Universal method for optimizing counter anion to improve hemostatic performance of biological material

Abstract

Disclosed herein is a universal method for optimizing counter anions to improve the hemostatic performance of a biological material. Method I is a method for regulating and controlling an electronegat

Description

[0001] The present invention belongs to the technical field of preparation of hemostatic materials for medical applications, and relates to universal methods for improving hemostatic properties of biomaterials by optimizing counter anions. BACKGROUND

[0002] Polymer materials, flexible and diverse in structure design, are widely studied in many fields such as hemostatic materials, antibacterial materials and drug carriers. Cationic polymers, which facilitate the interaction of negatively charged proteins and cells, are used in the development of various types of hemostatic materials, but their excessively high positive charges retard the generation of thrombin and adversely affect the blood coagulation. It is therefore necessary to improve the pro-coagulant properties of cationic polymers by modulating their structures. Compared to the modulation of cationic polymer structure through fine chemical synthesis, the introduction of negatively charged small molecules on cationic polymers through counter anion exchange is promising in achieving facile modulation of polymer structure. Based on this, some counter anions (such as hydroxide ion, and chloridion) are converted into the newly introduced negatively charged small molecules through the competitive interaction of the negatively charged small molecules with the original counter anions of the cationic polymer, thereby modulating the excessively high positive charges of the cationic polymers on the surface of a base material as well as the corresponding strong binding ability (which generally results in a decrease in blood coagulation function and prolongation of the endogenous blood coagulation time) with key blood coagulation factors / proteins in the blood coagulation process. For the preparation of hemostatic materials, the present invention further provides preparation techniques for transforming such negatively charged small molecule / cationic polymer composites into hemostatic materials, in which, the negatively charged small molecule / cationic polymer composites can be blended with hydrophilic uncharged polymers to obtain hemostatic gels or membranes, or they are used to modify polymer medical materials to obtain hemostatic materials (i.e. the positive charges of cationic polymers are modulated by negatively charged small molecules to modify different base materials with cationic polymers on the surfaces to obtain hemostatic materials with pro-coagulant surfaces). SUMMARY

[0003] In response to this, the present invention provides universal methods for improving hemostatic properties of biomaterials by optimizing counter anions. The following specific technical solutions are provided.

[0004] Universal methods for improving hemostatic properties of biomaterials by optimizing counter anions include two methods.

[0005] Method I involves one-pot modulation of a cationic polymer and negatively charged small molecules by the following specific steps:

[0006] 1) dissolving the cationic polymer and the negatively charged small molecules to obtain a blend solution, the cationic polymer and the negatively charged small molecules being in a mass ratio of 1:0.15-2, and

[0007] 2) preparing the blend solution obtained in step 1) into a gel or membrane, or modifying a base material with the blend solution to prepare a hemostatic material; and

[0008] Method 2 involves stepwise modulation of cationic surfaces by negatively charged small molecules by the following specific steps:

[0009] modifying a base material with cationic polymers on the surface (cationic surface) by immersing into a negatively charged small molecule solution to obtain a material with a pro-coagulant surface, including the following specific steps:

[0010] 1) formulating the negatively charged small molecule solution,

[0011] 2) immersing the base material with cationic surface into the solution of step 1), and

[0012] 3) performing washing and drying to obtain a negatively charged small molecule-modulated pro-coagulant surface,

[0013] the negatively charged small molecule being a mixture of one or more of sodium methyl sulfate, sodium methanesulfonate, sodium N-cy cl ohexyl sulfamate, sodium morpholine ethanesulfonate salt monohydrate, sodium 3-N(-morpholino) propanesulfonate, sodium 3-morpholino-2-hydroxypropanesulfonate, and gluconic acid.

[0014] Further, the blend solution in step 1) of Method I is an aqueous solution or a mixed solution formed by water, methanol, ethanol, ethyl acetate, and isopropanol, the aqueous solution being a glucose solution with a glucose concentration of 5%-10%; and the cationic polymer in step 1) of Method I is a mixture of one or more of polylysine, poly(dimethylaminoethyl methacrylate), poly(hexamethylenebiguanide hydrochloride), poly(hexamethyleneguanidine hydrochloride), and quatemized starch, and the cationic polymer being at a concentration of 0.1-3 mg / mL in a solution.

[0015] Further, the cationic polymer in step 1) of Method I is polylysine or poly(hexamethylenebiguanide hydrochloride); and the negatively charged small molecule in step 1) is sodium methyl sulfate or sodium methanesulfonate.

[0016] Further, the blend solution is prepared into the gel in step 2) of Method I by: adding a hydrophilic uncharged polymer to the blend solution obtained in step 1 of Method I to obtain a mixed gel, the hydrophilic uncharged polymer being a mixture of one or more of temperature-sensitive hydrophilic polymers including poloxamer, polylactide-glycolide-poly ethylene glycol-polylactide-glycolide, and poly(N-isopropylacrylamide); the blend solution is prepared into the membrane in step 2) of Method I by: adding a hydrophilic uncharged polymer to the blend solution obtained in step 1) of Method I, and drying an obtained mixed solution at high temperature to prepare a hemostatic membrane with a hemostatic property, the hydrophilic uncharged polymer being a mixture of one or more of hydrophilic cellulose derivatives including hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and methylcellulose; and the base material is modified with the blend solution in step 2) of Method I by: immersing the base material with the blend solution obtained in step 1) of Method I, and taking out the base material for drying, the selected base material being polymer medical materials including a gauze, a gelatin sponge, and an alginate nonwoven.

