static electricity The method of spraying controls the particle size of the hemostatic material, so that the hemostatic material has a smaller and uniform particle size; through
  • the cellulose includes natural fiber materials such as wood fiber, cotton fiber, bacterial cellulose, and regenerated cellulose.
  • the present invention provides a new type of degradable hemostatic material (microspheres), the hemostatic material is oxidized cellulose loaded with clotting factors, wherein the carboxyl content of the oxidized cellulose 20-30% (mass percentage), degree of polymerization of 200-400, particle size of 50-150 ⁇ m (preferably 90-120 ⁇ m), said coagulation factor accounts for 0.01-2 of said oxidized cellulose mass percentage %.
  • the coagulation factor is a soluble calcium salt, preferably calcium chloride.
  • the present invention provides a method for preparing the hemostatic material, including the following steps:
  • the pre-oxidation process the cellulose is crushed and swelled with sodium hydroxide solution in sequence;
  • Oxidation process selectively oxidize the hydroxyl group at the C 6 position of the cellulose molecule, and introduce a carboxyl group at the C 6 position to obtain oxidized cellulose; and assist ultrasonic treatment in the oxidation process;
  • Electrostatic spraying Dissolve the mixture obtained in step C in an alkali system, stir and filter, collect the filtrate, spray the filtrate into the coagulation liquid by means of electrostatic spray, collect the precipitate, and wash with water and/or ethanol until it is neutral;
  • step D The product obtained in step D is mechanically crushed or ground, dried, sieved, and sterilized to obtain it.
  • the coagulation liquid used in step D is ethanol, sulfuric acid solution or hydrochloric acid solution.
  • step A includes: adding a certain amount of cellulose to a high-pressure homogenizer (or dynamic high-pressure homogenizer), refiner, decomposer, pulverizer (low temperature pulverizer) or ultrasonic machine (high-intensity ultrasonic In the machine), pulverize until the fiber length is less than 1mm; filter the slurry to obtain the precipitate. After washing with water (wash the precipitate with deionized water several times), place it in 1-20% sodium hydroxide solution to moisten The swelling treatment is 20-120min, and the swollen cellulose is washed with water and/or ethanol to neutrality.
  • a high-pressure homogenizer or dynamic high-pressure homogenizer
  • refiner refiner
  • decomposer refiner
  • pulverizer low temperature pulverizer
  • ultrasonic machine high-intensity ultrasonic In the machine
  • step B includes: adding cellulose to the oxidation system for oxidation reaction, the oxidation reaction and ultrasonic treatment are performed simultaneously; after the reaction, the precipitate is collected by filtration or centrifugation, and washed with water and/or ethanol to neutrality to obtain oxidation Cellulose.
  • the oxidation system used in the present invention can be a NO 2 oxidation system, a hypochlorite oxidation system, a TEMPO oxidation system or a HNO 3 /H 3 PO 4 -NaNO 2 oxidation system.
  • the NO 2 oxidation system uses CCl 4 as a solvent and a prepared NO 2 oxidation solution with a concentration of 10-30%.
  • the hypochlorite oxidation system can be a 20-200% v/v NaClO solution.
  • the TEMPO oxidation system may be a TEMPO-NaClO-NaBr oxidation system.
  • the HNO 3 /H 3 PO 4 -NaNO 2 oxidation system may be a mixture of HNO 3 and H 3 PO 4 containing NaNO 2 .
  • the step B includes: adding 1-20 g of cellulose to 100-200 mL of the NO 2 oxidation system, the reaction temperature is 5-30°C, and the reaction time It is 8-100h; the oxidation reaction and the ultrasonic treatment are carried out simultaneously; after the reaction, the precipitate is collected by filtration or centrifugation, and the precipitate is washed with CCl 4 and ethanol successively to obtain oxidized cellulose.
  • the step B includes: suspending 5-20 g of cellulose in 100-200 mL of water, adjusting the pH of the suspension to 9-11, at 40-60°C Under the conditions, add 20 ⁇ 200mL of 20 ⁇ 100%v/v NaClO solution, and react for 2 ⁇ 10h; the oxidation reaction and ultrasonic treatment are carried out simultaneously; after the reaction, the precipitate is collected by filtration or centrifugation, and washed with water and/or ethanol to Neutral to obtain oxidized cellulose.
  • the step B includes: suspending 10-100 g of cellulose in 100-1000 mL of Na 2 CO 3 /NaHCO 3 buffer with a pH of 9-11, and stirring for 2-10 minutes Then, add TEMPO to make the final concentration of TEMPO 0.1 ⁇ 1.0mg/mL, then add NaBr to make the final concentration of NaBr 1 ⁇ 10mg/mL; control the temperature of the reaction system to 20 ⁇ 30°C, wait until TEMPO and NaBr are completely dissolved Afterwards, adjust the pH of the reaction system to 9-11, and then add saturated NaClO solution step by step, adding once every 30-60 min.
  • the volume of NaClO solution added is 20-100% of the total volume of the solution, and the reaction is 3-10h; oxidation reaction and The ultrasonic treatment is performed simultaneously; after the reaction, the precipitate is collected by filtration or centrifugation, and washed with water and/or ethanol to neutrality to obtain oxidized cellulose.
  • the molar ratio of Na 2 CO 3 and NaHCO 3 in the Na 2 CO 3 /NaHCO 3 buffer is 1:3 to 3:1 (preferably 1:2).
  • the step B includes: preparing HNO 3 and H 3 PO 4 analytical pure in a volume ratio of 0.5:1 to 3:1 To form a mixture, add cellulose to the above mixture to make the concentration 0.01 ⁇ 0.1g/mL, then add NaNO 2 powder to make the final concentration of NaNO 2 10 ⁇ 50mg/mL; at 20 ⁇ 50°C
  • the lower reaction is 8 to 48 hours; the oxidation reaction and the ultrasonic treatment are carried out simultaneously; after the reaction, the precipitate is collected by filtration or centrifugation, and washed with water and/or ethanol to neutrality to obtain oxidized cellulose.
  • the ultrasound can be a sink ultrasound or a probe ultrasound.
  • the ultrasonic conditions in step B are: ultrasonic frequency 20-40kHz, power 100-1000W.
  • step D the mixture obtained in step C is dissolved in an alkali system at -20 to -5°C.
  • the alkaline system is a 5-20% sodium hydroxide solution, a 5-20% sodium hydroxide solution containing 5-30% urea, or a 5-20% lithium hydroxide solution containing 5-30% urea.
  • the voltage of the electrostatic spray is 5-20 kV, and the feed rate is 20-400 ⁇ L/min.
  • the drying method adopted in step E is spray drying, supercritical drying, vacuum drying, blast drying or freeze drying.
  • the preparation method of the regenerated cellulose is as follows: natural fiber (such as wood fiber, cotton fiber, bacterial cellulose and other fibrous materials) is used as raw material, and the cellulose is made through alkalization, aging, sulfonation and other processes. .
  • natural fiber such as wood fiber, cotton fiber, bacterial cellulose and other fibrous materials
  • the present invention has at least the following advantages and beneficial effects:
  • the invention provides a novel degradable hemostatic material and a preparation method thereof, which have the advantages of continuous process, high efficiency, low cost, etc., and prepare an oxidized cellulose hemostatic material with uniform and controllable microspheres, high oxidation degree and strong hemostatic ability.
