WO2024045119A1 - 一种优化抗衡阴离子以改善生物材料止血性能的通用方法 - Google Patents

一种优化抗衡阴离子以改善生物材料止血性能的通用方法 Download PDF

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WO2024045119A1
WO2024045119A1 PCT/CN2022/116464 CN2022116464W WO2024045119A1 WO 2024045119 A1 WO2024045119 A1 WO 2024045119A1 CN 2022116464 W CN2022116464 W CN 2022116464W WO 2024045119 A1 WO2024045119 A1 WO 2024045119A1
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solution
substrate
electronegative
hemostatic
sodium
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French (fr)
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徐福建
胡杨
杨雪
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北京化工大学
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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
    • 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
    • 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
    • A61L24/06—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • 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
    • A61L24/10—Polypeptides; Proteins

Definitions

  • the invention belongs to the technical field of preparation of medical hemostatic materials and relates to a general method for optimizing counteranions to improve the hemostatic performance of biological materials.
  • cationic polymers are beneficial to the accumulation of negatively charged proteins and cells, and are used in the development of many types of hemostatic materials.
  • the effect of cationic polymers on coagulation is a double-edged sword, and their excessive positivity will also delay thrombin. of generation. Therefore, it is necessary to regulate the structure of cationic polymers to improve their procoagulant properties.
  • the introduction of electronegative small molecules onto cationic polymers through small molecule exchange is expected to achieve simpler polymer structure regulation.
  • the present invention further provides a preparation technology for converting this negatively charged small molecule/cationic polymer complex into a hemostatic material, that is, it can be combined with a hydrophilic uncharged polymer to obtain a hemostatic gel. Or film, it can also be used to modify polymer medical materials to obtain hemostatic materials; the positive electricity of cationic polymers can also be controlled by small negatively charged molecules, and modifications can be made on the surfaces of different substrates with cationic polymers. properties, resulting in a procoagulant surface.
  • the present invention provides a general method for optimizing counteranions to improve the hemostatic properties of biomaterials. Specifically, the following technical solutions are provided:
  • Method 1 is a one-pot control of electronegative small molecules and cationic polymers. The specific steps are:
  • step 2) Make the blended solution obtained in step 1) into a gel or film, or modify the blended solution on the base material to prepare a hemostatic material;
  • Method 2 involves step-by-step regulation of electronegative small molecules on the surface of cations.
  • the specific steps are:
  • the substrate with cations on the surface is modified by soaking in a solution of negatively charged small molecules to obtain a material with a procoagulant surface.
  • the specific steps are:
  • step 2 Immerse the substrate with cations on the surface into the solution in step 1);
  • the electronegative small molecules are sodium methyl sulfate, sodium methanesulfonate, sodium N-cyclohexyl sulfamate, morpholinoethanesulfonate sodium salt monohydrate, and 3-N(-morpholino)propanesulfonate.
  • the blended solution in step 1) of method 1 is an aqueous solution or a mixed solution of water and methanol, ethanol, ethyl acetate, and isopropyl alcohol;
  • the aqueous solution is a glucose solution, and the concentration of glucose is 5% ⁇ 10%;
  • the cationic polymer described in step 1) of method 1 is polylysine, polydimethylaminoethyl methacrylate, polyhexamethylene biguanide hydrochloride, polyhexamethylene guanidine hydrochloride , one or more mixtures of quaternized starch, the concentration range in the solution is 0.1 ⁇ 3mg/mL.
  • the cationic polymer described in step 1) of method 1 is polylysine or polyhexamethylene biguanide hydrochloride; the electronegative small molecule described in step 1) is sodium methyl sulfate or methylsulfonic acid. sodium.
  • the method for preparing a gel from the blended solution described in step 2) of method 1 is: adding a hydrophilic uncharged polymer to the blended solution obtained in step 1) to obtain a mixed gel.
  • Water-based uncharged polymers are temperature-sensitive hydrophilic polymers such as poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide, and poly(N-isopropylacrylamide).
  • One or more substances are mixed;
  • the method for preparing a film from the blended solution described in step 2) of method 1 is: adding a hydrophilic uncharged polymer to the blended solution obtained in step 1) of method 1 to obtain The mixed solution is dried at high temperature to form a hemostatic film with hemostatic properties.
  • the hydrophilic uncharged polymer is hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose, hydroxyethyl
  • One or more mixtures of hydrophilic cellulose derivatives such as cellulose and methylcellulose; the method for modifying the substrate by the blended solution described in step 2) of method 1 is: using the solution obtained in step 1) of method 1
  • the base material is soaked in the blending solution, and then the base material is taken out and dried.
  • the selected base material is gauze, gelatin sponge, alginic acid non-woven fabric and other polymer medical materials.
  • the step of making a gel from the blended solution described in Method 1 is as follows: adding a cationic polymer to the glucose solution so that the concentration range in the solution is 0.1 to 3 mg/mL, a small electronegative molecule, and adding a temperature-sensitive polymer.
  • thermosensitive polymer poloxamer polylactide-glycolide-polyethylene glycol-polylactide-glycolide or poly(N-isopropylacrylamide) makes the concentration of the thermosensitive polymer in the solution
  • the range is 100 ⁇ 400mg/mL
  • the steps of making a film from the blend solution described in method 1 are: adding a cationic polymer to the glucose solution so that the concentration range in the solution is 0.1 ⁇ 3mg/mL and the electronegative property is small Molecule, add hydroxypropyl cellulose, hydroxypropyl methyl cellulose or hydroxyethyl cellulose so that the concentration range in the solution is 20 to 200 mg/mL; the blending solution described in method 1 is used to modify the substrate.
  • the steps are: add cationic polymer to the glucose solution so that the concentration range in the solution is 0.1 to 3mg/mL, and electronegative small molecules, and fully penetrate the base material with the mixed solution of cationic polymer and electronegative small molecules
  • the concentration of the electronegative small molecule solution in method two is 0.5-50 mg/mL, and the soaking process time described in step 2) of method two is 0.5-10 h.
  • Substrate a is a cation-immobilized substrate obtained by self-assembly of a substrate that does not contain cations on its surface and a cationic polymer;
  • Substrate b is a surface cation-immobilized substrate obtained through a chemical reaction between a substrate that does not contain cations on its surface and a positively charged small molecule;
  • the substrate c is a substrate that contains a large number of cationic groups on its surface, and the cationic groups are one or more combinations of guanidine groups, primary amines, quaternary ammonium or tertiary amines.
  • the self-assembly described in the substrate a in method two is to coat the cationic polymer and the electronegative polymer on the surface through a layer-by-layer self-assembly method, and control the outermost layer to be a cationic polymer; the substrate in method two
  • the base material described in a that does not contain cations on its surface is 304 stainless steel, metal titanium nails, silicon wafers, gelatin sponges, polyvinyl alcohol sponges or medical collagen sponges;
  • the hydrochlorides are mixed, and the electronegative polymer is one or more of the poly(sodium 4-styrene sulfonate) and polymethacrylic acid.
  • the substrate whose surface does not contain cations as described in substrate b is a gelatin sponge, polyvinyl alcohol sponge, medical collagen sponge or gauze;
  • the chemical reaction described in substrate b in method two is quaternary
  • the ammonization reaction involves a ring-opening reaction between the hydroxyl, amino or carboxyl groups on the surface of the substrate that does not contain cations and the quaternary ammonium salt containing the epoxy group;
  • the quaternary ammonium salt containing the epoxy group is 2,3- Epoxypropyltrimethylammonium chloride or the product obtained by the reaction of epichlorohydrin and N,N-dimethyl ,fourteen.
  • method two is to soak 2,3-epoxypropyltrimethylquaternary ammonium salt-modified gelatin and sodium methyl sulfate to obtain a material with a procoagulant surface; poly(4-styrene sulfonate sodium) ) and poly(diallyldimethylammonium chloride) are alternately self-assembled and modified. After the cationic immobilized 304 stainless steel surface is soaked in sodium methyl sulfate, a material with a procoagulant surface can be obtained.
