Universal method for optimizing counter anion to improve hemostatic performance of biological material
Patent Information
- Application Number
- GB2024016434
- Authority / Receiving Office
- GB · GB
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2022-09-01
- Publication Date
- 2024-12-25
AI Technical Summary
The excessively high electropositivity of cationic polymers in hemostatic materials delays the generation of thrombin, leading to a decrease in coagulation function and a prolongation of the endogenous coagulation time of plasma. It is difficult to effectively regulate its structure to improve the procoagulant performance with existing technology.
By introducing the competition between negatively charged small molecules and cationic polymers, part of the counter anions are converted into negatively charged small molecules, and the strong positive charge of the cationic polymer and the binding ability of coagulation factors/proteins are controlled, combined with the hydrophilic uncharged polymerization Hemostatic gels or membranes can be prepared from materials, or the cationic polymer substrate can be modified by soaking in a solution of negatively charged small molecules to regulate its surface procoagulant properties.
It effectively eliminates the decrease in coagulation function caused by the excessively positive electricity of cationic polymers, improves the procoagulant performance of hemostatic materials, and simply improves the polymer structure to enhance the procoagulant performance of cationic polymer substrates.
Abstract
Description
A general approach to optimizing counteranions to improve hemostatic properties of biomaterials Technical Field
[0001] The invention belongs to the technical field of preparation of medical hemostatic materials and relates to a general method for optimizing counter anions to improve the hemostatic performance of biomaterials. Background Art
[0002] Due to their flexible and diverse structural design, polymer materials are widely studied in fields such as hemostasis, antibacterial agents, and drug delivery. Cationic polymers, among others, are known for their ability to aggregate negatively charged proteins and cells and are therefore being used in the development of various hemostatic materials. However, their effect on coagulation is a double-edged sword: their excessively high positive charge can also delay thrombin generation. Therefore, it is necessary to manipulate the structure of cationic polymers to improve their procoagulant properties. Compared to elaborate chemical synthesis reactions to manipulate cationic polymer structure, introducing negatively charged small molecules onto cationic polymers through small molecule exchange offers the potential for simpler polymer structure manipulation. Based on this, through the competitive interaction between negatively charged small molecules and the original counteranions of the cationic polymer, some counteranions (such as hydroxide and chloride ions) are converted into newly introduced negatively charged small molecules, thereby modulating the strong positive charge of the cationic polymer on the substrate surface and the accompanying strong binding and adhesion to key coagulation factors and proteins in the coagulation process (generally resulting in decreased coagulation function and prolonged plasma intrinsic clotting time). Regarding the preparation of hemostatic materials, the present invention further provides a preparation technology for converting this negatively charged small molecule / cationic polymer complex into a hemostatic material, which can be combined with a hydrophilic uncharged polymer to obtain a hemostatic gel or membrane, or it can be used to modify a polymer medical material to obtain a hemostatic material; the positive charge of the cationic polymer can also be regulated by the negatively charged small molecule, and the surface of different substrates with cationic polymers can be modified to obtain a pro-coagulant surface.
[0003] Summary of the Invention
[0004] In view of this, the present invention provides a general method for optimizing counter anions to improve the hemostatic properties of biomaterials. Specifically, the following technical solutions are provided:
[0005] A general approach to optimizing counteranions to improve the hemostatic properties of biomaterials involves two methods:
[0006] Method 1 is a one-pot regulation of negatively charged small molecules and cationic polymers. The specific steps are as follows:
[0007] 1) dissolving a cationic polymer and a negatively charged small molecule to obtain a blend solution, wherein the mass ratio of the cationic polymer to the negatively charged small molecule is 1:0.15-2;
[0008] 2) forming the blended solution obtained in step 1) into a gel or a film, or modifying a substrate with the blended solution to prepare a hemostatic material;
[0009] The second method is to regulate the negatively charged small molecules on the cationic surface in steps. The specific steps are as follows:
[0010] The substrate with cationic surface is modified by soaking in a negatively charged small molecule solution to obtain a material with a procoagulant surface. The specific steps are as follows:
[0011] 1) Prepare a negatively charged small molecule solution;
[0012] 2) immersing the substrate having the cations on its surface into the solution of step 1);
[0013] 3) washing and drying to obtain a pro-coagulant surface regulated by negatively charged small molecules;
[0014] The negatively charged small molecule is a mixture of one or more of sodium methyl sulfate, sodium methanesulfonate, sodium N-cyclohexylaminosulfonate, sodium morpholineethanesulfonic acid monohydrate, sodium 3-N(-morpholinyl)propanesulfonate, sodium 3-morpholine-2-hydroxypropanesulfonate, and gluconic acid.
[0015] Furthermore, the blended solution described in step 1) of method one is an aqueous solution or a mixed solution of water and methanol, ethanol, ethyl acetate, or isopropanol; the aqueous solution is a glucose solution, and the concentration of glucose is 5% to 10%; the cationic polymer described in step 1) of method one is a mixture of one or more of polylysine, polydimethylaminoethyl methacrylate, polyhexamethylene biguanide hydrochloride, polyhexamethyleneguanidine hydrochloride, and quaternized starch, and its concentration in the solution ranges from 0.1 to 3 mg / mL.
[0016] Furthermore, the cationic polymer in step 1) of method 1 is polylysine or polyhexamethylene biguanide hydrochloride; and the negatively charged small molecule in step 1) is sodium methyl sulfate or sodium methane sulfonate.