  • the new hemostatic material is suitable for hemorrhage, irregular parts and organ bleeding, and the material has good degradability and quick hemostasis.
  • the present invention is specifically optimized for the existing oxidized cellulose hemostatic material preparation process as follows:
  • the pre-oxidation process the fiber is crushed by a high-pressure homogenizer, refiner/decomposer, low-temperature pulverization, high-intensity ultrasound, and dynamic high-pressure homogenizer to activate cellulose, reduce crystallinity, and improve hydroxyl The degree is good for oxidation.
  • Oxidation process selective oxidation of cellulose C 6 position.
  • the oxidation system includes: NO 2 oxidation system, hypochlorite oxidation system, TEMPO oxidation system, HNO 3 /H 3 PO 4 ⁇ NaNO 2 oxidation system.
  • the simultaneous ultrasonic treatment and oxidation process can effectively increase the carboxyl content in cellulose.
  • Electrostatic spraying spraying the reaction liquid into the coagulation liquid by means of electrostatic spraying, so that the hemostatic material has a small and uniform particle size.
  • the invention provides a hemostatic product meeting the clinical requirements of a degradable hemostatic material and a preparation method thereof.
  • the specific plan is as follows:
  • Pre-treatment Add a certain amount of cellulose to one of the high-pressure homogenizer, refiner/decomposer, low-temperature pulverizer, high-intensity ultrasonic machine, and dynamic high-pressure homogenizer, and perform repeated pulverization operations until The fiber length is significantly shortened.
  • the slurry is filtered to obtain the precipitate. After washing the precipitate with deionized water for many times, it is swelled with a certain concentration of sodium hydroxide solution, and the swollen cellulose is filtered and washed with ethanol of different solubility to Standby after neutral.
  • CCl 4 as the solvent to prepare NO 2 oxidation solution; add the prepared cellulose to the above oxidation solution, the reaction temperature is 5-30 °C, the reaction time is 8-100h, the oxidation process and the ultrasonic process are carried out simultaneously. Including one of sink type or probe type ultrasound. After the reaction, the oxide is washed with CCl 4 several times, and then the product is washed with a certain concentration of ethanol aqueous solution until the reagent residue meets the requirements, and the lower layer is separated for use. .
  • the oxidation process is synchronized with the ultrasonic process. Including one of water tank type or probe type ultrasound. After the reaction, the product is repeatedly centrifuged and washed with deionized water or ethanol aqueous solution to separate the lower layer of precipitation for use.
  • HNO 3 and H 3 PO 4 analytical grades Take HNO 3 and H 3 PO 4 analytical grades and configure them into a mixed solution in a volume ratio of 0.5:1 to 3:1.
  • Electrostatic spray treatment dissolve the product obtained in step 3 at a low temperature in an alkali system, the low temperature is -20 ⁇ -5°C, the alkali system includes: sodium hydroxide system, sodium hydroxide/urea system, lithium hydroxide/urea system, hydrogen The solubility of sodium oxide or lithium hydroxide is 5-20% (m/v), and the concentration of urea is 5-30%. After stirring, it is filtered and the filtrate is sprayed into the coagulation liquid by electrostatic spraying.
  • the voltage in the medium is 5-20kV
  • the feed rate is 20-400 ⁇ L/min
  • the coagulation liquid is one of sulfuric acid aqueous solution, hydrochloric acid aqueous solution, and ethanol solution, and then the collected microspheres are washed repeatedly with ethanol to neutrality. spare.
  • Molding The product of the above process is crushed by one or more of ball mill, roller press, and hammer mill, and the processed product is dried.
  • the drying methods include spray drying, supercritical drying, vacuum drying, One of blast drying and freeze drying, and then the dried powder is sieved and sterilized to obtain oxidized cellulose hemostatic powder.
  • the mass percentage of ⁇ -cellulose accounts for more than 99% of the total fiber.
  • CCl 4 as the solvent, prepare 1000ml of oxidizing solution containing 10% NO 2 ; add cellulose to the above oxidizing solution, the reaction temperature is 25°C, the reaction time is 20h, the oxidation process and the water tank ultrasonic process are performed simultaneously. Ultrasonic frequency 28kHz, power 500W, after the reaction, the reactant is filtered, the oxide is washed 3 times with 500ml CCl 4 , then the product is washed 4 times with 500ml of anhydrous ethanol aqueous solution, and the precipitate is filtered off with suction for use;
  • step 4 Dissolve the product of step 3 in deionized water containing 10g of sodium hydroxide, 200g of urea and 2000ml at a temperature of -10°C. After the cellulose is fully dissolved, filter it, and spray the filtrate into the coagulation liquid by electrostatic spraying. , The voltage in the electrostatic spraying is 40kV, the feeding rate is 120 ⁇ L/min, the coagulation liquid is absolute ethanol, and then the collected microspheres are washed repeatedly with ethanol to neutrality, and the precipitate is filtered off by suction;
  • step 4 Dissolve the product of step 3 in deionized water containing 12g sodium hydroxide, 180g urea and 2000ml at a temperature of -12°C. After the cellulose is fully dissolved, filter it, and spray the filtrate into the coagulation liquid by electrostatic spraying. , The voltage in the electrostatic spraying is 20kV, the feeding rate is 150 ⁇ L/min, the coagulation liquid is absolute ethanol, and then the collected microspheres are washed repeatedly with ethanol until they are neutral for use;
  • the product is subjected to 6 hammer milling treatments, and then the product is freeze-dried.
  • the dried product is passed through a 100-mesh sieve (to remove particles larger than 150 ⁇ m) and a 300-mesh sieve (to remove particles smaller than 50 ⁇ m), 25kGy Sterilized by irradiation to obtain oxidized cellulose hemostatic powder (microspheres).
  • step 4 Dissolve the product of step 3 in deionized water containing 15g of sodium hydroxide, 250g of urea and 2000ml at a temperature of -15°C. After the cellulose is fully dissolved, filter, and spray the filtrate into the coagulation liquid by electrostatic spraying. , The voltage in the electrostatic spraying is 40kV, the feed rate is 100 ⁇ L/min, the coagulation liquid is absolute ethanol, and then the collected microspheres are washed repeatedly with ethanol until they are neutral for use;
  • the product is subjected to 4 ball milling treatments with a ball mill, and then spray-dried.
  • the dried product is passed through a 100-mesh sieve (to remove particles larger than 150 ⁇ m) and a 300-mesh sieve (to remove particles smaller than 50 ⁇ m), 25kGy radiation According to sterilization, oxidized cellulose hemostatic powder (microspheres) is obtained.
  • step 4 Dissolve the product of step 3 in deionized water containing 10g of sodium hydroxide, 20g of urea and 200ml of deionized water at a temperature of -12°C. After the cellulose is fully dissolved, filter, and spray the filtrate into the coagulation liquid by electrostatic spraying. In the electrostatic spraying, the voltage is 20kV, the feeding rate is 120 ⁇ L/min, and the coagulation liquid is absolute ethanol. Then the collected microspheres are washed repeatedly with ethanol until they are neutral for use;
  • the product is subjected to 5 times of ball milling treatment with a ball mill, and then vacuum dried.
  • the dried product is passed through a 100-mesh sieve (to remove particles larger than 150 ⁇ m) and a 300-mesh sieve (to remove particles smaller than 50 ⁇ m), 25kGy radiation According to sterilization, oxidized cellulose hemostatic powder (microspheres) is obtained.