  • the present invention provides a general method for optimizing counter anions to improve the hemostatic performance of biological materials.
  • a part of the counter anions such as hydrogen Oxygen radicals, chloride ions, etc.
  • the counter anions are transformed into newly introduced small negatively charged molecules, thereby regulating the strong positive charge of the cationic polymer on the surface of the substrate and the accompanying strong binding ability/adhesion with key coagulation factors/proteins in the coagulation process.
  • it will cause a decrease in coagulation function and a prolongation of the endogenous plasma coagulation time.
  • the present invention further provides a preparation technology for converting this negatively charged small molecule/cationic polymer complex into a hemostatic material, that is, it can be combined with a hydrophilic uncharged polymer to obtain a hemostatic gel. Or film, it can also be modified to polymer medical materials to obtain hemostatic materials.
  • the invention realizes the elimination of the negative impact on the endogenous coagulation pathway caused by the excessive electropositivity of the cationic polymer, and improves the coagulation-promoting performance of the cationic polymer through simple polymer structure regulation.
  • Method 2 of the present invention provides a general method for optimizing counter anions to improve the hemostatic performance of biological materials. By immersing the cationic polymer in a surface-fixed state in a solution of a negative small molecule, the negative small molecule and the cationic polymer can occur.
  • the original competition process of counteranions converts a part of the counteranions (such as hydroxide, chloride ions, etc.) into newly introduced small electronegative molecules, thereby regulating the strong electropositivity of the cationic polymer on the surface of the substrate and the accompanying
  • the strong binding ability/adhesion of key coagulation factors/proteins in the coagulation process (generally leads to a decrease in coagulation function and a prolongation of the endogenous plasma coagulation time).
  • the invention realizes the elimination of the negative impact on the endogenous coagulation pathway caused by the excessive electropositivity of the cationic polymer, and improves the coagulation-promoting performance of the substrate with the cationic polymer fixed on the surface through simple polymer structure regulation. Therefore, the present invention provides a simpler method for preparing a procoagulant surface compared to fine-tuning the polymer structure through chemical modification and synthesis reactions to develop substrates with procoagulant properties (polymers immobilized on the surface).
  • the method of the present invention provides a simpler method of using negatively charged small molecules to modify and enhance the cationic polymer structure compared to the precise chemical modification synthesis reaction to regulate the structure of the cationic polymer to develop polymer-based hemostatic materials and procoagulant surfaces. Methods based on hemostatic materials and procoagulant surfaces.
  • the poloxamer was prepared with 10% glucose solution to a concentration of 300 mg/mL to obtain gel Y8.
  • Hydroxypropyl cellulose was prepared with 10% glucose solution to a concentration of 75 mg/mL. 2 mL of the solution was fully spread in a 4.4 ⁇ 4.4 cm 2 mold and dried at 60°C for 6 hours to obtain polysaccharide film Y9.
  • the prepared procoagulant enhanced hemostatic materials X1 to X5 and Comparative Examples Y1 to Y10 were used for comparative experiments on in vitro coagulation effects.
  • Test method Take 50 ⁇ L of hemostatic gel and put it into a 2mL plastic centrifuge tube. Pre-solidify the gel at 37°C for 5 minutes. Weigh 5mg of the membrane and put 0.5 ⁇ 0.5cm2 of gauze into a 2mL plastic centrifuge tube. Add 100 ⁇ L of fresh anticoagulated blood from SD rats to 10 ⁇ L of calcium chloride solution (CaCl 2 ; 0.2M), mix well, immediately contact the prepared materials, and incubate in a constant temperature water bath at 37°C for 1 minute. Then slowly add 10 mL of deionized water and continue incubating for 3 minutes to fully lyse the blood cells that have not formed a blood clot and release hemoglobin.
  • CaCl 2 calcium chloride solution
  • Blood coagulation index % (BCI) (Abs sample/Abs blank) ⁇ 100%;
  • Abs sample is the absorbance of the experimental group at 545nm
  • Abs blank is the absorbance of the blank group at 545nm.
  • the BCI index of the examples and comparative examples is shown in Table 1:
  • the in vitro coagulation index BCI is an important indicator to characterize the in vitro procoagulant properties of materials and to screen hemostatic materials.
  • the BCI index of the hemostatic materials X1 to X5 obtained in Examples 1 to 5 of the present invention is significantly lower than the same type of materials in Comparative Examples Y1 to Y10. It can be seen that by using the preparation method of the present invention, hemostatic materials with excellent hemostatic properties can be obtained.
  • Comparative Example 1 is compared with Example 1.
  • preparation step 1) a small amount of sodium methyl sulfate and polylysine are blended to obtain a blended solution, and then poloxamer is added to the blended solution to prepare a hemostatic coagulant.
  • the BCI of Example 1 was 22.4%
  • the BCI of Comparative Example 1 was 46.8%. The results showed that the hemostatic performance of the gel obtained in Comparative Example 1 was worse than that of Example 1.
  • Comparative Example 2 is compared with Example 1.
  • preparation step 1) excess sodium methyl sulfate and polylysine are blended to obtain a blended solution, and then poloxamer is added to the blended solution to prepare a hemostatic coagulant.
  • the BCI of the gel in Example 1 was 22.4%, and the BCI in Comparative Example 2 was 53.9%.
  • the results showed that the hemostatic performance of the gel obtained in Comparative Example 2 was worse than that of Example 1. Therefore, it shows that adding excessive amounts of negatively charged small molecules will greatly weaken the positive charge of the cationic polymer, thereby affecting its ability to aggregate blood cells and platelets, making it impossible to effectively improve the hemostatic performance of the hemostatic material.
  • Comparative Example 3 is compared with Example 1.
  • the preparation step 1) no sodium methyl sulfate is added, polylysine is dissolved in 10% glucose solution to obtain a cationic polymer solution, and then the poloxamer is added
  • the hemostatic gel was prepared from the solution.
  • the BCI of Example 1 was 22.4% and the BCI of Comparative Example 3 was 65.0%.
  • the results showed that the hemostatic performance of the gel obtained in Comparative Example 3 was worse than that of Example 1. Therefore, it shows that cationic polymers that have not been regulated by small molecules have a negative impact on the endogenous coagulation pathway due to their excessively high electropositivity, resulting in a decrease in coagulation function, making it impossible to effectively improve the hemostatic performance of hemostatic materials. .
  • Comparative Example 4 is compared with Example 1.
  • preparation step 1) excess polylysine was dissolved in 10% glucose solution to obtain a cationic polymer solution, an equal mass of sodium methyl sulfate was added, and then polo Shamu was added to the solution to prepare a hemostatic gel.
  • the BCI of Example 1 was 22.4% and the BCI of Comparative Example 4 was 54.9%.
  • the results showed that the hemostatic performance of the gel obtained in Comparative Example 4 was worse than that of Example 1. Therefore, it shows that excessive cationic polymers are very positively charged and negatively charged small molecules have limited weakening effects. Therefore, they still have a negative impact on the endogenous coagulation pathway, resulting in a decrease in coagulation function, so that they cannot effectively improve hemostatic materials. hemostatic properties.
  • Comparative Example 5 is compared with Example 4.
  • the electronegative small molecule sodium methyl sulfate is not added, and polyhexamethylene biguanide hydrochloride is dissolved in 10% glucose solution to obtain a cationic polymer. solution, and then add poloxamer to the solution to prepare a hemostatic gel.
  • the BCI of Example 4 is 53.8%, and the BCI of Comparative Example 5 is 87.5%.
  • the results show that the hemostatic performance of the gel obtained in Comparative Example 5 is better than that of Example 4. difference. Therefore, it shows that cationic polymers that have not been regulated by small molecules have a negative impact on the endogenous coagulation pathway due to their excessively high electropositivity, so that they cannot effectively improve the hemostatic performance of hemostatic materials.
  • Comparative Example 6 is compared with Example 2.