[0017] Further, the blend solution is prepared into the gel in Method I by the steps of: adding a cationic polymer and the negatively charged small molecules in a glucose solution, making a concentration of the cationic polymer in the solution ranged from 0.1-3 mg / mL, and adding a temperature-sensitive hydrophilic polymer poloxamer, polylactide-glycolide-poly ethylene glycol-polylactide-glycolide, or poly(N-isopropylacrylamide), making a concentration of the temperature-sensitive polymer in the solution ranged from 100-400 mg / mL; the blend solution is prepared into the membrane in Method I by the steps of: adding a cationic polymer and the negatively charged small molecules in a glucose solution, making a concentration of the cationic polymer in the solution ranged from 0.1-3 mg / mL, and adding hydroxypropyl cellulose, hydroxypropylmethyl cellulose, or hydroxyethyl cellulose, making a concentration thereof in the solution ranged from 20-200 mg / mL; and the base material is modified by the blend solution in Method I by the steps of: adding a cationic polymer and the negatively charged small molecules in a glucose solution, making a concentration of the cationic polymer in the solution ranged from 0.1-3 mg / mL, and fully saturating the base material with a mixed solution of the cationic polymer and the negatively charged small molecule.

[0018] Further, the negatively charged small molecule solution in Method II is at a concentration of 0.5-50 mg / mL, and the immersing in step 2) of Method II is performed at a period of 0.5-10 h.

[0019] Further, three types of base materials with cationic surface in Method II are provided:

[0020] a base material a is a cation-immobilized base material obtained by self-assembly of a base material without cationic surface and a cationic polymer;

[0021] a base material b is a surface cation-immobilized base material obtained by chemical reaction between a base material without cationic surface and positively charged small molecule; and

[0022] a base material c is a base material containing a large number of cationic groups on its own surface, the cationic group being a combination of one or more of guanidino, primary amine, quaternary ammonium or tertiary amine.

[0023] Further, the self-assembly in the base material a refers to the encapsulation of a cationic polymer and a negatively charged polymer on a surface by a layer-by-layer self-assembly method, with control that the cationic polymer is on an outermost layer; the base material without cationic surface in the base material a in Method II is a 304 stainless steel, a metallic titanium nail, a silicon sheet, a gelatin sponge, a polyvinyl alcohol sponge or a medical collagen sponge; and the cationic polymer in the base material a in Method II is a mixture of one or more of poly(diallyldimethylammonium chloride) (PDADMAC), poly(N,N-dimethylaminoethyl methacrylate), polylysine, poly(hexamethylenebisguanidine) hydrochloride, poly(hexamethylenemonoguanidine) hydrochloride, and the negatively charged polymer is poly(sodium 4-styrenesulfonate) (PSS) or poly(methacrylic acid) or a mixture thereof.

[0024] Further, the base material without cationic surface in the base material b in Method II is a gelatin sponge, a polyvinyl alcohol sponge, a medical collagen sponge or a gauze; and the chemical reaction in the base material b in Method II is a quaternization reaction, in which a ring-opening reaction occurs between hydroxyl, amino or carboxyl on the surface of the base material without cationic surface and an epoxy-containing quaternary ammonium salt, the epoxy-containing quaternary ammonium salt being 2,3-epoxypropyltrimethyl ammonium chloride or a product obtained by reacting epoxy chloropropane and N,N-dimethyl X-amine, and the X being ethyl, propyl, butyl, hexyl, octyl, decyl, dodecyl, and tetradecyl.

[0025] Further, Method II involves that a 2,3-epoxypropyltrimethylquaternary ammonium salt-modified gelatin is soaked with sodium methyl sulfate to yield materials with pro-coagulant surfaces; and a cation-immobilized 304 stainless steel surface modified by alternating self-assembly of PSS and PDADMAC is soaked with sodium methyl sulfate to yield materials with pro-coagulant surfaces.

[0026] The present invention has the following beneficial effects: according to the universal methods for improving hemostatic properties of biomaterials by optimizing counter anions provided by the present invention, some counter anions (such as hydroxide ion and chloridion) are converted into the newly introduced negatively charged small molecules through the competitive interaction of the negatively charged small molecules with the original counter anions of the cationic polymer, thereby modulating the high positive charges of the cationic polymer on the surface of a base material as well as the concomitant strong binding ability / adhesion force (which generally results in a decrease in blood coagulation function and prolongation of the endogenous blood coagulation time) to key blood coagulation factors / proteins in the blood coagulation process. For the preparation of hemostatic materials, Method I of the present invention further provides preparation techniques for transforming such negatively charged small molecule / cationic polymer composites into hemostatic materials, either by blending them with hydrophilic uncharged polymers to obtain hemostatic gels or membranes, or by using them to modify polymeric medical materials to obtain hemostatic materials. The present invention achieves the elimination of the negative effects on the endogenous blood coagulation pathway caused by the excessively high positive charges of the cationic polymer, and the hemostatic property of the cationic polymer is improved by the facile modulation of polymer structure.

[0027] Method II of the present invention provides a universal method for improving hemostatic properties of biomaterials by optimizing counter anions, by immersing a cationic polymer in a surface-fixed state into a negatively charged small molecule solution, the competition occurs between the negatively charged small molecules and the original counter anions of the cationic polymer, so that some counter anions (such as hydroxide ion, and chloridion) are converted into the newly introduced negatively charged small molecules, thereby modulating the high positive charges of the cationic polymer on the surface of a base material as well as the concomitant strong binding ability / adhesion force (which generally results in a decrease in blood coagulation function and prolongation of the endogenous blood coagulation time) to key blood coagulation factors / proteins in the blood coagulation process. The present invention achieves the elimination of the negative effects on the endogenous blood coagulation pathway caused by the excessively high positive charges of the cationic polymer, and the hemostatic property of the base material of surface-immobilized cationic polymers is improved by the facile modulation of polymer structure. Thus, compared to the modulation of polymer structure to develop base materials (of surface-immobilized polymers) with pro-coagulant properties through the fine chemical modification synthesis, the present invention provides a more efficient method for preparing pro-coagulant surfaces.