  • the degree of polymerization was tested on the hemostatic materials prepared in the above embodiments, and the test method is as follows:
  • the test standard is ASTM (Part15D1795-62).
  • the specific steps are as follows: add 0.4 g of sample into an Erlenmeyer flask containing 25 mL of distilled water, and after the sample swells, add 25 mL of 1M copper ethylene diamine aqueous solution to it, seal the mouth of the Erlenmeyer flask, and stir for 30 min. After the sample is completely dissolved, the concentration of the sample is 0.8g/100mL. Then accurately transfer 10 mL of the above sample solution to a viscometer with a capillary inner diameter of 0.62 mm, and place it in a digital thermostat water bath, control the temperature at 25 ⁇ 0.5°C, and keep it constant for 15 minutes. Record the time T 1 for the sample solution to flow through the scale at both ends of the capillary. Also record the time T 0 for the solvent to flow through two graduations. Calculate the relative viscosity of the solution according to formula (1).
  • T 1 the time for the sample solution to flow through the scale at both ends of the capillary
  • T 0 the time for the solvent to flow through the scale at both ends of the capillary.
  • the intrinsic viscosity table By looking up the intrinsic viscosity table, find the corresponding [ ⁇ ]C value according to ⁇ r , where C is 0.008 g/mL. That is, the intrinsic viscosity can be obtained, and then the degree of polymerization DP can be calculated by formula (2).
  • Example 2 Example 3
  • Example 2 Example 3
  • Example 4 Particle size ⁇ m 50 ⁇ 150 50 ⁇ 150 50 ⁇ 150 50 ⁇ 150 Percentage content (%) 76 83 98 91
  • Percentage refers to the mass percentage of hemostatic materials with a particle size of 50-150 ⁇ m in all hemostatic materials.
  • the carboxyl group content of the hemostatic materials prepared in the above embodiments is determined, and the specific test process is as follows:
  • the acid-base neutralization titration method was used to determine the carboxyl group content in the hemostatic material.
  • V 1 the volume of NaOH consumed by the sample solution to be tested, L;
  • V 2 The volume of NaOH consumed by the blank solution, L;
  • Example Example 1 Example 2 Example 3 Example 4 Carboxyl content (%) 25 26 28 twenty two Control 1 Control 1-1 Control 1-2 Control 1-3 Control 1-4 Carboxyl content (%) 13 10 14 11 Control 2 Control 2-1 Control 2-2 Control 2-3 Control 2-4 Carboxyl content (%) 20 18 twenty three 15
  • control group 1 is the sample without pretreatment and direct oxidation in the corresponding embodiment
  • control group 2 is the sample without ultrasonic treatment in the corresponding embodiment.
  • a blood coagulation test was performed on the hemostatic materials prepared in the above embodiments, and the specific test process is as follows:
  • the whole blood coagulation index BCI (Blood Clotting Index) was used to evaluate its coagulability in vitro.
  • Example 1 Example 2
  • Example 3 Example 4 Control BCI value 0.22 0.19 0.17 0.21 0.25
  • the hemostatic time test is performed on the hemostatic materials prepared in the above embodiments, and the specific test process is as follows:
  • the animals needed for the experiment are 150-250g SD rats, which are raised in the experimental environment for one week before the experiment. 10 SD adult rats, half male and half male, were divided into 5 groups, including the Johnson & Johnson control group. The experimental groups were Example 1, Example 2, Example 3, and Example 4.
  • the experimental groups were Example 1, Example 2, Example 3, and Example 4.
  • 3% pentobarbital sodium anesthetic was injected intraperitoneally to anesthetize it. After successful anesthesia, his limbs were tied to the operating table. Cut it with scissors at a distance of 2 cm from the end of the tail. After the wound bleeds freely for 20 seconds, apply hemostatic powder immediately and cover the entire wound.
  • Example 2 Example 3
  • Example 4 Control Hemostasis time (s) 79 76 68 73 85
  • the dialysis bag is boiled in pure water for 10 minutes and then taken out. 25 dialysis bags are put into the corresponding number of hemostatic materials, and 7mL of newly prepared 3% hydrogen peroxide is added to immerse, after removing air bubbles, clamp the dialysis bag. Add 7 mL of freshly prepared 3% hydrogen peroxide solution to the remaining 5 dialysis bags as a blank control.
  • the above materials were placed in 30 250mL beakers according to their numbers, and 120mL of newly prepared 3% hydrogen peroxide solution was added. The mouth of the beaker was sealed and placed in a shaking incubator. The temperature was set to 37°C, the speed was 150rpm, and the temperature was kept for 14 days. At the same time, replace 100 mL of the newly prepared 3% hydrogen peroxide solution outside the dialysis bag every 24 hours.
  • W weight loss percentage, %
  • W t The dry weight of the hemostatic material at the specified time point, g.
  • Example 1 Example 2
  • Example 3 Example 4 Control example 2d 62 54 70 66 43 4d 80 83 92 85 75 6d 98 94 99 96 84 8d 97 97 98 98 96 14d 99 98 99 99 98
  • the hemostatic material provided by the present invention has a relatively short in vitro degradation time, which can effectively reduce the possibility of a series of immune reactions such as rejection and wrapping.
  • the cytotoxicity test was carried out on the hemostatic materials and instant yarns prepared in the above embodiments.
  • the specific test process is as follows:
  • Preparation of the extract Under aseptic conditions, accurately weigh the hemostatic material and instant yarn of the same quality of the example, add 50.00mg/mL serum and L-glutamine-free MEM medium at 37°C, Leaching in a constant temperature shaker at 100rpm for 24h. Centrifuge, take the upper layer liquid as the 100% leaching solution, and dilute sequentially to obtain 50%, 25%, and 12.5% material leaching solution. Before adding samples, each extract is supplemented with fetal bovine serum at 10% and L-glutamine at 1% to obtain.
  • the extract is tested in accordance with the MTT method specified in GB/T 16886.5-2017.
  • the test is based on the relative cell proliferation rate as a criterion.
  • the specific test results are shown in Table 7.
  • Example 2 Example 3
  • Example 4 Control group Original solution (100%) ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70% 50% extract ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70% 25% extract ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70% 12.5% extract ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70% ⁇ 70%
  • the present invention selects mechanical method to pretreat the cellulose raw material, which improves the accessibility of the cellulose active group hydroxyl group, and then swells it with alkali to further facilitate oxidation.
  • the cellulose is oxidized in different ways Treatment, the oxidation process assisted ultrasonic treatment is beneficial to increase the carboxyl content of the oxidation product, and then the coagulant is added to the oxidation product, which is conducive to the coagulation of the hemostatic material, and then the product is shaped by different physical methods.
  • the hemostatic material prepared by the above method It has the advantages of fast hemostasis, strong adhesion, good degradability, wide application range, simple operation and low cost.
  • the invention provides a novel degradable hemostatic material and a preparation method thereof.
  • the hemostatic material of the present invention is oxidized cellulose loaded with clotting factors, wherein the carboxyl content of the oxidized cellulose is 20-30%, the degree of polymerization is 200-400, the particle size is 50-150 ⁇ m, and the clotting factor accounts for the oxidized cellulose. 0.01 to 2% of the mass percentage.