[0017] Furthermore, the preparation method of the blend solution described in step 2) of method 1 as a gel is as follows: a hydrophilic non-charged polymer is added to the blend solution obtained in step 1) to obtain a mixed gel, wherein the hydrophilic non-charged polymer is a mixture of one or more thermosensitive hydrophilic polymers such as poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide, and poly(N-isopropylacrylamide); the preparation method of the blend solution described in step 2) of method 1 as a membrane is as follows: a hydrophilic non-charged polymer is added to the blend solution obtained in step 1) of method 1 to obtain a mixed gel. The obtained mixed solution is dried at high temperature to prepare a hemostatic membrane with hemostatic properties, wherein the hydrophilic non-charged polymer is a mixture of one or more hydrophilic cellulose derivatives such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and methyl cellulose; the method for modifying the substrate with the blended solution described in step 2) of method 1 is: soaking the substrate with the blended solution obtained in step 1) of method 1, and then taking out the substrate for drying. The selected substrate is a polymer medical material such as gauze, gelatin sponge, and alginate non-woven fabric.
[0018] Furthermore, the step of preparing a gel by the blend solution described in method 1 is as follows: adding a cationic polymer to a glucose solution so that its concentration in the solution ranges from 0.1 to 3 mg / mL, a negatively charged small molecule, and adding a thermosensitive polymer poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide or poly(N-isopropylacrylamide) so that the concentration of the thermosensitive polymer in the solution ranges from 100 to 400 mg / mL; the step of preparing a membrane by the blend solution described in method 1 is as follows: adding a cationic polymer to a glucose solution so that its concentration in the solution ranges from 0.1 to 3 mg / mL, a negatively charged small molecule, and adding hydroxypropyl cellulose, hydroxypropyl methylcellulose or hydroxyethyl cellulose so that its concentration in the solution ranges from 20 to 200 mg / mL; the step of modifying the substrate by the blend solution described in method 1 is as follows: adding a cationic polymer to a glucose solution so that its concentration in the solution ranges from 0.1 to 3 mg / mL, a negatively charged small molecule, and fully impregnating the substrate with the mixed solution of the cationic polymer and the negatively charged small molecule.
[0019] Furthermore, the concentration of the negatively charged small molecule solution in method 2 is 0.5-50 mg / mL, and the soaking process time in step 2) of method 2 is 0.5-10 h.
[0020] Furthermore, there are three types of substrates with cations on the surface described in Method 2:
[0021] 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;
[0022] Substrate b is a surface cation-immobilized substrate obtained by chemical reaction between a substrate that does not contain cations on its surface and a positively charged small molecule;
[0023] The substrate c is a substrate having 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 groups or tertiary amines.
[0024] Furthermore, the self-assembly described in substrate a in method 2 is to coat the cationic polymer and the negatively charged polymer on the surface through a layer-by-layer self-assembly method, and control the outermost layer to be a cationic polymer; the substrate a in method 2 that does not contain cations on its surface is 304 stainless steel, a metal titanium nail, a silicon wafer, a gelatin sponge, a polyvinyl alcohol sponge or a medical collagen sponge; the cationic polymer in substrate a in method 2 is a mixture of one or more of polydiallyldimethylammonium chloride, poly(N,N-dimethylaminoethyl methacrylate), polylysine, polyhexamethylene biguanide hydrochloride, and polyhexamethylene monoguanide hydrochloride, and the negatively charged polymer is a mixture of one or more of poly(sodium 4-styrene sulfonate) and polymethacrylic acid.
[0025] Furthermore, the substrate b in method 2, which does not contain cations on its surface, is a gelatin sponge, a polyvinyl alcohol sponge, a medical collagen sponge, or a gauze; the chemical reaction in method 2, which does not contain cations on its surface, is a quaternization reaction, wherein the hydroxyl, amino, or carboxyl groups on the surface of the substrate, which does not contain cations on its surface, react with an epoxy-containing quaternary ammonium salt to produce a ring-opening reaction; the epoxy-containing quaternary ammonium salt is 2,3-epoxypropyltrimethylammonium chloride or a product obtained by the reaction of epichlorohydrin and N,N-dimethyl-X-amine, where X is ethyl, propyl, butyl, hexyl, octyl, decyl, dodecayl, or tetradecyl.
[0026] Furthermore, method 2 is to obtain a material with a procoagulant surface by soaking gelatin modified with 2,3-epoxypropyltrimethyl quaternary ammonium salt in sodium methyl sulfate; and to obtain a material with a procoagulant surface by soaking a cation-immobilized 304 stainless steel surface modified by alternating self-assembly of poly(sodium 4-styrene sulfonate) and poly(diallyldimethylammonium chloride) in sodium methyl sulfate.
[0027] The beneficial effects of the present invention are as follows: the present invention provides a general method for optimizing counter anions to improve the hemostatic properties of biomaterials. Through the competitive effect between negatively charged small molecules and the original counter anions of cationic polymers, a portion of the counter anions (such as hydroxide, chloride ions, etc.) are converted into newly introduced negatively charged small molecules, thereby regulating the strong positive charge of the cationic polymer on the surface of the substrate and the accompanying strong binding ability / adhesion force with key coagulation factors / proteins in the coagulation process (generally resulting in a decrease in coagulation function and an extension of the plasma intrinsic coagulation time). With respect to the preparation of hemostatic materials, the present invention further provides a preparation technology for converting this negatively charged small molecule / cationic polymer complex into a hemostatic material, which can be combined with a hydrophilic uncharged polymer to obtain a hemostatic gel or membrane, or it can be used to modify a polymer medical material to obtain a hemostatic material. The present invention eliminates the negative effects of the intrinsic coagulation pathway caused by the excessively high positive charge of the cationic polymer, and improves the procoagulant properties of the cationic polymer through simple polymer structure regulation.