[0028] In summary, the present invention provides more efficient methods to enhance cationic polymer-based hemostatic materials and pro-coagulant surfaces using negatively charged small molecule modifications, as opposed to fine chemical modification synthesis to modulate the cationic polymer structures. DETAILED DESCRIPTION

[0029] Preferred examples of the present invention are described below in detail.

[0030] Experiments and data related to Method I are shown in Examples 1-5, Comparative Examples 1-10, Test Example 1, and Tables 1 and 2.

[0031] Experiments and data related to Method II are shown in Examples 6-8, Comparative Examples 11-13, Test Example 2, and Table 3. Example 1

[0032] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and sodium methyl sulfate which had a mass ratio of 1:1 with the polylysine was added to obtain a blend solution LI-Ml.

[0033] 2) A certain amount of poloxamer was added to the blend solution Ll-Ml, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel XI with a hemostatic property was obtained. Example 2

[0034] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and sodium methyl sulfate which had a mass ratio of 2:1 with the polylysine was added to obtain a blend solution L1-M2.

[0035] 2) A certain amount of hydroxypropyl cellulose was added to the blend solution LI-M2, making the hydroxypropyl cellulose to be at a concentration of 75 mg / mL in the solution, and 2 mL of a mixed solution was taken and fully spread in a mold of 4.4^4.4 cm2, and dried at 60°C for 6 h to obtain a membrane X2 with a hemostatic property. Example 3

[0036] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and sodium methyl sulfate which had a mass ratio of 0.25:1 with the polylysine was added to obtain a mixed solution LI-M0.25.

[0037] 2) 5 mL of the mixed solution was taken to fully saturate 4 layers of medical gauze of 8x6 cm2, and the medical gauze was air-dried for 24 h at normal temperature to obtain a modified gauze X3 with a hemostatic property. Example 4

[0038] 1) A certain amount of poly(hexamethylenebisguanidine) hydrochloride was added to 10% of glucose solution, making the poly(hexamethylenebisguanidine) hydrochloride to be at a concentration of 1 mg / mL, and sodium methyl sulfate which had a mass ratio of 1:1 with the poly(hexamethylenebisguanidine) hydrochloride was added to obtain a blend solution Hl-ML

[0039] 2) A certain amount of poloxamer was added to the blend solution Hl-Ml, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel X4 with a hemostatic property was obtained. Example 5

[0040] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and sodium methanesulfonate which had a mass ratio of 1:1 with the polylysine was added to obtain a blend solution LI-DI.

[0041] 2) A certain amount of poloxamer was added to the blend solution LI-DI, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel X5 with a hemostatic property was obtained. Comparative Example 1

[0042] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and sodium methyl sulfate which had a mass ratio of 0.1:1 with the polylysine was added to obtain a blend solution LI-M0.1.

[0043] 2) A certain amount of poloxamer was added to the blend solution L1-M0.1, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel Y1 was obtained. Comparative Example 2

[0044] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and sodium methyl sulfate which had a mass ratio of 6:1 with the polylysine was added to obtain a blend solution L1-M6.

[0045] 2) A certain amount of poloxamer was added to the blend solution L1-M6, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel Y2 was obtained. Comparative Example 3

[0046] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 1 mg / mL, and a solution LI was obtained.

[0047] 2) A certain amount of poloxamer was added to the solution LI, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel Y3 was obtained. Comparative Example 4

[0048] 1) A certain amount of polylysine was added to 10% of glucose solution, making the polylysine to be at a concentration of 5 mg / mL, and sodium methyl sulfate which had a mass ratio of 1:1 with the polylysine was added to obtain a blend solution L5-M5.

[0049] 2) A certain amount of poloxamer was added to the blend solution L5-M5, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel Y4 with a hemostatic property was obtained. Comparative Example 5

[0050] 1) A certain amount of poly(hexamethylenebisguanidine) hydrochloride was added to 10% of glucose solution, making the poly(hexamethylenebisguanidine) hydrochloride to be at a concentration of 1 mg / mL, and a solution Hl was obtained.

[0051] 2) A certain amount of poloxamer was added to the solution Hl, making the poloxamer to be at a concentration of 300 mg / mL in the solution, and a gel Y5 was obtained. Comparative Example 6

[0052] A certain amount of hydroxypropyl cellulose was added to the solution LI obtained in step 1) of Comparative Example 3, making the hydroxypropyl cellulose to be at a concentration of 75 mg / mL in the solution, and 2 mL of a mixed solution was taken and fully spread in a mold of 4.4x4.4 cm2, and dried at 60 °C for 6 h to obtain a membrane Y6. Comparative Example 7

[0053] 5 mL of the solution LI obtained in step 1) of Comparative Example 3 was taken to fully saturate 4 layers of medical gauze of 8x6 cm2, and the medical gauze was air-dried for 24 h at normal temperature to obtain a modified gauze Y7. Comparative Example 8

[0054] Poloxamer was prepared into a solution at a concentration of 300 mg / mL in 10% of glucose solution, and a gel Y8 was obtained. Comparative Example 9

[0055] Hydroxypropyl cellulose was prepared into a solution at a concentration of 75 mg / mL in 10% of glucose solution, and 2 mL of the solution was taken and fully spread in a mold of 4.4x4.4 cm2, and dried at 60°C for 6 h to obtain a polysaccharide membrane Y9. Comparative Example 10

[0056] An unmodified medical gauze Y10. Test Example 1

[0057] The prepared pro-coagulant-enhanced hemostatic materials X1-X5, and Y1-Y10 prepared in comparative examples were subjected to comparison experiments of in vitro blood coagulation effect.