  • the preparation method of the present invention includes the steps of oxidation pretreatment process, oxidation process, complexation of blood coagulation factors, electrostatic spraying and molding.
  • the invention has the advantages of continuous process, high efficiency, low cost, etc., and prepares an oxidized cellulose hemostatic material with uniform and controllable microspheres, high oxidation degree and strong hemostatic ability.
  • the new hemostatic material is suitable for massive hemorrhage, irregular parts and organ hemorrhage.
  • the material has good degradability, fast hemostasis, and has good economic value and application prospects.

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Abstract

一种新型可降解的止血材料及其制备方法。止血材料为负载有凝血因子的氧化再生纤维素，其中氧化再生纤维素的羧基含量为20％～30％，聚合度为200～400，粒径大小为50～150μm，凝血因子占氧化再生纤维素质量百分数的0.1～2％。制备方法包括氧化前处理过程、氧化过程、络合凝血因子、静电喷射及成型等步骤。该方法具有工艺连续、高效、成本低等优点，制备出微球粒均一可控、氧化程度高、止血能力强的氧化再生纤维素止血材料。该止血材料适用于大出血、不规则部位及脏器出血。

Description

新型可降解的止血材料及其制备方法
交叉引用
本申请要求2019年8月9日提交的专利名称为“新型可降解的止血材料及其制备方法”的第201910735681.9号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于医用敷料领域，具体地说，涉及一种新型可降解的止血材料及其制备方法。
背景技术
纤维素作为世界上储量最丰富的天然高分子材料，具有原料来源广泛、可再生性、生物可降解性、生物相容性等特点，被认为是可持续材料的一种重要资源。但随着科学技术的进步和人类生活品质的提高，纤维素自身的特性远不能满足人类的需求。氧化纤维素是纤维素经氧化处理成为纤维素酸的薄纱状或面布状的纤维材料，外观似棉纱，质地柔软而菲薄，具有良好的生物相容性、可降解性和安全性等特点。临床上氧化纤维素已成功应用于神经外科、耳鼻喉科、肝胆外科等手术止血中，术中操作简便、易行。目前临床上广泛使用的是速即纱(Surgicel)，是一种氧化再生纤维编织纱块，属于羧甲基纤维素类止血材料，它是一种水溶性全吸收中性纤维素，具备很好的水溶性，对水和盐水有极强的亲和力，当它溶于水后形成一定黏度的水性胶体，从而膨胀填补创面空隙或压迫血管达到止血目的。但由于止血纱形貌的特性，对于纱布无法填塞的部位，止血纱不能发挥作用，限制了其使用。目前针对氧化纤维素止血粉开展了研究，由此拓宽了氧化纤维素止血材料的临床应用范围。但现有工艺制备的氧化纤维素止血材料，均具有氧化程度低、成本高、粒径较大且不均一等缺点，亟待改进。
发明内容
本发明的目的是提供一种新型可降解的止血材料及其制备方法。
本发明构思如下：本发明通过对纤维素进行氧化前破碎处理、氧化前润胀处理以及氧化过程进行超声作用，提高了纤维素的氧化程度，使得氧化纤维素的羧基含量均可达到22％以上，符合氧化纤维素止血材料的羧基含量要求；本发明选用NO 2氧化体系、次氯酸盐氧化体系、TEMPO氧化体系、HNO 3/H 3PO 4～NaNO 2氧化体系对纤维素进行C 6选择性氧化，提高了氧化效率，副反应少，可有效控制氧化程度；本发明通过络合凝血因子，有效提高了止血材料的凝血效果，与阳性对照比，明显缩短了止血时间；本发明通过静电喷射的方式控制止血材料的粒径，使得止血材料具有较小且均一的粒径；通过不同的干燥方式及物理研磨工艺，进一步控制产品的粒径，使得产品在临床应用时喷洒均匀，能够有效、快速止血。
在本发明中，所述纤维素包括：木纤维、棉纤维、细菌纤维素等天然纤维材料以及再生纤维素。
为了实现本发明目的，第一方面，本发明提供一种新型可降解的止血材料(微球粒)，所述止血材料为负载有凝血因子的氧化纤维素，其中所述氧化纤维素的羧基含量为20～30％(质量百分含量)，聚合度为200～400，粒径大小为50～150μm(优选为90～120μm)，所述凝血因子占所述氧化纤维素质量百分数的0.01～2％。
所述凝血因子为可溶性钙盐，优选氯化钙。
第二方面，本发明提供所述止血材料的制备方法，包括以下步骤：
A、氧化前处理过程：将纤维素依次经过破碎处理和氢氧化钠溶液润胀处理；
B、氧化过程：对纤维素分子的C 6位羟基进行选择性氧化，在C 6位上引入羧基，得到氧化纤维素；并在氧化过程中辅以超声处理；
C、络合凝血因子：将氧化纤维素与凝血因子按不同超声时间进行络合；
D、静电喷射：将步骤C所得混合物溶于碱体系中，搅拌过滤，收集滤液，将滤液以静电喷射的方式喷射于凝固液中，收集沉淀，用水和/或乙醇 洗涤至中性；
E、成型：将步骤D所得产物进行机械破碎或研磨，干燥后，过筛，灭菌，即得。
其中，步骤D所用凝固液为乙醇、硫酸溶液或盐酸溶液。
前述的方法，步骤A包括：将一定量的纤维素加入到高压均质机(或动态高压均质机)、磨浆机、疏解机、粉碎机(低温粉碎机)或超声机(高强度超声机)中，进行粉碎处理，至纤维长度小于1mm；将浆液过滤，得沉淀物，水洗后(用去离子水将沉淀物洗涤多次)，放入1～20％的氢氧化钠溶液中润胀处理20～120min，将润胀后的纤维素用水和/或乙醇洗涤至中性。
前述的方法，步骤B包括：将纤维素加入到氧化体系中进行氧化反应，氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素。
本发明所用氧化体系可以是NO 2氧化体系、次氯酸盐氧化体系、TEMPO氧化体系或HNO 3/H 3PO 4～NaNO 2氧化体系。
所述NO 2氧化体系是以CCl 4为溶剂，配制的浓度为10～30％的NO 2氧化液。
所述次氯酸盐氧化体系可以是20～200％v/v的NaClO溶液。
所述TEMPO氧化体系可以是TEMPO-NaClO-NaBr氧化体系。
所述HNO 3/H 3PO 4～NaNO 2氧化体系可以是含NaNO 2的HNO 3和H 3PO 4混合液。