[0028] Method 2 of the present invention The present invention provides a general method for optimizing counter anions to improve the hemostatic properties of biomaterials. By immersing the cationic polymer in a surface-fixed state in a negatively charged small molecule solution, a competition process between the negatively charged small molecules and the original counter anions of the cationic polymer can occur, and a portion of the counter anions (such as hydroxide, chloride ions, etc.) is converted into newly introduced negatively charged small molecules, thereby regulating the strong positive charge of the cationic polymer on the surface of the substrate and the accompanying strong binding ability / adhesion force with key coagulation factors / proteins in the coagulation process (generally resulting in a decrease in coagulation function and an extension of the plasma intrinsic coagulation time). The present invention achieves the elimination of the negative impact of the intrinsic coagulation pathway caused by the excessively high positive charge of the cationic polymer, and improves the procoagulant properties of the substrate with the surface-fixed cationic polymer through simple polymer structure regulation. Therefore, the present invention provides a simpler method for preparing a procoagulant surface relative to the sophisticated chemical modification synthesis reaction to regulate the polymer structure to develop a substrate (surface-fixed polymer) with procoagulant properties.
[0029] In summary, compared with the sophisticated chemical modification synthesis reaction to regulate the cationic polymer structure to develop polymer-based hemostatic materials and pro-coagulant surfaces, the method of the present invention provides a simpler method for utilizing negatively charged small molecules to modify and improve cationic polymer-based hemostatic materials and pro-coagulant surfaces. DETAILED DESCRIPTION
[0030] The preferred embodiments of the present invention are described in detail below.
[0031] The experiments and data related to Method 1 are Examples 1-5, Comparative Examples 1-10, Test Example 1, Table 1 and Table 2.
[0032] The experiments and data related to Method 2 are Examples 6-8, Comparative Examples 11-13, Test Example 2 and Table 3.
[0033] Example 1
[0034] 1) Add a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL, and add sodium methyl sulfate at a mass ratio of 1:1 to polylysine to obtain a blend solution L1-M1;
[0035] 2) A certain amount of poloxamer was added to the blended solution L1-M1 so that its concentration in the solution was 300 mg / mL, thereby obtaining a gel X1 having hemostatic properties.
[0036] Example 2
[0037] 1) Adding a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL, and adding sodium methyl sulfate at a mass ratio of 2:1 to polylysine to obtain a blend solution L1-M2;
[0038] 2) Add a certain amount of hydroxypropyl cellulose to the blend solution L1-M2 to make its concentration in the solution 75 mg / mL, take 2 mL of the mixed solution and spread it fully on a 4.4×4.4 cm 2 The film was placed in a mold and dried at 60°C for 6 hours to obtain a membrane X2 with hemostatic properties.
[0039] Example 3
[0040] 1) Add a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL, and add sodium methyl sulfate at a mass ratio of 0.25:1 to polylysine to obtain a mixed solution L1-M0.25;
[0041] 2) Take 5mL of the mixed solution and fully soak 8×6cm 2 A total of 4 layers of medical gauze were air-dried at room temperature for 24 hours to obtain modified gauze X3 with hemostatic properties.
[0042] Example 4
[0043] 1) adding a certain amount of polyhexamethylene biguanide hydrochloride to a 10% glucose solution to obtain a concentration of 1 mg / mL, and adding sodium methyl sulfate at a mass ratio of 1:1 to polyhexamethylene biguanide hydrochloride to obtain a blend solution H1-M1;
[0044] 2) A certain amount of poloxamer was added to the blended solution H1-M1 so that its concentration in the solution was 300 mg / mL, thereby obtaining a gel X4 having hemostatic properties.
[0045] Example 5
[0046] 1) Adding a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL, and adding sodium methanesulfonate at a mass ratio of 1:1 to polylysine to obtain a blend solution L1-D1;
[0047] 2) A certain amount of poloxamer was added to the blended solution L1-D1 so that its concentration in the solution was 300 mg / mL, thereby obtaining a gel X5 having hemostatic properties.
[0048] Comparative Example 1
[0049] 1) Adding a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL, and adding sodium methyl sulfate at a mass ratio of 0.1:1 to polylysine to obtain a blend solution L1-M0.1;
[0050] 2) A certain amount of poloxamer was added to the blend solution L1-M0.1 so that its concentration in the solution was 300 mg / mL to obtain gel Y1.
[0051] Comparative Example 2
[0052] 1) Adding a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL, and adding sodium methyl sulfate at a mass ratio of 6:1 to polylysine to obtain a blend solution L1-M6;
[0053] 2) A certain amount of poloxamer was added to the blended solution L1-M6 so that its concentration in the solution was 300 mg / mL to obtain gel Y2.
[0054] Comparative Example 3
[0055] 1) Adding a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 1 mg / mL to obtain solution L1;
[0056] 2) A certain amount of poloxamer was added to solution L1 so that its concentration in the solution was 300 mg / mL to obtain gel Y3.