  • the preparation step 1) no electronegative small molecule sodium methyl sulfate is added, polylysine is dissolved in 10% glucose solution to obtain a cationic polymer solution, and then hydroxypropyl Cellulose was added to the solution to prepare a hemostatic film.
  • the BCI of Example 2 was 21.3%, and the BCI of Comparative Example 6 was 62.8%.
  • the results showed that the hemostatic performance of the film obtained in Comparative Example 6 was worse than that of Example 2. Therefore, it shows that cationic polymers that have not been regulated by electronegative small molecules have a negative impact on the endogenous coagulation pathway due to their excessive electropositivity, so that they cannot effectively improve the hemostatic performance of hemostatic materials.
  • Comparative Example 7 is compared with Example 3.
  • the preparation step 1) no electronegative small molecule sodium methyl sulfate is added. Polylysine is dissolved in 10% glucose solution to obtain a cationic polymer solution, and then the medical gauze is soaked. After modification, the BCI of Example 3 was 44.6%, and the BCI of Comparative Example 7 was 92.6%. The results showed that the hemostatic performance of the gauze obtained in Comparative Example 6 was worse than that of Example 4. Therefore, it shows that cationic polymers that have not been regulated by electronegative small molecules have a negative impact on the endogenous coagulation pathway due to their excessive electropositivity, so that they cannot effectively improve the hemostatic performance of hemostatic materials.
  • Comparative Example 8 does not add any modifying materials and uses poloxamer alone to prepare hydrogel.
  • the BCI of the hydrogel is 50.6 (much larger than the BCI of gels X1 and X5 modified by the method of the present invention). ), the hemostatic performance is not as good as that after adding modified materials. Therefore, it shows that using the method of the present invention to regulate cationic polymers with negatively charged small molecules will improve the hemostatic performance of the gel.
  • Comparative Example 9 does not add any modified materials and uses hydroxypropyl cellulose alone to prepare a membrane.
  • the BCI of the membrane is 58.1% (much larger than the BCI of membrane
  • the modified invention has good hemostatic properties. Therefore, it shows that using the method of the present invention to control the cationic polymer with electronegative small molecules will improve the hemostatic performance of the membrane.
  • Comparative Example 10 does not add any modified materials and uses medical gauze alone.
  • the BCI of the gauze is 76.5% (much greater than the BCI of the medical gauze X3 modified by the method of the present invention).
  • the hemostatic performance does not adopt the method of the present invention.
  • the performance after modification is good. Therefore, it shows that using the method of the present invention to regulate cationic polymers with electronegative small molecules will improve the hemostatic performance of the membrane.
  • the prepared procoagulant enhanced hemostatic material X1 and comparative examples Y1 and Y3 were subjected to activated partial thromboplastin time (APTT) comparative experiments.
  • Test method Take 50 ⁇ L of hemostatic gel and put it into a 2 mL plastic centrifuge tube, and pre-solidify the gel at 37°C for 5 minutes.
  • the anticoagulated whole blood of SD rats was centrifuged at 3000 rpm for 15 min, and the supernatant was taken out as platelet-poor plasma (PPP).
  • PPP platelet-poor plasma
  • the blank group is APTT without added materials, which is regarded as 100%.
  • APTT (%) is calculated by the following equation 2:
  • APTT refers to activated partial thromboplastin time, which is the most commonly used test in clinical practice to reflect the coagulation activity of the endogenous coagulation system.
  • the APTT time of normal plasma was taken as 100%. The longer the APTT time, the greater the impact on the intrinsic coagulation pathway and the less conducive to coagulation.
  • Comparative Example 1 is compared with Example 1.
  • preparation step 1) a small amount of sodium methyl sulfate and polylysine are blended to obtain a blended solution, and then poloxamer is added to the blended solution to prepare hemostasis.
  • Gel, APTT test results show that the APTT of Negatively charged small molecules + cationic polymers) have less impact on the endogenous coagulation pathway, while a small amount of negatively charged small molecules are not enough to weaken the negative impact of cationic polymers on the endogenous coagulation pathway, which is not conducive to coagulation.
  • Comparative Example 3 is compared with Example 1.
  • the preparation step 1) no sodium methyl sulfate is added, polylysine is dissolved in 10% glucose solution to obtain a cationic polymer solution, and then poloxamer is added to the solution.
  • the hemostatic gel was prepared.
  • the APTT test results showed that the APTT of X1 was 110% and that of Y3 was 157%. The time was significantly longer than that of normal plasma (100%). Therefore, it shows that only cationic polymers, without the use of electronegative small molecules for regulation, have a greater impact on the endogenous coagulation pathway and are not conducive to coagulation.
  • the substrate with cations on the surface is quaternized gelatin sponge
  • the cationic polymer is quaternized gelatin sponge
  • the electronegative small molecule is sodium methyl sulfate.
  • the substrate with cations on the surface is quaternized gelatin sponge
  • the cationic polymer is quaternized gelatin sponge
  • the electronegative small molecule is sodium methanesulfonate.
  • S 304 stainless steel sheet
  • isopropyl alcohol, ethanol, and water ultrasonically and then treat it with oxygen plasma after drying.
  • Use deionized water to prepare 1 mg/mL poly(diallyldimethylammonium chloride) (PDADMAC) solution and 1 mg/mL poly(4-styrene sodium sulfonate) (PSS) solution. Soak PDADMAC and PSS solutions alternately for 20 minutes each time, wash with deionized water for 1 minute, and blow nitrogen for 2 minutes to obtain S-PP 4.5 (the outermost layer of S-PP 4.5 is a cationic polymer polydiallyldimethylammonium chloride).
  • PDADMAC poly(diallyldimethylammonium chloride)
  • PSS poly(4-styrene sodium sulfonate)
  • the substrate with cations on the surface is a cation-immobilized substrate obtained by alternating self-assembly of 304 stainless steel sheets with poly(sodium 4-styrenesulfonate) and poly(diallyldimethylammonium chloride).
  • S-PP 4.5 the cationic polymer is poly(diallyldimethylammonium chloride), and the electronegative small molecule is sodium methyl sulfate.
  • the quaternized gelatin sponge obtained in step 1) in Example 6 was named Y11.
  • Example 8 The S-PP 4.5 in step 1) in Example 8 was named Y12.
  • the quaternized gelatin sponge in step 1) in Example 6 was soaked in a 21 mg/mL sodium methanesulfonate aqueous solution for 2 minutes, washed three times with deionized water for 30 minutes each time, and freeze-dried to obtain a hemostatic sponge Y13.
  • the soaking time is only 2 minutes, which is much shorter than the 2 hours in Embodiment 1.
  • the prepared procoagulant surface hemostatic materials X6 to X8 and Comparative Examples Y11 to Y13 were used for comparative experiments on in vitro coagulation effects.
  • Test method Take 5mg of the hemostatic sponge series and put it into a 2mL plastic centrifuge tube. Take 1 ⁇ 1cm2 of the stainless steel piece and put it into a 6-well plate. Add 100 ⁇ L of fresh anticoagulated blood from SD rats to 10 ⁇ L of calcium chloride solution (CaCl 2 ; 0.2M), mix well, immediately contact the prepared materials, and incubate in a constant temperature water bath at 37°C for 1 minute. Then slowly add 10 mL of deionized water and continue incubating for 3 minutes to fully lyse the blood cells that have not formed a blood clot and release hemoglobin.
  • CaCl 2 calcium chloride solution
  • Blood coagulation index % (BCI) (Abs sample /Abs blank ) ⁇ 100%.
  • Abs sample is the absorbance of the experimental group at 545nm
  • Abs blank is the absorbance of the blank group at 545nm.
  • the BCI index of the examples and comparative examples is shown in Table 1:
  • the in vitro coagulation index BCI is an important indicator to characterize the in vitro procoagulant properties of materials and to screen hemostatic materials.
  • Table 3 the BCI index of the hemostatic materials X6 to X8 obtained in Examples 6 to 8 of the present invention is significantly lower than the same type of materials in Comparative Examples Y11 to Y13. It can be seen that by using the preparation method of the present invention, a material surface with excellent procoagulant/hemostatic properties can be obtained.