进一步地，当所述氧化体系为NO 2氧化体系时，所述步骤B包括：将1～20g纤维素加入到100～200mL所述NO 2氧化体系中，反应温度为5～30℃，反应时间为8～100h；氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，依次用CCl 4、乙醇洗涤沉淀，得到氧化纤维素。
进一步地，当所述氧化体系为次氯酸盐氧化体系时，所述步骤B包括：将5～20g纤维素悬浮于100～200mL水中，调悬浮液pH至9～11，在40～60℃条件下，向其中加入20～100％v/v的NaClO溶液20～200mL，反应2～10h；氧化 反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素。
进一步地，当所述氧化体系为TEMPO氧化体系时，所述步骤B包括：将10～100g纤维素悬浮于pH9～11的Na 2CO 3/NaHCO 3缓冲液100～1000mL中，搅拌2～10min后，加入TEMPO，使TEMPO的终浓度为0.1～1.0mg/mL，再加入NaBr，使NaBr的终浓度为1～10mg/mL；控制反应体系温度为20～30℃，待TEMPO和NaBr完全溶解后，调反应体系pH至9～11，然后分步加入饱和NaClO溶液，每隔30～60min加一次，NaClO溶液加入的体积为溶液总体积的20～100％，反应3～10h；氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素。其中，所述Na 2CO 3/NaHCO 3缓冲液中Na 2CO 3和NaHCO 3的摩尔比为1:3～3:1(优选1:2)。
进一步地，当所述氧化体系为HNO 3/H 3PO 4～NaNO 2氧化体系时，所述步骤B包括：将HNO 3和H 3PO 4分析纯按体积比0.5:1～3:1配制成混合液，将纤维素加入到上述混合液中，使其浓度为0.01～0.1g/mL，再加入NaNO 2粉末，使NaNO 2的终浓度为10～50mg/mL；在20～50℃条件下反应8～48h；氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素。
前述的方法，超声可以是水槽式超声或探头式超声。步骤B中超声条件为：超声频率20～40kHz，功率100～1000W。
前述的方法，步骤D中，将步骤C所得混合物在-20～-5℃条件下溶于碱体系中。
优选地，所述碱体系为5～20％氢氧化钠溶液、含5～30％尿素的5～20％氢氧化钠溶液或含5～30％尿素的5～20％氢氧化锂溶液。
优选地，静电喷射的电压为5～20kV，供料速率为20～400μL/min。
优选地，步骤E中采用的干燥方式为喷雾干燥、超临界干燥、真空干燥、鼓风干燥或冷冻干燥。
本发明中，所述再生纤维素的制备方法如下：以天然纤维(如木纤维、棉纤维、细菌纤维素等纤维材料)为原料，经碱化、老化、磺化等工序制成的纤维素。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明提供一种新型可降解的止血材料及其制备方法，具有工艺连续、高效、成本低等优点，制备出微球粒均一可控、氧化程度高、止血能力强的氧化纤维素止血材料。该新型止血材料适用于大出血、不规则部位及脏器出血，该材料可降解性好，止血快。
本发明针对现有氧化纤维素止血材料制备工艺具体优化如下：
(一)氧化前处理过程：通过高压均质机、磨浆机/疏解机、低温粉碎、高强度超声、动态高压均质机对纤维进行破碎，活化纤维素，降低结晶度，提高羟基的可及度，利于氧化。
(二)氧化过程：对纤维素C 6位进行选择性氧化。氧化体系包括：NO 2氧化体系、次氯酸盐氧化体系、TEMPO氧化体系、HNO 3/H 3PO 4～NaNO 2氧化体系。超声波处理和氧化过程同步进行，可有效提高纤维素中的羧基含量。
(三)络合凝血因子：止血材料中络合钙离子，提高止血材料的凝血效果。
(四)静电喷射：将反应液以静电喷射的方式喷射于凝固液中，使得止血材料具有较小且均一的粒径。
(五)成型：将氧化产物通过喷雾干燥、或超临界干燥、真空干燥、鼓风干燥、冷冻干燥等方法进行干燥后，再用球磨机、压辊机、锤磨机进行成型处理，进一步控制产品的粒径，使得产品在临床应用时喷洒均匀，能够有效、快速止血。
具体实施方式
本发明提供一种具有满足可降解止血材料临床要求的止血产品及其制备方法。具体方案如下：
1、前处理：将一定量的纤维素加入到高压均质机、磨浆机/疏解机、低温粉碎机、高强度超声机、动态高压均质机中的一种，进行反复粉碎操作，至纤维长度明显缩短为止。将浆液过滤，得沉淀物，用去离子水将沉淀物洗涤多次后，用一定浓度的氢氧化钠溶液润胀，将润胀后的纤维素用不同溶度的乙醇进行滤洗，洗至中性后备用。
2、氧化：
(1)NO 2氧化纤维素
以CCl 4为溶剂，配制NO 2氧化液；将制备好的纤维素按加入到上述氧化液中，反应温度为5～30℃，反应时间为8～100h，氧化过程和超声过程同步进行，超声包括水槽式或探头式超声中的一种，反应结束后，氧化物先用CCl 4洗涤多次，再用一定浓度的乙醇水溶液洗涤产物，至试剂残留量符合要求为止，分离出下层沉淀，备用。
(2)次氯酸盐氧化体系
将制备好的纤维素按悬浮于去离子水中，用HCl和NaOH调节体系的pH＝9～11，温度设定为40～60℃，然后加入20～200％(v/v)的次氯酸钠溶液，反应2～10h，氧化过程和超声过程同步，超声包括水槽式或探头式超声中的一种，反应结束后，用乙醇水溶液洗涤抽滤多次，再用蒸馏水洗涤至中性，最后再用无水乙醇洗涤，分离出下层沉淀，备用。
(3)TEMPO氧化体系
将制备好的纤维素悬浮于pH＝9～11的Na 2CO 3/NaHCO 3缓冲液(Na 2CO 3和NaHCO 3的摩尔比为1:2)中，高速搅拌2～10min。加入TEMPO，使得TEMPO在溶液中的溶度为0.1～1.0mg/mL，再加入NaBr，溶度为1～10mg/mL，控制温度为20～30℃，待TEMPO和NaBr完全溶解后，用HCl溶液调pH＝9～11，然后分次加入饱和NaClO溶液，每隔30～60min加一次，NaClO加入的体积为溶液体积的20～100％，反应3～10h，氧化过程和超声过程同步，超声包括水槽式或探头式超声中的一种，反应结束后用去离子水或乙醇水溶液将产物反复离心洗涤，分离出下层沉淀，备用。
(4)HNO 3/H 3PO 4～NaNO 2体系
取HNO 3和H 3PO 4分析纯按体积比0.5:1～3:1的比例配置成混合液，将制备好的纤维素加入到混合液中，使得纤维素在混合液中的浓度为0.01～0.1g/mL，再加入NaNO 2粉末，NaNO 2在溶液中的浓度为10～50mg/mL，迅速搅拌均匀，反应温度为20～50℃，反应时间为8～48h，氧化过程和超声过程同步，超声包括水槽式或探头式超声中的一种，反应结束后用去离子水或乙醇水溶液将产物反复离心洗涤，分离出下层沉淀，备用。
3、络合凝血因子：将氧化物添加到过量的饱和氯化钙溶液中，超声10～60min，过滤得沉淀物。