[0057] Comparative Example 4
[0058] 1) Adding a certain amount of polylysine to a 10% glucose solution to obtain a concentration of 5 mg / mL, and adding sodium methyl sulfate at a mass ratio of 1:1 to polylysine to obtain a blend solution L5-M5;
[0059] 2) A certain amount of poloxamer was added to the blended solution L5-M5 so that its concentration in the solution was 300 mg / mL, thereby obtaining gel Y4 having hemostatic properties.
[0060] Comparative Example 5
[0061] 1) Adding a certain amount of polyhexamethylene biguanide hydrochloride to a 10% glucose solution to obtain a concentration of 1 mg / mL to obtain solution H1;
[0062] 2) A certain amount of poloxamer was added to solution H1 so that its concentration in the solution was 300 mg / mL to obtain gel Y5.
[0063] Comparative Example 6
[0064] A certain amount of hydroxypropyl cellulose was added to the L1 solution in step 1) of comparative example 3 to make its concentration in the solution 75 mg / mL. 2 mL of the mixed solution was spread on a 4.4×4.4 cm 2 The film was placed in a mold and dried at 60°C for 6h to obtain film Y6.
[0065] Comparative Example 7
[0066] Take 5mL of the L1 solution in step 1) of comparative example 3 and fully soak the 8×6cm 2 A total of 4 layers of medical gauze were air-dried at room temperature for 24 h to obtain modified gauze Y7.
[0067] Comparative Example 8
[0068] Poloxamer was prepared with 10% glucose solution to a concentration of 300 mg / mL to obtain gel Y8.
[0069] Comparative Example 9
[0070] Prepare a 75 mg / mL solution of hydroxypropyl cellulose with 10% glucose solution, take 2 mL of the solution and spread it on a 4.4 × 4.4 cm 2 The film was placed in a mold and dried at 60°C for 6 h to obtain polysaccharide film Y9.
[0071] Comparative Example 10
[0072] Unmodified medical gauze Y10.
[0073] Test Example 1
[0074] The prepared pro-coagulant enhanced hemostatic materials X1 to X5 and comparative examples Y1 to Y10 were subjected to an in vitro coagulation effect comparison experiment.
[0075] Test method: 50 μL of hemostatic gel was taken and placed in a 2 mL plastic centrifuge tube. The gel was solidified at 37°C for 5 min. 5 mg of the membrane was weighed and 0.5 × 0.5 cm of gauze was taken. 2Place in a 2mL plastic centrifuge tube. Add 100μL of fresh anticoagulated blood from SD rats to 10μL of calcium chloride solution (CaCl2; 0.2M) and mix thoroughly. Immediately contact the prepared materials and incubate in a 37°C constant temperature water bath for 1 minute. Then, slowly add 10mL of deionized water and continue incubating for 3 minutes to fully lyse the blood cells that have not formed blood clots and release hemoglobin. After 3 minutes, aspirate 100μL of the liquid and add it to a 96-well plate. Use an enzyme-linked immunosorbent assay (ELISA) to measure the absorbance at 545nm (Abs) to calculate the hemoglobin content. For the blank group, add 100μL of fresh anticoagulated blood to 10mL of deionized water, incubate in a 37°C constant temperature water bath for 3 minutes, and aspirate 100μL to measure the absorbance at 545nm (Abs). Finally, calculate the blood coagulation index (BCI) using the following formula 1.
[0076] Blood coagulation index % (BCI) = (Abs sample / Abs blank) × 100% ................... Formula 1
[0077] Where: Abs 样品 is the absorbance of the experimental group at 545 nm; Abs 空白 is the absorbance of the blank group at 545nm. The BCI index of the embodiment and the comparative example is shown in Table 1:
[0078] Table 1 In vitro coagulation index test results
[0079] Sample X1X2X3X4X5Y1Y2Y3BCI(%)22.421.344.653.830.346.853.965.0Sample Y4Y5Y6Y7Y8Y9Y10BCI(%)54.987.562.892.650.658.176.5
[0080] The in vitro coagulation index (BCI) is an important indicator for characterizing a material's in vitro procoagulant properties and for screening hemostatic materials. Lower BCI values indicate better in vitro procoagulant properties. As shown in Table 1, the BCI indices of hemostatic materials X1-X5 obtained in Examples 1-5 of the present invention are significantly lower than those of the same materials in Comparative Examples Y1-Y10. This demonstrates that the preparation method of the present invention can produce hemostatic materials with excellent hemostatic properties.
[0081] By adding negatively charged small molecules in different mass ratios to cationic polymers, the effect of decreased coagulation function (i.e., increased BCI index) caused by the strong positive charge of cationic polymers can be effectively regulated, the negative effects of the intrinsic coagulation pathway can be eliminated, and the procoagulant properties of cationic polymers can be effectively improved.
[0082] Comparative Example 1 is compared with Example 1. In preparation step 1), a small amount of sodium methyl sulfate and polylysine were blended to obtain a blended solution, and poloxamer was then added to the blended solution to prepare the hemostatic gel. The BCI of Example 1 was 22.4%, while that of Comparative Example 1 was 46.8%. The results showed that the hemostatic performance of the gel obtained in Comparative Example 1 was inferior to that of Example 1. This indicates that the addition of a small amount of negatively charged small molecules did not adequately shield the positive charge of the cationic polymer, resulting in an overly positive charge in the material, negatively impacting the intrinsic coagulation pathway and causing a decrease in coagulation function, thus failing to effectively improve the hemostatic performance of the hemostatic material.