  • the electropositivity of the cationic polymer can be effectively adjusted, and the adverse effects on the endogenous coagulation pathway caused by the excessive electropositivity of the cationic polymer can be effectively improved. Procoagulant properties.
  • Comparative Example 11 is a quaternized gelatin sponge modified without soaking in the electronegative small molecule sodium methyl sulfate.
  • the BCI of Y11 in Table 3 is larger than the BCI of Examples 6 and 7, indicating that the positive charge has not been regulated by the electronegative small molecule. Due to their excessively high electropositivity, cationic polymers have a negative impact on the intrinsic coagulation pathway and cannot effectively improve the procoagulant/hemostatic properties of the gelatin sponge surface.
  • Comparative Example 12 is a stainless steel sheet coated with a cationic polymer coating that is not soaked in sodium methyl sulfate for modification.
  • the BCI result of Y12 in Table 3 is much greater than that of Example 8, indicating that it has not been regulated by electronegative small molecules. Due to its excessively high electropositivity, the stainless steel surface of electropositive cationic polymers has a negative impact on the intrinsic coagulation pathway, making it impossible to effectively improve the procoagulant/hemostatic performance of the stainless steel surface.
  • Example 13 in the preparation step 2) of Example 6, the quaternized gelatin sponge was soaked in sodium methane sulfonate with a suitable concentration, but the soaking time was too short and was not conducive to the effective exchange of counterions.
  • the BCI of Y13 in Table 3 is greater than The BCI value of Hemostatic properties of hemostatic materials.

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Abstract

本发明公开了一种优化抗衡阴离子以改善生物材料止血性能的通用方法，方法一为负电性小分子和阳离子聚合物进行一锅法调控：将阳离子聚合物与负电性小分子溶解，得到共混溶液，所述的阳离子聚合物与负电性小分子的质量比为1:0.15～2，将得到的共混溶液制成凝胶或膜，或将共混溶液对基材进行改性；方法二为负电性小分子在阳离子表面进行分步调控：将表面具备阳离子的基材通过浸泡负电性小分子溶液进行改性：配制负电性小分子溶液，将表面具备阳离子的基材浸入负电性小分子溶液中，洗涤、干燥；负电性小分子为甲基硫酸钠、甲基磺酸钠、N-环己基氨基磺酸钠、吗啉乙磺酸钠盐一水合物、3-N(-吗啉基)丙磺酸钠、3-吗啉-2-羟基丙磺酸钠、葡萄糖酸的一种或多种混合。

Description

一种优化抗衡阴离子以改善生物材料止血性能的通用方法 技术领域
本发明属于医用止血材料的制备技术领域，涉及一种优化抗衡阴离子以改善生物材料止血性能的通用方法。
背景技术
高分子材料由于结构设计灵活多样，在止血、抗菌、药物载体等多领域研究广泛。其中阳离子聚合物有利于聚集负电性蛋白和细胞，被用于多类型止血材料的开发，但阳离子聚合物对凝血的作用是一把双刃剑，其过高的正电性也会延缓凝血酶的生成。因此有必要通过调控阳离子聚合物的结构以改善其促凝血性能。相对于精细的化学合成反应调控阳离子聚合物结构，通过小分子交换在阳离子聚合物上引入负电性小分子，将有望实现更加简便的聚合物结构调控。基于此，通过负电性小分子与阳离子聚合物原有抗衡阴离子的竞争作用，将一部分抗衡阴离子(如氢氧根、氯离子等)转变为新引入的负电性小分子，从而调控基材表面阳离子聚合物的强正电性以及伴随的与凝血过程中关键凝血因子/蛋白的强结合能力/黏附作用力(一般会造成凝血功能下降、血浆内源性凝血时间这一指标延长)。针对止血材料制备，本发明进一步提供了将这种负电性小分子/阳离子聚合物复合体转变为止血材料的制备技术，即可将其与亲水性不带电荷聚合物结合后得到止血凝胶或膜，也可以将其对高分子医用材料进行改性得到止血材料；还可以通过负电性小分子对阳离子聚合物的正电性进行调控，可在不同具备阳离子聚合物的基材表面进行改性，得到促凝血表面。
发明内容
有鉴于此，本发明提供一种优化抗衡阴离子以改善生物材料止血性能的通用方法。具体提供如下技术方案：
一种优化抗衡阴离子以改善生物材料止血性能的通用方法，有两种方法：
方法一为负电性小分子和阳离子聚合物进行一锅法调控，具体步骤为：
1)将阳离子聚合物与负电性小分子溶解，得到共混溶液，所述的阳离子聚合物与负电性小分子的质量比为1:0.15～2；
2)将步骤1)得到的共混溶液制成凝胶或膜，或将共混溶液对基材进行改性，制备得到止血材料；
方法二为负电性小分子在阳离子表面进行分步调控，具体步骤为：
将表面具备阳离子的基材通过浸泡负电性小分子溶液进行改性，得到具有促凝血表面的材料，具体步骤为：
1)配制负电性小分子溶液；
2)将表面具备阳离子的基材浸入步骤1)的溶液中；
3)洗涤、干燥，得到负电性小分子调控的促凝血表面；
所述的负电性小分子为甲基硫酸钠、甲基磺酸钠、N-环己基氨基磺酸钠、吗啉乙磺酸钠盐一水合物、3-N(-吗啉基)丙磺酸钠、3-吗啉-2-羟基丙磺酸钠、葡萄糖酸的一种或多种混合。
进一步，方法一的步骤1)所述的共混溶液为水溶液或水与甲醇、乙醇、乙酸乙酯、异丙醇形成的混合溶液；所述的水溶液为葡萄糖溶液，葡萄糖的浓度为5％～10％；方法一的步骤1)所述的阳离子聚合物为聚赖氨酸、聚甲基丙烯酸二甲氨基乙酯、聚六亚甲基双胍盐酸盐、聚六亚甲基胍盐酸盐、季铵化淀粉的一种或多种混合，其在溶液中的浓度范围在0.1～3mg/mL。