4、静电喷射处理：将步骤3所得产物用碱体系低温溶解，低温为-20～-5℃，碱体系包括：氢氧化钠体系、氢氧化钠/尿素体系、氢氧化锂/尿素体系，氢氧化钠或氢氧化锂的溶度为5～20％(m/v)，尿素的浓度为5～30％，搅拌均匀后进行过滤，将滤液以静电喷射的方法喷射于凝固液中，静电喷射中的电压为5～20kV，供料速率为20～400μL/min，凝固液为硫酸水溶液、盐酸水溶液、乙醇溶液中的一种，然后用乙醇将收集到的微球进行反复洗涤至中性，备用。
5、成型：将上述过程的产物用球磨机、压辊机、锤磨机中一种或多种进行粉碎处理，将处理后的产物进行干燥，干燥方式包括喷雾干燥、超临界干燥、真空干燥、鼓风干燥、冷冻干燥中的一种，然后将干燥后的粉末过筛，灭菌，得到氧化纤维素止血粉。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中使用的纤维素，其中α-纤维素的质量百分含量占总纤维的99％以上。
实施例1 新型可降解的止血材料的制备
1、取100g纤维素溶于500ml去离子水中，将混悬液加入到高压均质机 中，反复均质5次，将浆液过滤，得沉淀物，用去离子水将沉淀物洗涤5次后干燥，将干燥后的纤维素用500ml 5％的氢氧化钠润胀1h，将润胀后的纤维素用500ml 80％乙醇进行滤洗，洗至中性后备用；
2、以CCl 4为溶剂，配制1000ml含10％NO 2的氧化液；将纤维素加入到上述氧化液中，反应温度为25℃，反应时间为20h，氧化过程和水槽式超声过程同步进行，超声频率28kHz，功率500W，反应结束后，将反应物过滤，先用500ml CCl 4洗涤氧化物3次，再用500ml无水乙醇水溶液洗涤产物4次，抽滤得沉淀物备用；
3、将沉淀物添加到过量的饱和的氯化钙溶液中，超声10min，过滤得沉淀物；
4、将步骤3的产物溶于含10g氢氧化钠、200g尿素和2000ml的去离子水中，温度为-10℃，纤维素充分溶解后进行过滤，将滤液以静电喷射的方法喷射于凝固液中，静电喷射中的电压为40kV，供料速率为120μL/min，凝固液为无水乙醇，然后用乙醇将收集到的微球进行反复洗涤至中性，抽滤得沉淀物备用；
5、将上述产物用球磨机进行球磨处理，然后将产物进行真空干燥，将干燥产物分别过100目筛(去除粒径大于150μm的颗粒)和300目筛(去除粒径小于50μm的颗粒)，25kGy辐照灭菌，得氧化纤维素止血粉(微球粒)。
实施例2 新型可降解的止血材料的制备
1、将100g纤维素加入到低温粉碎机中，进行反复粉碎处理，粉碎次数6次。将粉碎干燥后的纤维素用8％的氢氧化钠润胀2h，将润胀后的纤维素用500ml无水乙醇进行滤洗，备用；
2、将纤维素悬浮于1000ml去离子水中，用HCl和NaOH调节体系的pH＝10，温度设定为40℃，然后加入1500ml饱和次氯酸钠溶液，反应3h，氧化过程和水槽式超声同步，超声频率40kHz，功率1000W，反应结束后，用50％乙醇洗涤抽滤3次，再用蒸馏水洗涤至中性，最后再无水乙醇洗涤2 次，抽滤得沉淀物备用；
3、将沉淀物添加到过量的饱和的氯化钙溶液中，超声20min，过滤得沉淀物；
4、将步骤3的产物溶于含12g氢氧化钠、180g尿素和2000ml的去离子水中，温度为-12℃，纤维素充分溶解后进行过滤，将滤液以静电喷射的方法喷射于凝固液中，静电喷射中的电压为20kV，供料速率为150μL/min，凝固液为无水乙醇，然后用乙醇将收集到的微球进行反复洗涤至中性，备用；
5、将产物进行6次锤磨处理，然后将产物进行冷冻干燥，将干燥产物分别过100目筛(去除粒径大于150μm的颗粒)和300目筛(去除粒径小于50μm的颗粒)，25kGy辐照灭菌，得氧化纤维素止血粉(微球粒)。
实施例3 新型可降解的止血材料的制备
1、将50g纤维素加入500ml的去离子中，将悬浮液加入到磨浆机中，进行反复磨浆处理，磨浆10次。将浆液过滤，得沉淀物，用500ml去离子水将沉淀物洗涤8次后干燥，将干燥后的纤维素用500ml 5％的氢氧化钠润胀2h，将润胀后的纤维素用80％乙醇进行滤洗，备用；
2、将纤维素悬浮于1000ml pH＝10的Na 2CO 3/NaHCO 3缓冲液(Na 2CO 3和NaHCO 3的摩尔比为1:2)中，高速搅拌5min。加0.3g TEMPO，再加入4gNaBr，控制温度为25℃，待TEMPO和NaBr完全溶解后，用0.5mol/L HCl溶液调pH＝9，然后每隔30min分4次加入40ml饱和NaClO溶液，反应5h，氧化过程和水槽超声过程同步，超声频率40kHz，功率1000W，反应结束后用500ml去离子水反复离心洗涤，分离出下层沉淀，抽滤沉淀物备用；
3、将沉淀物添加到过量的饱和的氯化钙溶液中，超声30min，过滤得沉淀物；
4、将步骤3的产物溶于含15g氢氧化钠、250g尿素和2000ml的去离子水中，温度为-15℃，纤维素充分溶解后进行过滤，将滤液以静电喷射的方法喷射于凝固液中，静电喷射中的电压为40kV，供料速率为100μL/min， 凝固液为无水乙醇，然后用乙醇将收集到的微球进行反复洗涤至中性，备用；
5、将产物用球磨机进行4次球磨处理，然后进行喷雾干燥，将干燥产物分别过100目筛(去除粒径大于150μm的颗粒)和300目筛(去除粒径小于50μm的颗粒)，25kGy辐照灭菌，得氧化纤维素止血粉(微球粒)。
实施例4 新型可降解的止血材料的制备
1、将50g纤维素加入到1000ml去离子水中，将悬浮液加入到动态高压均质机中，进行10次反复均质。将浆液过滤，得沉淀物，用去离子水将沉淀物洗涤5次后干燥，将干燥后的纤维素用500ml 1％的氢氧化钠润胀1h，将润胀后的纤维素用无水乙醇进行滤洗，备用；
2、取100ml HNO 3和50ml H 3PO 4配制成混合液，将纤维素加入到混合液中，再加入0.5g NaNO 2粉末，迅速搅拌均匀，反应温度为40℃，反应时间为24h，氧化过程和水槽式超声过程同步，超声频率20kHz，功率200W，反应结束后用去离子水将产物反复离心洗涤，分离出下层沉淀，抽滤得沉淀物备用；
3、将沉淀物添加到过量的饱和的氯化钙溶液中，超声10min，过滤得沉淀物；
4、将步骤3的产物溶于含10g用氢氧化钠、20g尿素和200ml的去离子水中，温度为-12℃，纤维素充分溶解后进行过滤，将滤液以静电喷射的方法喷射于凝固液中，静电喷射中的电压为20kV，供料速率为120μL/min，凝固液为无水乙醇，然后用乙醇将收集到的微球进行反复洗涤至中性，备用；
5、将产物用球磨机进行5次球磨处理，然后进行真空干燥，将干燥产物分别过100目筛(去除粒径大于150μm的颗粒)和300目筛(去除粒径小于50μm的颗粒)，25kGy辐照灭菌，得氧化纤维素止血粉(微球粒)。
实施例5 止血材料聚合度的测试
对上述各实施例制备的止血材料进行聚合度测试，测试方法如下：
测试标准为ASTM(Part15D1795-62)。具体步骤如下：在盛有25mL蒸馏水的锥形瓶中加入0.4g样品，待样品溶胀后，再向其中加入25mL1M的铜乙二胺水溶液，封住锥形瓶口，搅拌30min。样品全部溶解后，样品的浓度即为0.8g/100mL。然后准确移取10mL上述样品溶液至毛细管内径为0.62mm的粘度计中，并置于数显恒温水浴中，控制温度为25±0.5℃，恒温15min。记录样品溶液流经毛细管两端刻度所用的时间T 1。同样记录溶剂流经两刻度的时间T 0。根据公式(1)计算出溶液的相对粘度。