[0083] Comparative Example 2 compares Example 1. In step 1), an excess of sodium methylsulfate and polylysine were blended to form a blended solution, and poloxamer was then added to the blended solution to prepare the hemostatic gel. The BCI of Example 1 was 22.4%, while that of Comparative Example 2 was 53.9%. The results showed that the hemostatic performance of the gel obtained in Comparative Example 2 was inferior to that of Example 1. This demonstrates that the addition of excessive negatively charged small molecules significantly weakens the positive charge of the cationic polymer, thereby affecting its ability to aggregate blood cells and platelets, and thus failing to effectively improve the hemostatic performance of the hemostatic material.
[0084] Comparative Example 3, compared to Example 1, omitted sodium methylsulfate in step 1) of preparation. Instead, polylysine was dissolved in a 10% glucose solution to obtain a cationic polymer solution, and poloxamer was added to the solution to prepare the hemostatic gel. The BCI of Example 1 was 22.4%, while that of Comparative Example 3 was 65.0%. The results showed that the hemostatic performance of the gel obtained in Comparative Example 3 was inferior to that of Example 1. This suggests that the excessively high positive charge of cationic polymers without small molecule manipulation of positive charge negatively impacts the intrinsic coagulation pathway, resulting in decreased coagulation function and, consequently, an inability to effectively improve the hemostatic performance of the hemostatic material.
[0085] Comparative Example 4, compared to Example 1, differs from Example 1 in that, in preparation step 1), an excess amount of polylysine was dissolved in a 10% glucose solution to obtain a cationic polymer solution, an equal amount of sodium methylsulfate was added, and poloxamer was then added to the solution to prepare a hemostatic gel. The BCI of Example 1 was 22.4%, while that of Comparative Example 4 was 54.9%. The results showed that the hemostatic performance of the gel obtained in Comparative Example 4 was inferior to that of Example 1. This suggests that the excess cationic polymer has a strong positive charge, while the negatively charged small molecule has a limited weakening effect on it. Therefore, it still negatively affects the intrinsic coagulation pathway, resulting in a decrease in coagulation function and an inability to effectively improve the hemostatic performance of the hemostatic material.
[0086] Comparative Example 5, compared to Example 4, omitted the addition of the negatively charged small molecule sodium methylsulfate in step 1). Instead, polyhexamethylene biguanide hydrochloride was dissolved in a 10% glucose solution to obtain a cationic polymer solution, and poloxamer was then added to the solution to prepare a hemostatic gel. The BCI of Example 4 was 53.8%, while that of Comparative Example 5 was 87.5%. The results showed that the hemostatic performance of the gel obtained in Comparative Example 5 was inferior to that of Example 4. This suggests that the excessively high positive charge of cationic polymers, which have not been manipulated by small molecules for positive charge, negatively impacts the intrinsic coagulation pathway, rendering them ineffective in improving the hemostatic performance of hemostatic materials.
[0087] Comparative Example 6, compared to Example 2, omitted the addition of the negatively charged small molecule sodium methylsulfate in step 1) of preparation. Instead, polylysine was dissolved in a 10% glucose solution to obtain a cationic polymer solution, and hydroxypropyl cellulose was added to the solution to prepare a hemostatic membrane. The BCI of Example 2 was 21.3%, while that of Comparative Example 6 was 62.8%. The results showed that the hemostatic performance of the membrane obtained in Comparative Example 6 was inferior to that of Example 2. This suggests that the excessively high positive charge of the positively charged cationic polymer, which has not been modulated by a negatively charged small molecule, negatively impacts the intrinsic coagulation pathway, rendering it ineffective in improving the hemostatic performance of the hemostatic material.
[0088] Comparative Example 7, compared to Example 3, omitted the addition of the negatively charged small molecule sodium methylsulfate in step 1). Instead, polylysine was dissolved in a 10% glucose solution to obtain a cationic polymer solution, which was then used to immerse medical gauze for modification. The BCI of Example 3 was 44.6%, while that of Comparative Example 7 was 92.6%. The results showed that the hemostatic performance of the gauze obtained in Comparative Example 6 was inferior to that of Example 4. This suggests that the excessively high positive charge of the positively charged cationic polymer, which has not been modulated by the negatively charged small molecule, negatively affects the intrinsic coagulation pathway, rendering it ineffective in improving the hemostatic performance of the hemostatic material.
[0089] Comparative Example 8, in which poloxamer alone was used to prepare a hydrogel without any modifying materials, showed a BCI of 50.6 (much higher than the BCIs of gels X1 and X5 modified using the method of the present invention). However, the hemostatic performance was not as good as that achieved with the addition of the modifying materials. This demonstrates that the method of the present invention, using negatively charged small molecules to modulate cationic polymers, improves the hemostatic performance of the gel.
[0090] Comparative Example 9, in which no modifying materials were added, used hydroxypropyl cellulose alone to prepare a membrane. The membrane had a BCI of 58.1% (much higher than that of membrane X2 prepared using the method of the present invention), and its hemostatic performance was inferior to that of the membrane modified using the method of the present invention. This demonstrates that the use of cationic polymers modulated by negatively charged small molecules using the method of the present invention improves the hemostatic performance of the membrane.
[0091] Comparative Example 10, in which medical gauze alone was used without any modified materials, had a BCI of 76.5% (much higher than the BCI of medical gauze X3 modified using the method of the present invention). However, the hemostatic performance was not as good as that of the modified gauze using the method of the present invention. This demonstrates that the hemostatic properties of the membrane can be enhanced by using negatively charged small molecules to modulate cationic polymers using the method of the present invention.