进一步，方法一的步骤1)所述的阳离子聚合物为聚赖氨酸或聚六亚甲基双胍盐酸盐；步骤1)所述的负电性小分子为甲基硫酸钠或甲基磺酸钠。
进一步，方法一步骤2)所述的共混溶液制成凝胶的制备方法为：将亲水性不带电荷聚合物加入步骤1)得到的共混溶液中，得到混合凝胶，所述亲水性不带电荷聚合物为泊洛沙姆、聚丙交酯-乙交酯-聚乙二醇-聚丙交酯-乙交酯、聚(N-异丙基丙烯酰胺)等温敏亲水性聚合物一种或多种混合；方法一步骤2)所述的共混溶液制成膜的制备方法为：将亲水性不带电荷聚合物加入方法一步骤1)得到的共混溶液中，得到的混合溶液高温干燥，制成具有止血性能的止血膜，所述的亲水性不带电荷聚合物为羟丙基纤维素、羟丙基甲基纤维素、羟甲基纤维素、羟乙基纤维素、甲基纤维素等亲水性纤维素衍生物的一种或多种混合；方法一步骤2)所述的共混溶液对基材改性方法为：利用方法一步骤1)得到的共混溶液对基材进行浸泡，然后取出基材进行干燥，所选的基材为纱布、明胶海绵、海藻酸无纺布等高分子医用材料。
进一步，方法一所述的共混溶液制成凝胶的步骤为：在葡萄糖溶液中，加入阳离子聚合物使其在溶液中的浓度范围为0.1～3mg/mL、负电性小分子，加入温敏性聚合物泊洛沙姆、聚丙交酯-乙交酯-聚乙二醇-聚丙交酯-乙交酯或聚(N-异丙基丙烯酰胺，使温敏性聚合物在溶液中的浓度范围为100～400mg/mL；方法一所述的共混溶液制成膜的步骤为：在葡萄糖溶液中，加入阳离子聚合物使其在溶液中的浓度范围为0.1～3mg/mL、负电性小分子，加入羟丙基纤维素、羟丙基甲基纤维素或羟乙基纤维素使其在溶液中的浓度范围为20～200mg/mL；方法一所述的共混溶液对基材进行改性的步骤为：在葡萄糖溶液中，加入阳离子聚合物使其在溶液中的浓度范围为0.1～3mg/mL、负电性小分子，将阳离子聚合物和负电性小分子的混合溶液充分浸透基材。
进一步，方法二的负电性小分子溶液的浓度为0.5-50mg/mL，方法二步骤2)所述的浸泡过程时间为0.5-10h。
进一步，方法二中所述的表面具备阳离子的基材有三种：
基材a为本身表面不含有阳离子的基材与阳离子聚合物经自组装得到的阳离子固定化基材；
基材b为本身表面不含有阳离子的基材与正电性小分子经化学反应得到的表面阳离子固定化基材；
基材c为本身表面含有大量阳离子基团的基材，阳离子基团为胍基、伯胺、季铵或叔胺的一种或多种组合。
进一步，方法二中基材a中所述的自组装为将阳离子聚合物和负电性聚合物通过层层自组装方法包覆在表面，并控制最外层是阳离子聚合物；方法二中基材a中所述的本身表面不含有阳离子的基材为304不锈钢、金属钛钉、硅片、明胶海绵、聚乙烯醇海绵或医用胶原蛋白海绵；方法二中基材a中所述的阳离子聚合物为聚二烯丙基二甲基氯化铵、聚甲基丙烯酸N,N-二甲基氨基乙酯、聚赖氨酸、聚六亚甲基双胍盐酸盐、聚六亚甲基单胍盐酸盐中的一种或多种混合，所述的负电性聚合物为聚(4-苯乙烯磺酸钠)、聚甲基丙烯酸中的一种或多种混合。
进一步，方法二中基材b中所述的本身表面不含有阳离子的基材为明胶海绵、聚乙烯醇海绵、医用胶原蛋白海绵或纱布；方法二中基材b中所述的化学反应为季铵化反应，通过本身表面不含有阳离子的基材表面的羟基、氨基或羧基与含环氧基的季铵盐发生开 环反应；所述的含环氧基的季铵盐为2,3-环氧丙基三甲基氯化铵或由环氧氯丙烷和N,N-二甲基X胺反应得到的产物，所述的X为乙、丙、丁、己、辛、癸、十二、十四。
进一步，方法二为2，3-环氧丙基三甲基季铵盐改性的明胶和甲基硫酸钠进行浸泡后，可以得到具有促凝血表面的材料；聚(4-苯乙烯磺酸钠)和聚(二烯丙基二甲基氯化铵)交替自组装改性得到的阳离子固定化304不锈钢表面和甲基硫酸钠进行浸泡后，可以得到具有促凝血表面的材料。
本发明的有益效果在于：本发明提供了一种优化抗衡阴离子以改善生物材料止血性能的通用方法，通过负电性小分子与阳离子聚合物原有抗衡阴离子的竞争作用，将一部分抗衡阴离子(如氢氧根、氯离子等)转变为新引入的负电性小分子，从而调控基材表面阳离子聚合物的强正电性以及伴随的与凝血过程中关键凝血因子/蛋白的强结合能力/黏附作用力(一般会造成凝血功能下降、血浆内源性凝血时间这一指标延长)。针对止血材料制备，本发明进一步提供了将这种负电性小分子/阳离子聚合物复合体转变为止血材料的制备技术，即可将其与亲水性不带电荷聚合物结合后得到止血凝胶或膜，也可以将其对高分子医用材料进行改性得到止血材料。本发明实现了对阳离子聚合物过高正电性造成内源性凝血途径负面影响的消除，通过简便的聚合物结构调控改善了阳离子聚合物的促凝血性能。
本发明方法二本发明提供了一种优化抗衡阴离子以改善生物材料止血性能的通用方法，通过表面固定状态下的阳离子聚合物浸泡于负电性小分子溶液，可发生负电性小分子与阳离子聚合物原有的抗衡阴离子的竞争过程，将一部分抗衡阴离子(如氢氧根、氯离子等)转变为新引入的负电性小分子，从而调控基材表面阳离子聚合物的强正电性以及伴随的与凝血过程中关键凝血因子/蛋白的强结合能力/黏附作用力(一般会造成凝血功能下降、血浆内源性凝血时间这一指标延长)。本发明实现了对阳离子聚合物过高正电性造成内源性凝血途径负面影响的消除，通过简便的聚合物结构调控改善了表面固定阳离子聚合物的基材的促凝血性能。因此，本发明相对于精细的化学修饰合成反应调控聚合物结构以开发具有促凝血性能的(表面固定高分子的)基材，提供了一种更简便的制备促凝血表面的方法。
综上，本发明的方法相对于精细的化学修饰合成反应调控阳离子聚合物结构以开发高分子基止血材料及促凝血表面，提供了一种更简便的利用负电性小分子改性提升阳离子聚合物基止血材料及促凝血表面的方法。
具体实施方式
下面对本发明的优选实施例进行详细的描述。
方法一相关的实验和数据是实施例1-5，对比例1-10，测试例1，表1和表2。
方法二相关的实验和数据是实施例6-8，对比例11-13，测试例2和表3。
实施例1
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，加入与聚赖氨酸质量比为1：1的甲基硫酸钠，得到共混溶液L1-M1；
2)将一定量的泊洛沙姆加入共混溶液L1-M1中，使其在溶液中的浓度为300mg/mL，得到具有止血性能的凝胶X1。
实施例2
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，加入与聚赖氨酸质量比为2：1的甲基硫酸钠，得到共混溶液L1-M2；
2)将一定量的羟丙基纤维素加入共混溶液L1-M2中，使其在溶液中的浓度为75mg/mL，取2mL混合溶液充分铺展于4.4×4.4cm 2的模具中，60℃烘干6h，得到具有止血性能的膜X2。
实施例3
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，加入与聚赖氨酸质量比为0.25：1的甲基硫酸钠，得到混合溶液L1-M0.25；
2)取5mL混合溶液充分浸透8×6cm 2共4层医用纱布，常温环境下风干24h，得到具有止血性能的改性纱布X3。
实施例4
1)在10％葡萄糖溶液中加入一定量的聚六亚甲基双胍盐酸盐使其浓度为1mg/mL，加入与聚六亚甲基双胍盐酸盐质量比为1：1的甲基硫酸钠，得到共混溶液H1-M1；
2)将一定量的泊洛沙姆加入共混溶液H1-M1中，使其在溶液中的浓度为300mg/mL，得到具有止血性能的凝胶X4。
实施例5
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，加入与聚赖氨酸质量比为1：1的甲基磺酸钠，得到共混溶液L1-D1；
2)将一定量的泊洛沙姆加入共混溶液L1-D1中，使其在溶液中的浓度为300mg/mL， 得到具有止血性能的凝胶X5。
对比例1