η r＝K·T 1/T 0      公式(1)
式中：η r——相对黏度；
K——仪器常数；
T 1——样品溶液流经毛细管两端刻度的时间；
T 0——溶剂流经毛细管两端刻度的时间。
通过查找特性粘度表，根据η r找到对应的[η]C值，其中C为0.008g/mL。即可以得到特性粘度，再利用公式(2)计算得到聚合度DP。
DP 0.9＝0.75[η]        公式(2)
式中：η——特性粘度；
DP——样品聚合度。
实施例1～4所得止血材料的聚合度测试结果见表1。
表1 止血材料聚合度
  实施例1 实施例2 实施例3 实施例4
聚合度 320 280 390 370
实施例6 止血材料粒径测试
对上述各实施例制备的止血材料进行粒径测试，测试设备为欧美克LS～909。结果见表2。
表2 止血材料粒径及百分含量
  实施例1 实施例2 实施例3 实施例4
粒径μm 50～150 50～150 50～150 50～150
百分含量(％) 76 83 98 91
注：百分含量是指粒径为50～150μm的止血材料占全部止血材料的质量百分数。
实施例7 止血材料羧基含量测定
对上述各实施例制备的止血材料进行羧基含量测定，具体测试过程如下：
采用酸碱中和滴定法测定止血材料中的羧基含量。在锥形瓶中加入50mL的2％醋酸钙溶液，将精确称量的0.5g止血材料浸入锥形瓶中，超声波振荡20～30min。然后以酚酞为指示剂，用0.1mol/L的NaOH溶液滴定，空白溶液校正，则止血材料的羧基含量可按公式(3)进行计算。
Figure PCTCN2019106172-appb-000001
式中：V 1-待测样品溶液消耗的NaOH体积，L；
V 2-空白溶液消耗的NaOH体积，L；
m-止血材料干燥后质量，g。
测定结果见表3。
表3 止血材料羧基含量
实施例 实施例1 实施例2 实施例3 实施例4
羧基含量(％) 25 26 28 22
对照1 对照1-1 对照1-2 对照1-3 对照1-4
羧基含量(％) 13 10 14 11
对照2 对照2-1 对照2-2 对照2-3 对照2-4
羧基含量(％) 20 18 23 15
注：对照组1为相应实施例未进行前处理，直接氧化的样品；对照组2 为相应实施例未进行超声处理。
实施例8 止血材料凝血测试
对上述各实施例制备的止血材料进行凝血测试，具体测试过程如下：
按照Shih M F的实验方法，采用全血凝血指数BCI(Blood Clotting Index)对其体外凝血性进行评价。首先，抽取新鲜兔血，加入3.8％的枸橼酸钠抗凝剂备用，将0.5g样品置于50ml离心管中并保证样品全部置于离心管底部。将离心管放入37℃恒温摇床孵育5min后，将200μL的新鲜抗凝兔血滴加到样品上，随即加入20μl 0.2M的CaCl 2溶液，然后在37℃恒温摇床孵育。10min后，缓缓地加入25ml去离子水于离心管中，37℃恒温摇床振摇5min，转速50rpm。然后取出部分溶液，用紫外分光光度计在波长545nm处测其吸光度。另取200μL新鲜抗凝兔血于离心管中，然后加入25ml去离子水稀释，在同样的波长下测其吸光度，并将其设定为参考值。BCI的具体计算方法见如下公式(4)：
BCI＝A 样品/A 空白×100％    公式(4)
凝血指数BCI值越小，则表示材料的凝血效果越好。具体测试结果见表4。
表4 止血材料BCI值
  实施例1 实施例2 实施例3 实施例4 对照
BCI值 0.22 0.19 0.17 0.21 0.25
注：对照选用强生
Figure PCTCN2019106172-appb-000002
Powder Absorbable Hemostat。
实施例9 止血材料止血时间测试
对上述各实施例制备的止血材料进行止血时间测试，具体测试过程如下：
实验所需用动物为150～250g SD大鼠，实验前先在实验环境下饲养一周。SD成年大鼠10只，雌雄各半，分为5组，包括强生对照组，实验组为实施例1、实施例2、实施例3和实施例4。首先按照30mg/kg的剂量，采用3％的戊巴比妥钠麻醉剂腹腔注射对其麻醉。麻醉成功后将其四肢绑缚在手 术台上。在距离尾部末端2cm处用剪刀一次性剪断，伤口自由出血20s后，立即施以止血粉，并且覆盖整个伤口。开始计时，随后用镊子夹住大鼠尾根部，1min后松开镊子观察出血情况，直至止血成功为止，停止计时。记录每组止血时间，取平均止血时间。具体测试结果见表5。
表5 止血材料止血时间
  实施例1 实施例2 实施例3 实施例4 对照
止血时间(s) 79 76 68 73 85
注：对照选用强生
Figure PCTCN2019106172-appb-000003
Powder Absorbable Hemostat。
实施例10 止血材料体外降解时间测试
对上述各实施例制备的止血材料进行体外降解时间的测试，具体测试过程如下：
各实施例和对照品(速即纱)均称取质量相同的止血材料5份，透析袋30片，分为1、2、3、4四个实验组、对照组和空白组。
透析袋经纯水煮沸10min后取出，将25只透析袋中放入对应编号的止血材料，并加7mL新配3％过氧化氢浸没，排除气泡后，夹紧透析袋。将其余5只透析袋加7mL新配3％过氧化氢溶液，作为空白对照。以上材料按编号分别置于30个250mL烧杯中，再加120mL新配3％过氧化氢溶液，将烧杯口密封并置于震荡培养箱，设置温度37℃、转速150rpm，保持14d。同时每隔24h透析袋外更换100mL新配3％过氧化氢溶液。
失重百分率的计算
在第2d、4d、6d、8d、14d分别取出各组，真空干燥至恒重，根据公式(5)计算失重百分率：
Figure PCTCN2019106172-appb-000004
W——失重百分率，％；
W 0——止血材料原始重量，g；
W t——指定时间点止血材料干燥后的重量，g。
具体测试结果见表6。
表6 止血材料体外降解失重百分率
失重百分率 实施例1 实施例2 实施例3 实施例4 对照例
2d 62 54 70 66 43
4d 80 83 92 85 75
6d 98 94 99 96 84
8d 97 97 98 98 96
14d 99 98 99 99 98
从表6可以看出，与对照例相比，本发明提供的止血材料体外降解时间相对较短，可有效减少排异、包裹等一系列免疫反应发生的可能性。
实施例11 止血材料的细胞毒性
对上述各实施例制备的止血材料和速即纱进行细胞毒性的测试，具体测试过程如下：
浸提液的制备：无菌条件下，精密称取同质量的实施例止血材料和速即纱，按50.00mg/mL加不含血清和L一谷氨酰胺的MEM培养液，于37℃、100rpm恒温振荡器中浸提24h。离心，取上层液作为100％浸提液，依次稀释得50％、25％、12.5％材料浸提液。加样前，各浸提液按10％补加胎牛血清、按1％补加L-谷氨酰胺，即得。
取浸提液按照GB/T 16886.5-2017中规定的MTT法进行试验。测试以细胞相对增殖率为评判标准，具体测试结果见表7。
表7 止血材料细胞毒性
实施倍数 实施例1 实施例2 实施例3 实施例4 对照组