[0092] Test Example 2
[0093] An activated partial thromboplastin time (APTT) comparison experiment was performed on the prepared pro-coagulant enhanced hemostatic material X1 and comparative examples Y1 and Y3.
[0094] Test method: 50 μL of hemostatic gel was drawn into a 2 mL plastic centrifuge tube and the gel was solidified at 37°C for 5 minutes. Anticoagulated whole blood of SD rats was centrifuged at 3000 rpm for 15 minutes, and the supernatant platelet-poor plasma (PPP) was taken out. 50 μL of PPP was mixed with 50 μL of APTT reagent and immediately added to the gel material. After incubation in a 37°C water bath for 3 minutes, 50 μL of calcium chloride solution was added and the plasma coagulation time was immediately observed and recorded. The blank group was the APTT without adding material, which was taken as 100%. APTT (%) was calculated by the following formula 2:
[0095] APTT (%) = (APTT material group / APTT blank group) × 100% ................... Formula 2
[0096] Table 2 Activated partial thromboplastin time test results
[0097] Sample X1Y1Y3APTT (%) 110216157
[0098] APTT, or activated partial thromboplastin time, is the most commonly used clinical test to measure the activity of the intrinsic coagulation system. The APTT time of normal plasma is taken as 100%. A longer APTT time indicates a greater impact on the intrinsic coagulation pathway, impairing coagulation.
[0099] Comparative Example 1 is compared with Example 1. In the 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 gel. The APTT test results show that the APTT of X1 is 110%, the APTT of Y1 is 216%, and the Y1 time is significantly prolonged compared with normal plasma (100%). Therefore, it is shown that X1 (appropriate amount of negatively charged small molecules + cationic polymers) obtained by the method of the present invention has less effect on the intrinsic coagulation pathway, and a small amount of negatively charged small molecules is not enough to weaken the negative effect of cationic polymers on the intrinsic coagulation pathway, which is not conducive to coagulation.
[0100] Comparative Example 3, compared to Example 1, omitted sodium methylsulfate in step 1) of the preparation process. Instead, polylysine was dissolved in a 10% glucose solution to obtain a cationic polymer solution, and poloxamer was then added to the solution to prepare the hemostatic gel. The APTT test results showed that the APTT for X1 was 110% and that for Y3 was 157%, significantly prolonged compared to normal plasma (100%). This suggests that the cationic polymer alone, without the modulation of a negatively charged small molecule, significantly impacts the intrinsic coagulation pathway, hindering coagulation.
[0101] The following are examples related to method 2.
[0102] Example 6
[0103] 1) Dissolve 300 mg of sodium hydroxide in 10 mL of deionized water and 1200 mg of 2,3-epoxypropyltrimethylammonium salt (GTA) in 40 mL of deionized water. Soak gelatin sponge with the mixture for 24 h. Wash with deionized water three times for 10 min each time and freeze-dry to obtain quaternized gelatin sponge (QGS).
[0104] 2) The quaternized gelatin sponge prepared in 1) was soaked in a 21 mg / mL aqueous sodium methylsulfate solution for 2 h, washed with deionized water three times for 30 min each time, and freeze-dried to obtain hemostatic sponge X6.
[0105] In this embodiment, the substrate with cations on the surface is quaternized gelatin sponge, the cationic polymer is quaternized gelatin sponge, and the negatively charged small molecule is sodium methyl sulfate.
[0106] Example 7
[0107] The quaternized gelatin sponge prepared in step 1) of Example 1 was soaked in a 45 mg / mL aqueous solution of sodium methanesulfonate for 2 h, washed with deionized water three times for 30 min each time, and freeze-dried to obtain hemostatic sponge X7.
[0108] In this embodiment, the substrate with cations on the surface is quaternized gelatin sponge, the cationic polymer is quaternized gelatin sponge, and the negatively charged small molecule is sodium methanesulfonate.
[0109] Example 8
[0110] 1) 304 stainless steel sheet (abbreviated as S) cut into 1×1cm 2, continuously ultrasonically cleaned with isopropanol, ethanol, and water, dried, and treated with oxygen plasma. 1 mg / mL poly (diallyldimethylammonium chloride) (PDADMAC) solution and 1 mg / mL poly (sodium 4-styrenesulfonate) (PSS) solution were prepared with deionized water. Alternately soak the PDADMAC and PSS solutions for 20 minutes each, washed with deionized water for 1 minute, and blown with nitrogen for 2 minutes to obtain S-PP 4.5 (S-PP 4.5 The outermost layer is a cationic polymer (polydiallyldimethylammonium chloride).
[0111] 2) Replace the S-PP in 1) 4.5 The material was immersed in a 1 mg / mL sodium methylsulfate aqueous solution for 1 hour, washed with deionized water once for 1 minute each time, and dried with nitrogen to obtain hemostatic material X8.
[0112] In this embodiment, the substrate with cations on the surface is a cation-immobilized substrate S-PP obtained by alternating self-assembly of 304 stainless steel sheet, poly (sodium 4-styrene sulfonate) and poly (diallyldimethylammonium chloride). 4.5 , the cationic polymer is poly (diallyldimethylammonium chloride) and the negatively charged small molecule is sodium methyl sulfate.
[0113] Comparative Example 11
[0114] The quaternized gelatin sponge obtained in step 1) of Example 6 was named Y11.
[0115] Comparative Example 12
[0116] S-PP in step 1) of Example 8 4.5 , named Y12.