[0058] Test method: 50 pL of hemostatic gel was aspirated into a 2 mL plastic centrifuge tube, the gel was pre-cured for 5 min at 37°C, and 5 mg of a membrane and a gauze of 0.5x0.5 cm2 were put into the 2 mL plastic centrifuge tube. 100 pL of fresh anticoagulation blood from Sprague-Dawley (SD) rats was mixed in 10 pL of calcium chloride solution (CaCk; 0.2 M), and the mixed solution was immediately contacted with the pre-prepared material, followed by incubating for 1 min in a 37°C constant-temperature water bath. 10 mL of deionized water was added slowly and incubation was continued for 3 min to fully lyse the blood cells that did not form a blood clot to release hemoglobin. After 3 min, 100 pL of liquid was aspirated and added to a 96-well plate, and the absorbance (Abs) at 545 nm was tested by an enzyme-linked immunoassay to calculate the hemoglobin content. In a blank group, 100 pL of fresh anti coagulation blood was added to 10 mL of deionized water, followed by incubating for 3 min in a 37°C constant-temperature water bath, and 100 pL was aspirated for testing the Abs at 545 nm. Finally, a blood coagulation index (BCI) is calculated by the following Formula 1.

[0059] BCI (%) = (Abssampie / Absbiank) x 100% Formula 1.

[0060] In the formula, AbsSamPie represents the absorbance at 545 nm in the experimental group; and Absbiank represents the absorbance at 545 nm in the blank group. The BCI for the examples and the comparative examples are shown in Table 1: Table 1 In vitro BCI test results Sample XI X2 X3 X4 X5 Y1 Y2 Y3 22.4 21.3 44.6 53.8 30.3 46.8 53.9 65.0 (%) Sample Y4 Y5 Y6 Y7 Y8 Y9 Y10 BCI 54.9 87.5 62.8 92.6 50.6 58.1 76.5 (%)

[0061] In vitro BCI is an important index to evaluate the in vitro pro-coagulant property of materials and to screen for hemostatic materials, and the smaller the BCI value, the better the in vitro hemostatic property of the material. As can be seen from Table 1, the BCI of the hemostatic materials X1-X5 obtained in Examples 1-5 of the present invention is significantly lower than that of the same kind of materials Y1-Y10 in comparative examples. It can be seen that by using the preparation method of the present invention, hemostatic materials with excellent hemostatic properties can be obtained.

[0062] By adding negatively charged small molecules with different mass ratios to the cationic polymer, the effect of decreased blood coagulation function (i.e., increased BCI) caused by the high positive charges of the cationic polymer can be effectively regulated, eliminating the negative effects on the endogenous blood coagulation pathway, and effectively improving the pro-coagulant property of the cationic polymer.

[0063] Comparative Example 1 differs from Example 1 in that, in preparation step 1), a small amount of sodium methyl sulfate is blended with polylysine to obtain a blend solution, which is added with poloxamer to prepare a hemostatic gel. The BCI in Example 1 is 22.4% while that in Comparative Example 1 is 46.8%, showing that the hemostatic property of the gel obtained in Comparative Example 1 is worse than that in Example 1. Therefore, it indicates that the addition of a small amount of negatively charged small molecules is not sufficient to shield the positive charges of the cationic polymer, which results in the material being excessively positively charged, negatively affecting the endogenous blood coagulation pathway, and resulting in a decrease in the blood coagulation function to the extent that it is unable to effectively improve the hemostatic property of the hemostatic material.

[0064] Comparative Example 2 differs from Example 1 in that, in preparation step 1), an excessive amount of sodium methyl sulfate is blended with polylysine to obtain a blend solution, which is added with poloxamer to prepare a hemostatic gel. The BCI in Example 1 is 22.4% while that in Comparative Example 2 is 53.9%, showing that the hemostatic property of the gel obtained in Comparative Example 2 is worse than that in Example 1. Thus, it is shown that the addition of excessive amount of negatively charged small molecules greatly weakens the positive charges of the cationic polymer, thereby affecting its properties such as aggregation of blood cells and platelets, to the extent that the hemostatic property of the hemostatic material cannot be effectively improved.

[0065] Comparative Example 3 differs from Example 1 in that, in preparation step 1), without adding sodium methyl sulfate, polylysine is dissolved in 10% of glucose solution to obtain a cationic polymer solution, which is added with poloxamer to prepare a hemostatic gel. The BCI in Example 1 is 22.4% while that in Comparative Example 3 is 65.0%, showing that the hemostatic property of the gel obtained in Comparative Example 3 is worse than that in Example 1. Thus, it is shown that cationic polymer whose positive charges are not modulated by small molecules negatively affects the endogenous blood coagulation pathway due to excessively high positive charges, resulting in a decrease in the blood coagulation function to the extent that the hemostatic property of the hemostatic material cannot be improved effectively.

[0066] Comparative Example 4 differs from Example 1 in that, in preparation step 1), an excessive amount of polylysine is dissolved in 10% of glucose solution to obtain a cationic polymer solution, which is added with equal mass of sodium methyl sulfate, followed by adding poloxamer to prepare a hemostatic gel. The BCI in Example 1 is 22.4% while that in Comparative Example 4 is 54.9%, showing that the hemostatic property of the gel obtained in Comparative Example 4 is worse than that in Example 1. Thus, it is shown that the excessive amount of cationic polymers is so positively charged that the weakening effect of the negatively charged small molecules is limited, and therefore still negatively affect the endogenous blood coagulation pathway, resulting in a decrease in blood coagulation function to the extent that the hemostatic property of the hemostatic material cannot be effectively improved.

[0067] Comparative Example 5 differs from Example 4 in that, in preparation step 1), without adding negatively charged molecule sodium methyl sulfate, poly(hexamethylenebisguanidine) hydrochloride is dissolved in 10% of glucose solution to obtain a cationic polymer solution, which is added with poloxamer to prepare a hemostatic gel. The BCI in Example 4 is 53.8% while that in Comparative Example 5 is 87.5%, showing that the hemostatic property of the gel obtained in Comparative Example 5 is worse than that in Example 4. Thus, it is shown that cationic polymer whose positive charges are not modulated by small molecules negatively affects the endogenous blood coagulation pathway due to excessively high positive charges to the extent that the hemostatic property of the hemostatic material cannot be effectively improved.