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，加入与聚赖氨酸质量比为0.1：1的甲基硫酸钠，得到共混溶液L1-M0.1；
2)将一定量的泊洛沙姆加入共混溶液L1-M0.1中，使其在溶液中的浓度为300mg/mL，得到凝胶Y1。
对比例2
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，加入与聚赖氨酸质量比为6：1的甲基硫酸钠，得到共混溶液L1-M6；
2)将一定量的泊洛沙姆加入共混溶液L1-M6中，使其在溶液中的浓度为300mg/mL，得到凝胶Y2。
对比例3
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为1mg/mL，得到溶液L1；
2)将一定量的泊洛沙姆加入溶液L1中，使其在溶液中的浓度为300mg/mL，得到凝胶Y3。
对比例4
1)在10％葡萄糖溶液中加入一定量的聚赖氨酸使其浓度为5mg/mL，加入与聚赖氨酸质量比为1：1的甲基硫酸钠，得到共混溶液L5-M5；
2)将一定量的泊洛沙姆加入共混溶液L5-M5中，使其在溶液中的浓度为300mg/mL，得到具有止血性能的凝胶Y4。
对比例5
1)在10％葡萄糖溶液中加入一定量的聚六亚甲基双胍盐酸盐使其浓度为1mg/mL，得到溶液H1；
2)将一定量的泊洛沙姆加入溶液H1中，使其在溶液中的浓度为300mg/mL，得到凝胶Y5。
对比例6
将一定量的羟丙基纤维素加入对比例3步骤1)中的L1溶液，使其在溶液中的浓度为75mg/mL，取2mL混合溶液充分铺展于4.4×4.4cm 2的模具中，60℃烘干6h，得到膜Y6。
对比例7
取5mL对比例3步骤1)中的L1溶液充分浸透8×6cm 2共4层医用纱布，常温环境下风干24h，得到改性纱布Y7。
对比例8
将泊洛沙姆用10％葡萄糖溶液配制成浓度为300mg/mL的溶液，得到凝胶Y8。
对比例9
将羟丙基纤维素用10％葡萄糖溶液配制成浓度为75mg/mL的溶液，取2mL溶液充分铺展于4.4×4.4cm 2的模具中，60℃烘干6h，得到多糖膜Y9。
对比例10
未改性医用纱布Y10。
测试例1
制备的促凝血增强型止血材料X1～X5，对比例Y1～Y10进行体外凝血效果对比实验。
测试方法：止血凝胶吸取50μL放入2mL塑料离心管，在37℃预先使凝胶固化5min，膜称取5mg，纱布取0.5×0.5cm 2放入2mL塑料离心管。将100μL SD大鼠的新鲜抗凝血加入10μL的氯化钙溶液(CaCl 2；0.2M)中混匀，立刻与预先准备的材料接触，在37℃的恒温水浴锅中孵育1分钟。然后再缓慢的加入10mL去离子水，继续孵育3分钟，将没有形成血凝块的血细胞充分裂解，释放血红蛋白。3min后吸取100μL液体加入96孔板，用酶联免疫检测仪测试545nm处的吸光度Abs来计算血红蛋白含量。空白组是取100μL新鲜抗凝血加入10mL去离子水，37℃的恒温水浴锅中孵育3分钟，吸取100μL测试545nm处的吸光度Abs。最后通过以下公式1来计算凝血指数(BCI)。
凝血指数％(BCI)＝(Abs样品/Abs空白)×100％...................式1
式中：Abs 样品是实验组在545nm处的吸光度；Abs 空白是空白组在545nm处的吸光度。实施例及对比例的BCI指数如表1所示：
表1体外凝血指数测试结果
样品 X1 X2 X3 X4 X5 Y1 Y2 Y3
BCI(％) 22.4 21.3 44.6 53.8 30.3 46.8 53.9 65.0
样品 Y4 Y5 Y6 Y7 Y8 Y9 Y10  
BCI(％) 54.9 87.5 62.8 92.6 50.6 58.1 76.5  
体外凝血指数BCI是表征材料体外促凝血性能、筛选止血材料的重要指标，BCI值越小，表明材料的体外促凝血性能越好。从表1可以看出，本发明实施例1～5得到的止血材料X1～X5的BCI指数明显低于对比例Y1～Y10中的同种类材料。由此可见，采用本发明的制备方法，可以得到具有优异止血性能的止血材料。
通过加入与阳离子聚合物不同质量比的负电性小分子，可有效调节阳离子聚合物的强正电性造成的凝血功能下降(即BCI指数增加)的影响，消除内源性凝血途径负面的影响，有效的提高阳离子聚合物的促凝血性能。
对比例1是跟实施例1比，在制备步骤1)中，是将少量的甲基硫酸钠与聚赖氨酸共混得到共混溶液，再将泊洛沙姆加入共混溶液制备止血凝胶，实施例1的BCI为22.4％，对比例1的BCI为46.8％，结果表明，对比例1得到的凝胶的止血性能比实施例1的差。因此说明，加入少量负电性小分子对阳离子聚合物的正电性屏蔽不够，导致材料正电性过强，对内源性凝血途径产生负面影响，造成凝血功能下降，以至于无法有效的提高止血材料的止血性能。
对比例2是跟实施例1比，在制备步骤1)中，是将过量的甲基硫酸钠与聚赖氨酸共混得到共混溶液，再将泊洛沙姆加入共混溶液制备止血凝胶，实施例1的BCI为22.4％，对比例2的BCI为53.9％，结果表明，对比例2得到的凝胶的止血性能比实施例1的差。因此说明，加入过量的负电性小分子会极大的减弱阳离子聚合物的正电性，从而影响其聚集血细胞、血小板等性能，以至于无法有效的提高止血材料的止血性能。
对比例3是跟实施例1比，是在制备步骤1)中，不添加甲基硫酸钠，将聚赖氨酸溶解于10％葡萄糖溶液中得到阳离子聚合物溶液，再将泊洛沙姆加入溶液制备止血凝胶，实施例1的BCI为22.4％，对比例3的BCI为65.0％，结果表明，对比例3得到的凝胶的止血性能比实施例1的差。因此说明，未经过小分子调控正电性的阳离子聚合物由于其过高的正电性，对内源性凝血途径产生负面影响，造成凝血功能下降，以至于无法有效的提高止血材料的止血性能。
对比例4是跟实施例1比，是在制备步骤1)中，将过量聚赖氨酸溶解于10％葡萄糖溶液中得到阳离子聚合物溶液，添加等质量的甲基硫酸钠，再将泊洛沙姆加入溶液制备止血凝胶，实施例1的BCI为22.4％，对比例4的BCI为54.9％，结果表明，对比例4得到的凝胶的止血性能比实施例1的差。因此说明，过量阳离子聚合物的正电性很强，负电性小分子对其的削弱作用有限，因此仍对内源性凝血途径产生负面影响，造成凝血 功能下降，以至于无法有效的提高止血材料的止血性能。
对比例5是跟实施例4比，是在制备步骤1)中，不添加负电性小分子甲基硫酸钠，将聚六亚甲基双胍盐酸盐溶解于10％葡萄糖溶液中得到阳离子聚合物溶液，再将泊洛沙姆加入溶液制备止血凝胶，实施例4的BCI为53.8％，对比例5的BCI为87.5％，结果表明，对比例5得到的凝胶的止血性能比实施例4的差。因此说明，未经过小分子调控正电性的阳离子聚合物由于其过高的正电性，对内源性凝血途径产生负面影响，以至于无法有效的提高止血材料的止血性能。
对比例6是跟实施例2比，是制备步骤1)中，不添加负电性小分子甲基硫酸钠，将聚赖氨酸溶解于10％葡萄糖溶液中得到阳离子聚合物溶液，再将羟丙基纤维素加入溶液制备止血膜，实施例2的BCI为21.3％，对比例6的BCI为62.8％，结果表明，对比例6得到的膜的止血性能比实施例2的差。因此说明，未经过负电性小分子调控正电性的阳离子聚合物由于其过高的正电性，对内源性凝血途径产生负面影响，以至于无法有效的提高止血材料的止血性能。
对比例7是跟实施例3比，是制备步骤1)中，不添加负电性小分子甲基硫酸钠，将聚赖氨酸溶解于10％葡萄糖溶液中得到阳离子聚合物溶液，再浸泡医用纱布进行改性，实施例3的BCI为44.6％，对比例7的BCI为92.6％，结果表明，对比例6得到的纱布的止血性能比实施例4的差。因此说明，未经过负电性小分子调控正电性的阳离子聚合物由于其过高的正电性，对内源性凝血途径产生负面影响，以至于无法有效的提高止血材料的止血性能。