[0117] Comparative Example 13
[0118] The quaternized gelatin sponge prepared in step 1) of Example 6 was soaked in a 21 mg / mL aqueous solution of sodium methanesulfonate for 2 min, washed with deionized water three times for 30 min each time, and freeze-dried to obtain hemostatic sponge Y13.
[0119] In this embodiment, the soaking time is only 2 minutes, which is much shorter than the 2 hours in Example 1.
[0120] Test Example 2
[0121] The prepared pro-coagulant surface hemostatic materials X6 to X8 and comparative examples Y11 to Y13 were subjected to an in vitro coagulation effect comparison experiment.
[0122] Test method: Take 5mg of hemostatic sponge and put it into 2mL plastic centrifuge tube, take 1×1cm of stainless steel sheet 2Place in a 6-well plate. Add 100 μL of fresh anticoagulated blood from SD rats to 10 μL of calcium chloride solution (CaCl2; 0.2 M) and mix well. Immediately contact with the pre-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 blood clots and release hemoglobin. After 3 minutes, draw 100 μL of liquid and add it to a 96-well plate. Use an enzyme-linked immunosorbent assay to measure the absorbance Abs at 545 nm to calculate the hemoglobin content. For the blank group, take 100 μL of fresh anticoagulated blood and add 10 mL of deionized water. Incubate in a constant temperature water bath at 37°C for 3 minutes. Draw 100 μL to measure the absorbance Abs at 545 nm. Finally, calculate the coagulation index (BCI) using the following formula.
[0123] Blood coagulation index% (BCI) = (Abs 样品 / Abs 空白 )×100%...................formula
[0124] Where: Abs 样品 is the absorbance of the experimental group at 545 nm; Abs 空白 is the absorbance of the blank group at 545nm. The BCI index of the embodiment and the comparative example is shown in Table 1:
[0125] Table 3 In vitro coagulation index test results
[0126] Sample X6X7X8Y11Y12Y13BCI(%)42.038.1%40.849.189.971.2
[0127] The in vitro coagulation index (BCI) is an important indicator for characterizing a material's in vitro procoagulant properties and for screening hemostatic materials. Lower BCI values indicate better in vitro procoagulant properties. Table 3 shows that the BCI indices of hemostatic materials X6-X8 obtained in Examples 6-8 of the present invention are significantly lower than those of the same materials in Comparative Examples Y11-Y13. This demonstrates that the preparation method of the present invention can produce a material surface with excellent procoagulant / hemostatic properties.
[0128] By soaking in negatively charged small molecules of different concentrations, the positive charge of the cationic polymer can be effectively adjusted, eliminating the adverse effects of the excessive positive charge of the cationic polymer on the intrinsic coagulation pathway, and effectively improving the procoagulant performance of the cationic polymer.
[0129] Comparative Example 11 is a quaternized gelatin sponge that is not modified by soaking in the negatively charged small molecule sodium methylsulfate. The BCI of Y11 in Table 3 is larger than the BCI of Examples 6 and 7, indicating that the positively charged cationic polymer that has not been regulated by the negatively charged small molecule has a negative impact on the intrinsic coagulation pathway due to its excessively high positive charge, so that the pro-coagulant / hemostatic properties of the gelatin sponge surface cannot be effectively improved.
[0130] Comparative Example 12 is a stainless steel sheet coated with a cationic polymer coating that is not modified by soaking in sodium methyl sulfate. The BCI result of Y12 in Table 3 is much larger than the BCI of Example 8, indicating that the stainless steel surface without the positively charged cationic polymer regulated by negatively charged small molecules has a negative impact on the intrinsic coagulation pathway due to its excessively high positive charge, making it impossible to effectively improve the procoagulant / hemostatic properties of the stainless steel surface.
[0131] In Comparative Example 13, in the preparation step 2) of Example 6, the quaternized gelatin sponge was soaked in sodium methanesulfonate at an appropriate concentration, but the soaking time was too short, which was not conducive to the effective exchange of counterions. The BCI of Y13 in Table 3 was greater than the BCI value of X6, indicating that the inappropriate negatively charged small molecule regulation process did not adequately shield the positive charge of the cationic polymer, resulting in the material being too positively charged, which had a negative impact on the intrinsic coagulation pathway, making it impossible to effectively improve the hemostatic performance of the hemostatic material.
[0132] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.
Claims
1. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials, characterized in that: There are two methods: Method 1 is a one-pot regulation of negatively charged small molecules and cationic polymers. The specific steps are as follows: 1) dissolving a cationic polymer and a negatively charged small molecule to obtain a blend solution, wherein the mass ratio of the cationic polymer to the negatively charged small molecule is 1:0.15-2; 2) forming the blended solution obtained in step 1) into a gel or a film, or modifying a substrate with the blended solution to prepare a hemostatic material; The second method is to regulate the negatively charged small molecules on the cationic surface in steps: The substrate with cationic surface is modified by soaking in a negatively charged small molecule solution to obtain a material with a procoagulant surface. The specific steps are as follows: 1) Prepare a negatively charged small molecule solution; 2) immersing the substrate having the cations on its surface into the solution of step 1); 3) washing and drying to obtain a pro-coagulant surface regulated by negatively charged small molecules; The negatively charged small molecule is a mixture of one or more of sodium methyl sulfate, sodium methanesulfonate, sodium N-cyclohexylaminosulfonate, sodium morpholineethanesulfonic acid monohydrate, sodium 3-N(-morpholinyl)propanesulfonate, sodium 3-morpholine-2-hydroxypropanesulfonate, and gluconic acid.
2. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The blend solution described in step 1) of method 1 is an aqueous solution or a mixed solution of water and methanol, ethanol, ethyl acetate, or isopropanol; the aqueous solution is a glucose solution, and the concentration of glucose is 5% to 10%; the cationic polymer described in step 1) of method 1 is a mixture of one or more of polylysine, polydimethylaminoethyl methacrylate, polyhexamethylene biguanide hydrochloride, polyhexamethyleneguanidine hydrochloride, and quaternized starch, and its concentration in the solution ranges from 0.1 to 3 mg / mL.
3. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The cationic polymer in step 1) of method 1 is polylysine or polyhexamethylene biguanide hydrochloride; the negatively charged small molecule in step 1) is sodium methyl sulfate or sodium methane sulfonate.
4. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The method for preparing a gel from the blended solution described in step 2) of method 1 is as follows: adding a hydrophilic uncharged polymer to the blended solution obtained in step 1) to obtain a mixed gel, wherein the hydrophilic uncharged polymer is a mixture of one or more temperature-sensitive hydrophilic polymers such as poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide, and poly(N-isopropylacrylamide); the method for preparing a membrane from the blended solution described in step 2) of method 1 is as follows: adding a hydrophilic uncharged polymer to the blended solution obtained in step 1) of method 1, and drying the obtained mixed solution at high temperature to form a hemostatic membrane having hemostatic properties, wherein the hydrophilic uncharged polymer is a mixture of one or more hydrophilic cellulose derivatives such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and methyl cellulose; the method for preparing a membrane from step 2) of method 1 is as follows: adding a hydrophilic uncharged polymer to the blended solution obtained in step 1) of method 1, and drying the obtained mixed solution at high temperature to form a hemostatic membrane having hemostatic properties, wherein the hydrophilic uncharged polymer is a mixture of one or more hydrophilic cellulose derivatives such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and methyl cellulose; The method for modifying the substrate with the blend solution is as follows: soaking the substrate with the blend solution obtained in step 1) of method 1, and then taking out the substrate for drying. The selected substrate is a polymer medical material such as gauze, gelatin sponge, and alginate non-woven fabric.
5. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The step of preparing a gel by the blend solution described in method 1 is as follows: adding a cationic polymer to a glucose solution so that the concentration range of the solution is 0.1-3 mg / mL, a negatively charged small molecule, and adding a thermosensitive polymer poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide or poly(N-isopropylacrylamide) so that the concentration range of the thermosensitive polymer in the solution is 100-400 mg / mL; the step of preparing a membrane by the blend solution described in method 1 is as follows: adding a cationic polymer to a glucose solution so that the concentration range of the solution is 0.1-3 mg / mL, a negatively charged small molecule, and adding hydroxypropyl cellulose, hydroxypropyl methylcellulose or hydroxyethyl cellulose so that the concentration range of the solution is 20-200 mg / mL; the step of modifying a substrate by the blend solution described in method 1 is as follows: adding a cationic polymer to a glucose solution so that the concentration range of the solution is 0.1-3 mg / mL, a negatively charged small molecule, and fully impregnating the substrate with the mixed solution of the cationic polymer and the negatively charged small molecule.
6. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The concentration of the negatively charged small molecule solution in method 2 is 0.5-50 mg / mL, and the soaking time in step 2) of method 2 is 0.5-10 h.
7. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: There are three types of substrates with cationic surfaces as described in Method 2: 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 by 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 having 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 groups or tertiary amines.
8. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The self-assembly described in substrate a in method 2 is to coat the cationic polymer and the negatively charged polymer on the surface through a layer-by-layer self-assembly method, and control the outermost layer to be the cationic polymer; the substrate described in substrate a in method 2 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 cationic polymer described in substrate a in method 2 is a mixture of one or more of polydiallyldimethylammonium chloride, poly(N,N-dimethylaminoethyl methacrylate), polylysine, polyhexamethylene biguanide hydrochloride, and polyhexamethylene monoguanide hydrochloride, and the negatively charged polymer is a mixture of one or more of poly(sodium 4-styrene sulfonate) and polymethacrylic acid.
9. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: The substrate b in method 2, which does not contain cations on its surface, is a gelatin sponge, a polyvinyl alcohol sponge, a medical collagen sponge, or a gauze. The chemical reaction in substrate b in method 2 is a quaternization reaction, wherein a ring-opening reaction occurs between hydroxyl groups, amino groups, or carboxyl groups on the surface of the substrate which does not contain cations on its surface and an epoxy-containing quaternary ammonium salt. The epoxy-containing quaternary ammonium salt is 2,3-epoxypropyltrimethylammonium chloride or a product obtained by the reaction of epichlorohydrin and N,N-dimethyl-X-amine, where X is ethyl, propyl, butyl, hexyl, octyl, decyl, dodecamethyl, or tetradecamethyl.
10. A general method for optimizing counter anions to improve the hemostatic properties of biomaterials according to claim 1, characterized in that: After soaking gelatin modified with 2,3-epoxypropyltrimethyl quaternary ammonium salt in sodium methyl sulfate, a material with a procoagulant surface can be obtained; after soaking the cation-immobilized 304 stainless steel surface modified by alternating self-assembly of poly(sodium 4-styrene sulfonate) and poly(diallyldimethylammonium chloride) in sodium methyl sulfate, a material with a procoagulant surface can be obtained.