[0068] Comparative Example 6 differs from Example 2 in that, in preparation step 1), without adding negatively charged molecule sodium methyl sulfate, polylysine is dissolved in 10% of glucose solution to obtain a cationic polymer solution, which is added with hydroxypropyl cellulose to prepare a hemostatic membrane. The BCI in Example 2 is 21.3% while that in Comparative Example 6 is 62.8%, showing that the hemostatic property of the membrane obtained in Comparative Example 6 is worse than that in Example 2. Thus, it is shown that the cationic polymer whose positive charges are not modulated by the small molecules negatively affects the endogenous blood coagulation pathway due to excessive positive charges to the extent that the hemostatic property of the hemostatic material cannot be effectively improved.

[0069] Comparative Example 7 differs from Example 3 in that, in preparation step 1), without adding negatively charged small molecule sodium methyl sulfate, polylysine is dissolved in 10% of glucose solution to obtain a cationic polymer solution, in which, a medical gauze is immersed to be modified. The BCI in Example 3 is 44.6% while that in Comparative Example 7 is 92.6%, showing that the hemostatic property of the gauze obtained in Comparative Example 7 is worse than that in Example 3. Thus, it is shown that cationic polymer whose positive charges are not modulated by the negatively charged small molecules negatively affects the endogenous blood coagulation pathway due to excessive positive charges to the extent that the hemostatic property of the hemostatic material cannot be effectively improved.

[0070] In Comparative Example 8, a hydrogel is prepared using poloxamer alone without adding any modifying materials. The BCI of the hydrogel is 50.6 (much greater than that of the gels XI and X5 modified using the method of the present invention), and the hemostatic property is not as good as that obtained after the addition of the modifying materials. It is therefore shown that the modulation of cationic polymer with negatively charged small molecules using the method of the present invention enhances the hemostatic property of the gel-

[0071] In Comparative Example 9, a membrane is prepared using hydroxypropyl cellulose alone without adding any modifying materials. The BCI of the membrane is 58.1% (much greater than that of the membrane X2 obtained by the method of the present invention), and the hemostatic property is not as good as that obtained after the addition of the modifying materials of the present invention. It is therefore shown that the modulation of cationic polymer with negatively charged small molecules using the method of the present invention enhances the hemostatic property of the membrane.

[0072] In Comparative Example 10, a medical gauze is used alone without adding any modifying materials. The BCI of the gauze is 76.5% (much greater than that of the medical gauze X3 modified using the method of the present invention), and the hemostatic property is not as good as that obtained after the addition of the modifying materials. It is therefore shown that the modulation of cationic polymer with negatively charged small molecules using the method of the present invention enhances the hemostatic property of the medical gauze. Test Example 2

[0073] The prepared pro-coagulant-enhanced hemostatic material XI, and Y1 and Y3 prepared in comparative examples were subjected to comparative experiments of activated partial thromboplastin time (APTT).

[0074] Test method: 50 pL of hemostatic gel was aspirated into a 2 mL plastic centrifuge tube, the gel was pre-cured for 5 min at 37°C, anticoagulation whole blood from SD rats was centrifuged at 3000 rpm for 15 min, and platelet-poor plasma (PPP) was extracted from supernatant. 50 pL of PPP and 50 pL of APTT reagent were mixed before being immediately added to the gel material, followed by incubating in a 37°C water bath for 3 min, 50 pL of calcium chloride solution was added, and the plasma coagulation time was immediately observed and recorded. In blank group, APTT without materials added was taken as 100%. APTT (%) was calculated by the following Formula 2:

[0075] APTT (To) (APTTmaterial group / APTTblank group) * I 00To Formula 2. Table 2 APTT test results Sample XI Y1 Y3 APTT (%) 110 216 157

[0076] APTT is the most commonly used clinical test to evaluate the blood coagulation activity of the endogenous blood coagulation system. The APTT of normal plasma is taken as 100%, and a longer APTT represents a more detrimental effect on the endogenous blood coagulation pathway and is less favorable to blood coagulation.

[0077] Comparison Examples 1 differs from Example 1 in that, in preparation step 1), a small amount of sodium methyl sulfate and polylysine are blended to obtain a blend solution, which is added with poloxamer to prepare a hemostatic gel. The APTT test results show that the APTT of XI is 110% while that of Y1 is 216%, indicating that the time of Y1 is significantly prolonged in comparison with that of normal plasma (100%). Thus, it is shown that the XI (moderate amount of negatively charged small molecules + cationic polymer) obtained using the method of the present invention has a much smaller effect on the endogenous blood coagulation pathway, whereas a small amount of negatively charged small molecules is not sufficient to weaken the negative effect of the cationic polymer on the endogenous blood coagulation pathway, which is detrimental to blood coagulation.

[0078] Comparative Example 3 differs from Example 1 in that, in preparation step 1), without adding sodium methyl sulfate, polylysine is dissolved in 10% of glucose solution to obtain a cationic polymer solution, which is added with poloxamer to prepare a hemostatic gel. The APTT test results show that the APTT of XI is 110% while that of Y3 is 157%, indicating that the time of Y3 is significantly prolonged compared to that of normal plasma (100%). Thus, it is shown that the cationic polymer alone, without being modulated by the negatively charged small molecules, has a greater effect on the endogenous blood coagulation pathway and is detrimental to blood coagulation.