对比例8是是不添加任何改性材料，单独使用泊洛沙姆制备水凝胶，水凝胶的BCI为50.6(远远大于采用本发明的方法改性得到的凝胶X1和X5的BCI)，止血性能没有添加改性材料之后的性能好。因此说明，采用本发明的方法用负电性小分子调控阳离子聚合物会提升凝胶的止血性能。
对比例9是不添加任何改性材料，单独使用羟丙基纤维素制备膜，膜的BCI为58.1％(远远大于采用本发明的方法制备得到的膜X2的BCI)，止血性能没有采用本发明改性后的止血性能好。因此说明，采用本发明的方法用负电性小分子调控的阳离子聚合物会提升膜的止血性能。
对比例10是不添加任何改性材料，单独使用医用纱布，纱布的BCI为76.5％(远远大于采用本发明的方法改性得到的医用纱布X3的BCI)，止血性能没有采用本发明的方 法改性之后的性能好。因此说明，采用本发明的方法用负电性小分子调控阳离子聚合物会提升膜的止血性能。
测试例2
将制备的促凝血增强型止血材料X1，对比例Y1、Y3进行活化部分凝血活酶时间(APTT)对比实验。
测试方法：止血凝胶吸取50μL放入2mL塑料离心管，在37℃预先使凝胶固化5min。SD大鼠抗凝全血在3000rpm离心15min，取出上清液贫血小板血浆(PPP)。取50μL PPP与50μL APTT试剂混合后立刻加入凝胶材料中，在37℃水浴孵育3min后，加入50μL氯化钙溶液，立即观察记录血浆凝固时间。空白组为不添加材料的APTT，将其作为100％。APTT(％)通过下式2计算：
APTT(％)＝(APTT材料组/APTT空白组)×100％...................式2
表2活化部分凝血活酶时间测试结果
样品 X1 Y1 Y3
APTT(％) 110 216 157
APTT指活化部分凝血活酶时间，是临床上最常用的反映内源性凝血系统凝血活性的测试。以正常血浆的APTT时间作为100％。APTT时间越长，代表对内源性凝血途径影响越大，越不利于凝血。
对比例1是跟实施例1比，在是制备步骤1)中，是将少量的甲基硫酸钠与聚赖氨酸共混得到共混溶液，再将泊洛沙姆加入共混溶液制备止血凝胶，APTT测试结果显示，X1的APTT为110％，Y1的APTT为216％，Y1时间相较于正常血浆(100％)明显延长，因此说明，采用本发明的方法的得到的X1(适量负电性小分子+阳离子聚合物)对内源性凝血途径的影响更小，而少量负电性小分子不够削弱阳离子聚合物对内源性凝血途径的负面影响，不利于凝血。
对比例3是跟实施例1比，是制备步骤1)中，不添加甲基硫酸钠，将聚赖氨酸溶解于10％葡萄糖溶液中得到阳离子聚合物溶液，再将泊洛沙姆加入溶液制备止血凝胶，APTT测试结果显示，X1的APTT为110％，Y3的APTT为157％，时间相较于正常血浆(100％)明显延长。因此说明，只有阳离子聚合物，不用负电性小分子进行调节，对内源性凝血途径的影响较大，不利于凝血。
以下是方法二相关的案例。
实施例6
1)300mg氢氧化钠溶于10mL去离子水，1200mg 2，3-环氧丙基三甲基季铵盐(GTA)溶于40mL去离子水，二者混合浸泡明胶海绵24h，去离子水洗3次，每次10min，冷冻干燥得到季铵化明胶海绵(QGS)。
2)将1)中的季铵化明胶海绵浸泡在21mg/mL的甲基硫酸钠水溶液中2h，去离子水洗3次，每次30min，冷冻干燥得到止血海绵X6。
本实施例子中，表面具备阳离子的基材为季铵化明胶海绵，阳离子聚合物为季铵化明胶海绵，负电性小分子为甲基硫酸钠。
实施例7
将实施例1中步骤1)的季铵化明胶海绵浸泡在45mg/mL的甲基磺酸钠水溶液中2h，去离子水洗3次，每次30min，冷冻干燥得到止血海绵X7。
本实施例中，表面具备阳离子的基材为季铵化明胶海绵，阳离子聚合物为季铵化明胶海绵，负电性小分子为甲基磺酸钠。
实施例8
1)304不锈钢片(简称S)裁成1×1cm 2，用异丙醇、乙醇、水连续超声清洗，干燥后用氧等离子体处理。用去离子水配制1mg/mL聚(二烯丙基二甲基氯化铵)(PDADMAC)溶液，1mg/mL聚(4-苯乙烯磺酸钠)(PSS)溶液。交替浸泡PDADMAC和PSS溶液，每次20min，去离子水洗1min，氮气吹2min，得到S-PP 4.5(S-PP 4.5最外层是阳离子聚合物聚二烯丙基二甲基氯化铵)。
2)将1)中的S-PP 4.5浸泡在1mg/mL的甲基硫酸钠水溶液中1h，去离子水洗1次，每次1min，氮气吹干得到止血材料X8。
本实施例中，表面具备阳离子的基材是304不锈钢片与聚(4-苯乙烯磺酸钠)和聚(二烯丙基二甲基氯化铵)交替自组装得到的阳离子固定化基材S-PP 4.5，阳离子聚合物为聚(二烯丙基二甲基氯化铵)，负电性小分子为甲基硫酸钠。
对比例11
实施例6中步骤1)得到的季铵化明胶海绵，命名为Y11。
对比例12
实施例8中步骤1)中的S-PP 4.5，命名为Y12。
对比例13
将实施例6中步骤1)的季铵化明胶海绵浸泡在21mg/mL的甲基磺酸钠水溶液中2min，去离子水洗3次，每次30min，冷冻干燥得到止血海绵Y13。
本实施例中，浸泡时间仅为2分钟，远远短于实施例1的2小时。
测试例2
制备的促凝血表面止血材料X6～X8，对比例Y11～Y13进行体外凝血效果对比实验。
测试方法：止血海绵系列取5mg放入2mL塑料离心管，不锈钢片取1×1cm 2放入6孔板。将100μL SD大鼠的新鲜抗凝血加入10μL的氯化钙溶液(CaCl 2；0.2M)中混匀，立刻与预先准备的材料接触，在37℃的恒温水浴锅中孵育1分钟。然后再缓慢的加入10mL去离子水，继续孵育3分钟，将没有形成血凝块的血细胞充分裂解，释放血红蛋白。3min后吸取100μL液体加入96孔板，用酶联免疫检测仪测试545nm处的吸光度Abs来计算血红蛋白含量。空白组是取100μL新鲜抗凝血加入10mL去离子水，37℃的恒温水浴锅中孵育3分钟，吸取100μL测试545nm处的吸光度Abs。最后通过以下公式来计算凝血指数(BCI)。
凝血指数％(BCI)＝(Abs 样品/Abs 空白)×100％...................式
式中：Abs 样品是实验组在545nm处的吸光度；Abs 空白是空白组在545nm处的吸光度。实施例及对比例的BCI指数如表1所示：
表3体外凝血指数测试结果
样品 X6 X7 X8 Y11 Y12 Y13
BCI(％) 42.0 38.1％ 40.8 49.1 89.9 71.2
体外凝血指数BCI是表征材料体外促凝血性能、筛选止血材料的重要指标，BCI值越小，表明材料的体外促凝血性能越好。从表3可以看出，本发明实施例6～8得到的止血材料X6～X8的BCI指数明显低于对比例Y11～Y13中的同种类材料。由此可见，采用本发明的制备方法，可以得到具有优异促凝血/止血性能的材料表面。
通过浸泡不同浓度的负电性小分子，可有效调节阳离子聚合物的正电性，消除阳离子聚合物过强正电性带来的对内源性凝血途径的不利影响，可以有效的提高阳离子聚合物的促凝血性能。
对比例11是季铵化明胶海绵不浸泡负电性小分子甲基硫酸钠进行改性，表3的Y11 的BCI比实施例6和7的BCI都大，说明未经过负电性小分子调控正电性的阳离子聚合物由于其过高的正电性，对内源性凝血途径产生负面影响，以至于无法有效的提高明胶海绵表面的促凝血/止血性能。
对比例12是包覆阳离子聚合物涂层的不锈钢片不浸泡甲基硫酸钠进行改性，表3的Y12的BCI结果比实施例8的BCI大的多，说明，未经过负电性小分子调控正电性的阳离子聚合物的不锈钢表面由于其过高的正电性，对内源性凝血途径产生负面影响，以至于无法有效的提高不锈钢表面的促凝血/止血性能。
对比例13是在实施例6的制备步骤2)中，对季铵化明胶海绵浸泡浓度合适的甲基磺酸钠、但浸泡时间过短不利于抗衡离子有效交换，表3的Y13的BCI大于X6的BCI值，说明不合适的负电性小分子调控过程对阳离子聚合物的正电性屏蔽不够，导致材料正电性过强，对内源性凝血途径产生负面影响，以至于无法有效的提高止血材料的止血性能。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