原液(100％) <70％ <70％ <70％ <70％ <70％
50％浸提液 ≥70％ ≥70％ ≥70％ ≥70％ ＜70％
25％浸提液 ≥70％ ≥70％ ≥70％ ≥70％ ≥70％
12.5％浸提液 ≥70％ ≥70％ ≥70％ ≥70％ ≥70％
从表7可以看出，与对照例相比，本发明提供的止血材料细胞毒性明 显优于对照组，生物安全性更高。
本发明选用机械法对纤维素原材料进行预处理，提高了纤维素活性基团羟基的可及度，然后用碱对其进行润胀，进一步利于氧化，预处理后对纤维素进行不同方式的氧化处理，氧化过程辅助超声处理有利于提高氧化产物羧基含量，然后向氧化产物中添加凝血剂，有利于止血材料凝血，再通过不同的物理方法对产物进行成型处理，通过上述方法制备的止血材料，具有止血快、粘附性强、可降解性好、适用范围广、操作简单、成本低等优点。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种新型可降解的止血材料及其制备方法。本发明所述止血材料为负载有凝血因子的氧化纤维素，其中氧化纤维素的羧基含量为20～30％，聚合度为200～400，粒径大小为50～150μm，凝血因子占氧化纤维素质量百分数的0.01～2％。本发明所述制备方法包括氧化前处理过程、氧化过程、络合凝血因子、静电喷射及成型等步骤。本发明具有工艺连续、高效、成本低等优点，制备出微球粒均一可控、氧化程度高、止血能力强的氧化纤维素止血材料。该新型止血材料适用于大出血、不规则部位及脏器出血，该材料可降解性好，止血快，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种新型可降解的止血材料，其特征在于，所述止血材料为负载有凝血因子的氧化纤维素，其中所述氧化纤维素的羧基含量为20～30％，聚合度为200～400，粒径大小为50～150μm，所述凝血因子占所述氧化纤维素质量百分数的0.01～2％。
  2. 根据权利要求1所述的止血材料，其特征在于，所述凝血因子为可溶性钙盐。
  3. 权利要求1或2所述止血材料的制备方法，其特征在于，包括以下步骤：
    A、将纤维素依次经过破碎处理和氢氧化钠溶液润胀处理，制备纤维素；
    B、对纤维素分子的C 6位羟基进行选择性氧化，在C 6位上引入羧基，得到氧化纤维素；并在氧化过程中辅以超声处理；
    C、将氧化纤维素与凝血因子按不同条件进行络合；
    D、将步骤C所得混合物溶于碱体系中，搅拌过滤，收集滤液，将滤液以静电喷射的方式喷射于凝固液中，收集沉淀，用水和/或乙醇洗涤至中性；
    E、将步骤D所得产物进行机械破碎或研磨，干燥后，过筛，灭菌，即得；
    其中，步骤D所用凝固液为乙醇、硫酸溶液或盐酸溶液。
  4. 根据权利要求3所述的方法，其特征在于，步骤A包括：将一定量的纤维素加入到高压均质机、磨浆机、疏解机、粉碎机或超声机中，进行粉碎处理，至纤维长度小于1mm；将浆液过滤，得沉淀物，水洗后，放入1～20％的氢氧化钠溶液中润胀处理20～120min，将润胀后的纤维素用水和/或乙醇洗涤至中性。
  5. 根据权利要求3所述的方法，其特征在于，步骤B包括：将纤维素加入到氧化体系中进行氧化反应，氧化反应和超声处理同步进行；反应 结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素；
    其中，所述氧化体系为NO 2氧化体系、次氯酸盐氧化体系、TEMPO氧化体系或HNO 3/H 3PO 4～NaNO 2氧化体系；
    所述NO 2氧化体系是以CCl 4为溶剂，配制的浓度为10～30％的NO 2氧化液；
    所述次氯酸盐氧化体系为20～200％v/v的NaClO溶液；
    所述TEMPO氧化体系为TEMPO-NaClO-NaBr氧化体系；
    所述HNO 3/H 3PO 4～NaNO 2氧化体系为含NaNO 2的HNO 3和H 3PO 4混合液。
  6. 根据权利要求5所述的方法，其特征在于，当所述氧化体系为NO 2氧化体系时，所述步骤B包括：将1～20g纤维素加入到100～200mL所述NO 2氧化体系中，反应温度为5～30℃，反应时间为8～100h；氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，依次用CCl 4、乙醇洗涤沉淀，得到氧化纤维素；
    当所述氧化体系为次氯酸盐氧化体系时，所述步骤B包括：将5～20g纤维素悬浮于100～200mL水中，调悬浮液pH至9～11，在40～60℃条件下，向其中加入20～100％v/v的NaClO溶液20～200mL，反应2～10h；氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素；
    当所述氧化体系为TEMPO氧化体系时，所述步骤B包括：将10～100g纤维素悬浮于pH9～11的Na 2CO 3/NaHCO 3缓冲液100～1000mL中，搅拌2～10min后，加入TEMPO，使TEMPO的终浓度为0.1～1.0mg/mL，再加入NaBr，使NaBr的终浓度为1～10mg/mL；控制反应体系温度为20～30℃，待TEMPO和NaBr完全溶解后，调反应体系pH至9～11，然后分步加入饱和NaClO溶液，每隔30～60min加一次，NaClO溶液加入的体积为溶液总体积的20～100％，反应3～10h；氧化反应和超声处理同步进行；反应 结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素；其中，所述Na 2CO 3/NaHCO 3缓冲液中Na 2CO 3和NaHCO 3的摩尔比为1:3～3:1；
    当所述氧化体系为HNO 3/H 3PO 4～NaNO 2氧化体系时，所述步骤B包括：将HNO 3和H 3PO 4分析纯按体积比0.5:1～3:1配制成混合液，将纤维素加入到上述混合液中，使其浓度为0.01～0.1g/mL，再加入NaNO 2粉末，使NaNO 2的终浓度为10～50mg/mL；在20～50℃条件下反应8～48h；氧化反应和超声处理同步进行；反应结束后，过滤或离心收集沉淀，用水和/或乙醇洗涤至中性，得到氧化纤维素。
  7. 根据权利要求3所述的方法，其特征在于，步骤B中超声条件为：超声频率20～40kHz，功率100～1000W。
  8. 根据权利要求3所述的方法，其特征在于，所述步骤D中，将步骤C所得混合物在-20～-5℃条件下溶于碱体系中；所述碱体系为5～20％氢氧化钠溶液、含5～30％尿素的5～20％氢氧化钠溶液或含5～30％尿素的5～20％氢氧化锂溶液。
  9. 根据权利要求3所述的方法，其特征在于，所述步骤D中，静电喷射的电压为5～20kV，供料速率为20～400μL/min。
  10. 根据权利要求3-9中任一项所述的方法，其特征在于，步骤E中采用的干燥方式为喷雾干燥、超临界干燥、真空干燥、鼓风干燥或冷冻干燥。
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