[0079] Examples for Method II are shown as follows. Example 6

[0080] 1) 300 mg of sodium hydroxide was dissolved in 10 mL of deionized water, 1200 mg of 2,3-epoxypropyltrimethyltrimethylquaternary ammonium salt was dissolved in 40 mL of deionized water, the two solutions were mixed to soak a gelatin sponge for 24 h, the gelatin sponge after being soaked was washed with deionized water three times for 10 min each time, and the gelatin sponge after being washed was subjected to freeze drying to obtain a quaternized gelatin sponge (QGS).

[0081] 2) The QGS in 1) was immersed in 21 mg / mL of aqueous solution of sodium methyl sulfate for 2 h, the QGS after being immersed was washed with deionized water three times with each time for 30 min, and the QGS after being washed was subjected to freeze drying to obtain a hemostatic sponge X6.

[0082] In this example, the base material with cationic surface was QGS, the cationic polymer was QGS, and the negatively charged small molecule was sodium methyl sulfate. Example 7

[0083] The QGS in step 1) of Example 6 was immersed in 45 mg / mL of aqueous solution of sodium methanesulfonate for 2 h, the QGS after being immersed was washed with deionized water three times with each time for 30 min, and the QGS after being washed was subjected to freeze drying to obtain a hemostatic sponge X7.

[0084] In this example, the base material with cationic surface was QGS, the cationic polymer was QGS, and the negatively charged small molecule was sodium methanesulfonate. Example 8

[0085] 1) a 304 stainless steel sheet (referred to as S) was cut into 1 x 1 cm2 before being subjected to continuous ultrasonic cleaning with isopropanol, ethanol, and water, and the 304 stainless steel sheet after being cleaned was treated with an oxygen plasma before being dried. 1 mg / mL of PDADMAC solution and 1 mg / mL of PSS solution were prepared in deionized water. The 304 stainless steel sheet was alternatively immersed in the PDADMAC and PSS solutions for 20 min each time, washed with deionized water for 1 min, and purged with nitrogen for 2 min, to obtain S-PP4.5 (the outermost layer of S-PP4.5 was the cationic polymer PDADMAC).

[0086] 2) S-PP4.5 in 1) was immersed in 1 mg / mL aqueous solution of sodium methyl sulfate for 1 h, S-PP4.5 after being immersed was washed with deionized water once for 1 min, and S-PP4.5 after being washed was dried by nitrogen purging to obtain a hemostatic material X8.

[0087] In this example, the base material with cationic surface was the cation-immobilized base material S-PP4.5 obtained by alternating self-assembly of 304 stainless steel sheet with PSS and PDADMAC, the cationic polymer was PDADMAC, and the negatively charged small molecule was sodium methyl sulfate. Comparative Example 11

[0088] The QGS obtained from step 1) in Example 6 was named Y11. Comparative Example 12

[0089] S-PP4.5 obtained from step 1) in Example 8 was named Y12. Comparative Example 13

[0090] The QGS in step 1) of Example 6 was immersed in 21 mg / mL of aqueous solution of sodium methanesulfonate for 2 min, the QGS after being immersed was washed with deionized water three times with each time for 30 min, and the QGS after being washed was subjected to freeze drying to obtain a hemostatic sponge Y13.

[0091] In this example, the immersing time was only 2 min, much shorter than the 2 h in Example 1. Test Example 2

[0092] The prepared pro-coagulant surface hemostatic materials X6-X8, and Y11-Y13 prepared in comparative examples were subjected to comparative experiments of in vitro blood coagulation effect.

[0093] Test method: 5 mg of hemostatic sponge series was taken into a 2 mL plastic centrifuge tube, and Pl cm2 of stainless steel sheet was put into a 6-well plate. 100 pL of fresh anticoagulation blood from SD rats was mixed uniformly in 10 pL of calcium chloride solution (CaCh; 0.2 M), and the mixed solution was immediately contacted with the pre-prepared material, followed by incubating for 1 min in a 37°C constant-temperature water bath. 10 mL of deionized water was added slowly and incubation was continued for 3 min to fully lyse the blood cells that did not form a blood clot to release hemoglobin. After 3 min, 100 pL of liquid was aspirated and added to a 96-well plate, and the Abs at 545 nm was tested by an enzyme-linked immunoassay to calculate the hemoglobin content. In a blank group, 100 pL of fresh anti coagulation blood was added to 10 mL of deionized water, followed by incubating for 3 min in a 37°C constant-temperature water bath, and 100 pL was aspirated for testing the Abs at 545 nm. Finally, BCI is calculated by the following formula.

[0094] BCI (%) = (AbSsampie / AbSbiank) * 100% Formula.

[0095] In the formula, AbsSampie represents the absorbance at 545 nm in the experimental group; and Absbiank represents the absorbance at 545 nm in the blank group. The BCI for the examples and the comparative examples are shown in Table 3: Table 3 In vitro BCI test results Sample X6 X7 X8 Yll Y12 Y13 BCI (%) 42.0 38.1 40.8 49.1 89.9 71.2

[0096] In vitro BCI is an important index to eva uate the in vitro pro-coagu ant property of materials and to screen for hemostatic materials, the smaller the BCI value, the better the in vitro pro-coagulant property of the material. As can be seen from Table 3, the BCI of the hemostatic materials X6-X8 obtained in Examples 6-8 of the present invention is significantly lower than that of the same kind of materials Y11-Y13 obtained in comparative examples. Thus, it can be seen that the material surface with excellent pro-coagulant / hemostatic property can be obtained using the preparation method of the present invention.

[0097] By immersing into different concentrations of negatively charged small molecules, the positive charges of the cationic polymer can be effectively modulated, eliminating the adverse effects on the endogenous blood coagulation pathway brought about by the excessively high positive charges of the cationic polymer, and effectively improving the pro-coagulant property of the cationic polymer.