Claims (10)

  1. 一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，有两种方法：方法一为负电性小分子和阳离子聚合物进行一锅法调控，具体步骤为：
    1)将阳离子聚合物与负电性小分子溶解，得到共混溶液，所述的阳离子聚合物与负电性小分子的质量比为1:0.15～2；
    2)将步骤1)得到的共混溶液制成凝胶或膜，或将共混溶液对基材进行改性，制备得到止血材料；
    方法二为负电性小分子在阳离子表面进行分步调控，步骤为：
    将表面具备阳离子的基材通过浸泡负电性小分子溶液进行改性，得到具有促凝血表面的材料，具体步骤为：
    1)配制负电性小分子溶液；
    2)将表面具备阳离子的基材浸入步骤1)的溶液中；
    3)洗涤、干燥，得到负电性小分子调控的促凝血表面；
    所述的负电性小分子为甲基硫酸钠、甲基磺酸钠、N-环己基氨基磺酸钠、吗啉乙磺酸钠盐一水合物、3-N(-吗啉基)丙磺酸钠、3-吗啉-2-羟基丙磺酸钠、葡萄糖酸的一种或多种混合。
  2. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法一的步骤1)所述的共混溶液为水溶液或水与甲醇、乙醇、乙酸乙酯、异丙醇形成的混合溶液；所述的水溶液为葡萄糖溶液，葡萄糖的浓度为5％～10％；方法一的步骤1)所述的阳离子聚合物为聚赖氨酸、聚甲基丙烯酸二甲氨基乙酯、聚六亚甲基双胍盐酸盐、聚六亚甲基胍盐酸盐、季铵化淀粉的一种或多种混合，其在溶液中的浓度范围在0.1～3mg/mL。
  3. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法一的步骤1)所述的阳离子聚合物为聚赖氨酸或聚六亚甲基双胍盐酸盐；步骤1)所述的负电性小分子为甲基硫酸钠或甲基磺酸钠。
  4. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法一步骤2)所述的共混溶液制成凝胶的制备方法为：将亲水性不带电荷聚合物加入步骤1)得到的共混溶液中，得到混合凝胶，所述亲水性不带电荷聚合物为泊洛沙姆、聚丙交酯-乙交酯-聚乙二醇-聚丙交酯-乙交酯、聚(N-异丙基丙烯酰胺)等温敏亲水性聚合物一种或多种混合；方法一步骤2)所述的共混溶液制成膜的制备方法为：将亲水性不带电荷聚合物加入方法一步骤1)得到的共混溶液中，得到的混合溶液高温干燥，制成具有止血性能的止血膜，所述的亲水性不带电荷聚合物为羟丙基纤维素、羟丙基甲基纤维素、羟甲基纤维素、羟乙基纤维素、甲基纤维素等亲水性纤维素衍生物的一种或多种混合；方法一步骤2) 所述的共混溶液对基材改性方法为：利用方法一步骤1)得到的共混溶液对基材进行浸泡，然后取出基材进行干燥，所选的基材为纱布、明胶海绵、海藻酸无纺布等高分子医用材料。
  5. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法一所述的共混溶液制成凝胶的步骤为：在葡萄糖溶液中，加入阳离子聚合物使其在溶液中的浓度范围为0.1～3mg/mL、负电性小分子，加入温敏性聚合物泊洛沙姆、聚丙交酯-乙交酯-聚乙二醇-聚丙交酯-乙交酯或聚(N-异丙基丙烯酰胺，使温敏性聚合物在溶液中的浓度范围为100～400mg/mL；方法一所述的共混溶液制成膜的步骤为：在葡萄糖溶液中，加入阳离子聚合物使其在溶液中的浓度范围为0.1～3mg/mL、负电性小分子，加入羟丙基纤维素、羟丙基甲基纤维素或羟乙基纤维素使其在溶液中的浓度范围为20～200mg/mL；方法一所述的共混溶液对基材进行改性的步骤为：在葡萄糖溶液中，加入阳离子聚合物使其在溶液中的浓度范围为0.1～3mg/mL、负电性小分子，将阳离子聚合物和负电性小分子的混合溶液充分浸透基材。
  6. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法二的负电性小分子溶液的浓度为0.5-50mg/mL，方法二步骤2)所述的浸泡过程时间为0.5-10h。
  7. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法二中所述的表面具备阳离子的基材有三种：
    基材a为本身表面不含有阳离子的基材与阳离子聚合物经自组装得到的阳离子固定化基材；
    基材b为本身表面不含有阳离子的基材与正电性小分子经化学反应得到的表面阳离子固定化基材；
    基材c为本身表面含有大量阳离子基团的基材，阳离子基团为胍基、伯胺、季铵或叔胺的一种或多种组合。
  8. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法二中基材a中所述的自组装为将阳离子聚合物和负电性聚合物通过层层自组装方法包覆在表面，并控制最外层是阳离子聚合物；方法二中基材a中所述的本身表面不含有阳离子的基材为304不锈钢、金属钛钉、硅片、明胶海绵、聚乙烯醇海绵或医用胶原蛋白海绵；方法二中基材a中所述的阳离子聚合物为聚二烯丙基二甲基氯化铵、聚甲基丙烯酸N,N-二甲基氨基乙酯、聚赖氨酸、聚六亚甲基双胍盐酸盐、聚六亚甲基单胍盐酸盐中的一种或多种混合，所述的负电性聚合物为聚(4-苯乙烯磺酸钠)、聚甲基丙烯酸中的一种或多种混合。
  9. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，方法二中基材b中所述的本身表面不含有阳离子的基材为明胶海绵、聚乙烯醇海绵、医用胶原蛋白海绵或纱布；方法二中基材b中所述的化学反应为季铵化反应，通过本身表面不含有阳离子的基材表面的羟基、氨基或羧基与含环氧基的季铵盐发生开环反应；所述的含环氧基的季铵盐为2,3-环氧丙基三甲基氯化铵或由环氧氯丙烷和N,N-二甲基X胺反应得到的产物，所述的X为乙、丙、丁、己、辛、癸、十二、十四。
  10. 根据权利要求1所述的一种优化抗衡阴离子以改善生物材料止血性能的通用方法，其特征在于，2，3-环氧丙基三甲基季铵盐改性的明胶和甲基硫酸钠进行浸泡后，可以得到具有促凝血表面的材料；聚(4-苯乙烯磺酸钠)和聚(二烯丙基二甲基氯化铵)交替自组装改性得到的阳离子固定化304不锈钢表面和甲基硫酸钠进行浸泡后，可以得到具有促凝血表面的材料。
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CN1448189A (zh) * 2003-04-28 2003-10-15 浙江大学 采用静电自组装制备抗凝血生物材料的方法
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CN107362387A (zh) * 2017-07-30 2017-11-21 成都优瑞商务服务有限公司 一种医用止血材料及其制备方法
CN113975447A (zh) * 2021-11-24 2022-01-28 四川大学 一种抗菌海藻酸盐敷料及其制备方法和用途

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CN1448189A (zh) * 2003-04-28 2003-10-15 浙江大学 采用静电自组装制备抗凝血生物材料的方法
CN106474530A (zh) * 2015-08-24 2017-03-08 中国科学院金属研究所 一种基于壳寡糖的聚电解质海绵止血敷料的制备方法
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