[0098] In Comparative Example 11, the QGS is not modified by immersing into the negatively charged small molecule sodium methyl sulfate. The BCI of Yll in Table 3 is larger than that in both Examples 6 and 7, indicating that the cationic polymer whose high positive charges are not modulated by negatively charged small molecules negatively affects the endogenous blood coagulation pathway due to its excessively high interaction with key blood coagulation factors / proteins in the blood coagulation process to the extent that the pro-coagulant / hemostatic property of the gelatin sponge cannot be effectively improved.

[0099] In Comparative Example 12, a stainless steel sheet coated with a cationic polymer coating is not modified by immersing into sodium methyl sulfate. The BCI of Y12 in Table 3 is much larger than that in Example 8, indicating that the stainless steel surface of the cationic polymer without being modulated by negatively charged small molecules negatively affects the endogenous blood coagulation pathway due to its excessively high positive charges to the extent that the pro-coagulant / hemostatic property of the stainless steel surface cannot be effectively improved.

[00100] In Comparative Example 13, in preparation step 2) of Example 6, the QGS is soaked with an appropriate concentration of sodium methanesulfonate, but the soaking time is too short to the effective exchange of counter ions. The BCI of Y13 in Table 3 is greater than that of X6, indicating that the inappropriate negatively charged small molecule regulation process is not sufficient to shield the positive charges of the cationic polymer, which leads to the material being excessively positively charged, negatively affecting the endogenous blood coagulation pathway to the extent that the hemostatic property of the hemostatic material cannot be effectively improved.

[00101] Finally, it is to be noted that the above preferred examples only serve to illustrate the technical solutions of the present invention rather than limiting the present invention. Although the present invention has been described in detail by means of the above preferred examples, it is to be understood by those skilled in the art that a wide variety of changes can be made to the present invention in terms of form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Universal methods for improving hemostatic properties of biomaterials by optimizing counter anions, comprising two methods, whereinMethod I involves one-pot modulation of a cationic polymer and negatively charged small molecules by the following specific steps:1) dissolving the cationic polymer and the negatively charged small molecules to obtain a blend solution, the cationic polymer and the negatively charged small molecules being in a mass ratio of 1:0.15-2, and2) preparing the blend solution obtained in step 1) into a gel or membrane, or modifying a base material with the blend solution to prepare a hemostatic material; andMethod 2 involves stepwise modulation of cationic surfaces by negatively charged small molecules by the following steps:modifying a base material with cationic surface by immersing into a negatively charged small molecule solution to obtain a material with a pro-coagulant surface, comprising the following specific steps:1) formulating the negatively charged small molecule solution,2) immersing the base material with cationic surface into the solution of step 1), and3) performing washing and drying to obtain a negatively charged small molecule-modulated pro-coagulant surface,the negatively charged small molecule being a mixture of one or more of sodium methyl sulfate, sodium methanesulfonate, sodium N-cyclohexylsulfamate, sodium morpholine ethanesulfonate salt monohydrate, sodium 3-N(-morpholino) propanesulfonate, sodium 3-morpholino-2-hydroxypropanesulfonate, and gluconic acid.

2. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the blend solution in step 1) of Method I is an aqueous solution or a mixed solution formed by water, methanol, ethanol, ethyl acetate, and isopropanol, the aqueous solution being a glucose solution with a glucose concentration of 5%-10%; and the cationic polymer in step 1) of Method I is a mixture of one or more of polylysine, poly(dimethylaminoethyl methacrylate), poly(hexamethylenebiguanide hydrochloride), poly(hexamethyleneguanidine hydrochloride), and quaternized starch, and the cationic polymer being at a concentration of 0.1 -3 mg / mL in a solution.

3. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the cationic polymer in step 1) of Method I is polylysine or poly(hexamethylenebiguanide hydrochloride); and the negatively charged small molecule in step 1) is sodium methyl sulfate or sodium methanesulfonate.

4. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the blend solution is prepared into the gel in step 2) of Method I by: adding a hydrophilic uncharged polymer to the blend solution obtained in step 1 of Method I to obtain a mixed gel, the hydrophilic uncharged polymer being a mixture of one or more of temperature-sensitive hydrophilic polymers comprising poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide, and poly(N-isopropylacrylamide); the blend solution is prepared into the membrane in step 2) of Method I by: adding a hydrophilic uncharged polymer to the blend solution obtained in step 1) of Method I, and drying an obtained mixed solution at high temperature to prepare a hemostatic membrane with a hemostatic property, the hydrophilic uncharged polymer being a mixture of one or more of hydrophilic cellulose derivatives comprising hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and methylcellulose; and the base material is modified with the blend solution in step 2) of Method I by: immersing the base material with the blend solution obtained in step 1) of Method I, and taking out the base material for drying, the selected base material being polymer medical materials comprising a gauze, a gelatin sponge, and an alginate nonwoven.

5. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the blend solution is prepared into the gel in Method I by the steps of: adding a cationic polymer and the negatively charged small molecule in a glucose solution, making a concentration of the cationic polymer in the solution ranged from 0.1-3 mg / mL, and adding a temperature-sensitive hydrophilic polymer poloxamer, polylactide-glycolide-polyethylene glycol-polylactide-glycolide, or poly(N-isopropylacrylamide), making a concentration of the temperature-sensitive polymer in the solution ranged from 100-400 mg / mL; the blend solution is prepared into the membrane in Method I by the steps of: adding a cationic polymer and the negatively charged small molecule in a glucose solution, making a concentration of the cationic polymer in the solution ranged from 0.1-3 mg / mL, and adding hydroxypropyl cellulose, hydroxypropylmethyl cellulose, or hydroxyethyl cellulose, making a concentration thereof in the solution ranged from 20-200 mg / mL; and the base material is modified by the blend solution in Method I by the steps of: adding a cationic polymer and the negatively chargedsmall molecule in a glucose solution, making a concentration of the cationic polymer in the solution ranged from 0.1-3 mg / mL, and fully saturating the base material with a mixed solution of the cationic polymer and the negatively charged small molecule.

6. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the negatively charged small molecule solution in Method II is at a concentration of 0.5-50 mg / mL, and the immersing in step 2) of Method II is performed at a period of 0.5-10 h.

7. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein three types of base materials with cationic surface in Method II are provided:a base material a is a cation-immobilized base material obtained by self-assembly of a base material without cationic surface and a cationic polymer;a base material b is a surface cation-immobilized base material obtained by chemical reaction between a base material without cationic surface and positively charged small molecules; anda base material c is a base material containing a large number of cationic groups on its own surface, the cationic group being a combination of one or more of guanidino, primary amine, quaternary ammonium or tertiary amine.

8. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the self-assembly in the base material a refers to the encapsulation of a cationic polymer and a negatively charged polymer on a surface by a layer-by-layer self-assembly method, with control that the cationic polymer is on an outermost layer; the base material without cationic surface in the base material a in Method II is a 304 stainless steel, a metallic titanium nail, a silicon sheet, a gelatin sponge, a polyvinyl alcohol sponge or a medical collagen sponge; and the cationic polymer in the base material a in Method II is a mixture of one or more of poly(diallyldimethylammonium chloride) (PDADMAC), poly(N,N-dimethylaminoethyl methacrylate), polylysine, poly (hexamethylenebisguanidine) hydrochloride, poly(hexam ethylenemonoguanidine)hydrochloride, and the negatively charged polymer is poly(sodium 4-styrenesulfonate) (PSS) or poly(methacrylic acid) or a mixture thereof.

9. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein the base material without cationic surface in the base material b in Method II is a gelatin sponge, a polyvinyl alcohol sponge, a medical collagen sponge or a gauze; and the chemical reaction in the base material b inMethod II is a quaternization reaction, in which a ring-opening reaction occurs between hydroxyl, amino or carboxyl on the surface of the base material without cationic surface and an epoxy-containing quaternary ammonium salt, the epoxy-containing quaternary ammonium salt being 2,3-epoxypropyltrimethylammonium chloride or a product obtained by reacting 5 epoxy chloropropane and N,N-dimethyl X-amine, and the X being ethyl, propyl, butyl, hexyl, octyl, decyl, dodecyl, and tetradecyl.

10. The universal methods for improving hemostatic properties of biomaterials by optimizing counter anions according to claim 1, wherein a 2,3-epoxypropyltrimethylquaternary ammonium salt-modified gelatin is soaked with sodium 10 methyl sulfate to yield materials with pro-coagulant surfaces; and a cation-immobilized 304 stainless steel surface modified by alternating self-assembly of PSS and PDADMAC is soaked with sodium methyl sulfate to yield materials with pro-coagulant surfaces.INTERNATIONAL SEARCH REPORT International application No. PCT / CN2022 / 116464 A. CLASSIFICATION OF SUBJECT MATTER A61L 24 / 04(2006.01)i;A61L 24 / 06(2006.01)i;A61L 24 / 08(2006.01)i;A61L 24 / 08(2006.01)i;A61L 24 / 10(2006.01)i;A61L 24 / 00(2006.0 l)i According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHEDMinimum documentation searched (classification system followed by classification symbols)Documentation searched other than minimum documentation to the extent that such documents are included in the fields searchedA61LElectronic data base consulted during the international search (name of data base and, where practicable, search terms used)WPABS; CNABS; CNKI; CJFD; CNTXT; WOTXT; AiNU'-. DUXIU; WANFANG; STN; USTXT; EPTXT:Sulfonate, Polymer, Gluconic AcidC. DOCUMENTS CONSIDERED TO BE RELEVANTCategory* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X CN 113975447 A (SICHUAN UNIVERSITY) 28 January 2022 (2022-01-28) claim 1, and description, paragraphs 0019 and 0060-0062 1-10 A CN 1448189 A (ZHEJIANG UNIVERSITY) 15 October 2003 (2003-10-15) entire document 1-10 — — A CN 107362387 A (CHENGDU YOURUI BUSINESS SERVICE CO., LTD.) 21 November 2017 (2017-11-21) entire document 1-10 A CN 106474530 A (INSTITUTE OF METAL RESEARCH, CHINESE ACADEMY OF SCIENCES) 08 March 2017 (2017-03-08) entire document 1-10| | Further documents are listed in the continuation of Box C. | J | See patent family annex.* Special categories of cited documents:“A” document defining the general state of the art which is not considered to be of particular relevanceTT’ document cited by the applicant in the international application“E” earlier application or patent but published on or after the international filing date“L” document which may throw doubts on priority claim(s) or which is cited to establish the publication date of another citation or other special reason (as specified)“O” document referring to an oral disclosure, use, exhibition or other means“P” document published prior to the international filing date but later than the priority date claimed“T” later document published after the international filing dace or priority date and not in conflict with the application but cited to understand the principle or theory underlying the invention“X” document of particular relevance; the claimed invention cannot be considered novel or cannot be considered to involve an inventive step when the document is taken alone“Y” document of particular relevance; the claimed invention cannot be considered to involve an inventive step when the document is combined with one or more other such documents, such combination being obvious to a person skilled in the artdocument member of the same patent familyDate of the actual completion of the international searchDate of mailing of the international search report11 May 202325 May 2023Name and mailing address of the ISA / CNAuthorized officerChina National Intellectual Property Administration (ISA / CN)China No. 6, Xitucheng Road. Jimenqiao, Haidian District.Beijing 100088Telephone No.INTERNATIONAL SEARCH REPORT International application No.Information on patent family members „ „H 3 PCT / CN2022 / 116464Patent document cited in search report Publication date (day / month / year) Patent family member(s) Publication date (day / month / year) CN 113975447 A 28 January 2022 None CN 1448189 A 15 October 2003 CN 1257753 C 31 May 2006 CN 107362387 A 21 November 2017 None CN 106474530 A 08 March 2017 None