Rapid blood-triggered hyperelastic starch-based hemostatic freezing gel

Abstract

The invention provides a starch-based hemostatic material. Specifically, the invention provides the starch-based efficient hemostatic frozen gel with superelasticity, and the hemostatic frozen gel is composed of oxidized quaternized starch and methacrylated gelatin, is prepared through photo-initiation and a gel freezing process, and has multiple cross-linked networks. The freezing gel disclosed by the invention has excellent physical, hemostatic, antibacterial and biodegradable properties. Aiming at incompressible massive hemorrhage wounds, the frozen gel disclosed by the invention can be used for clinical compression injection filling and rapid blood absorption by virtue of the superelasticity of the frozen gel, so that physical plugging hemostasis is realized. Meanwhile, the frozen gel has an excellent shape recovery characteristic and can accelerate a blood coagulation cascade reaction, rapid active coagulation acceleration is realized, and the influence of bleeding on a wounded person is reduced to the greatest extent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically, relates to a starch-based cryogel with superelasticity and active blood coagulation promotion. Background Art

[0002] More than 30% of trauma deaths globally are caused by excessive blood loss. Timely, simple and effective hemostasis can effectively reduce the mortality rate of the injured. At present, a variety of hemostatic agents have been developed for emergency wound hemostasis, such as zeolite powder, porous starch microspheres, combat gauze, fibrin glue and other products. However, the above-mentioned hemostatic agents are difficult to exert their effects in a timely manner when facing massive bleeding, especially bleeding from penetrating and incompressible wounds. Cryogel is a new type of sponge material. Compared with the generally unsatisfactory strength and liquid absorption capacity of traditional sponge materials, cryogel materials have advantages such as stable structure and high liquid absorption, showing broad application prospects.

[0003] The term superelasticity is used to describe a special phenomenon in the shape change cycle of materials. Superelastic materials can spontaneously or quickly achieve a high shape recovery rate (≥90%) with the help of absorbing liquid under high compressive deformation (≥80%), and can withstand a load more than 50,000 times their own weight. Combining superelasticity with cryogel materials has broad application prospects in the field of hemostatic materials, especially suitable for emergency rescue scenarios such as battlefield conflicts and traffic accidents, to achieve rapid and effective hemostasis of incompressible wounds.

[0004] Starch is a plant polysaccharide with a wide range of sources, having advantages such as good biocompatibility, biodegradability, and low cost, and is an ideal matrix for biomedical materials. However, current starch-based hemostatic materials are limited to porous powder formulations and can only cope with small-scale superficial wound bleeding; a few starch sponge hemostatic materials have defects such as easy breakage and dispersion, inability to be injection-filled, and single hemostasis mechanism, and are difficult to be used for hemostasis of larger wounds. If starch-based materials are applied to cryogel materials and superelasticity is achieved, giving play to the chemical activity advantages of starch molecules, the application potential of starch-based hemostatic materials will be greatly improved. At present, there are no reports on starch hemostatic cryogel, mainly because of the lack of starch blood coagulation promotion and chemical cross-linking modification processes, making it difficult to achieve effective and stable loading of starch in cryogel.

[0005] Therefore, there is an urgent need in this technical field to develop a superelastic starch-based hemostatic cryogel that is simple to prepare, biocompatible and biodegradable. Summary of the Invention

[0006] The object of the present invention is to provide a superelastic starch-based highly efficient hemostatic cryogel that is simple to prepare, has high production efficiency, is biocompatible and biodegradable, and has antibacterial and multifunctional properties, and a preparation method thereof.

[0007] In a first aspect of the present invention, there is provided a starch-based hemostatic material, which comprises oxidized quaternized starch and methacrylated gelatin.

[0008] In another preferred embodiment, the hemostatic material is prepared by photocrosslinking, Schiff base crosslinking and cryogelation of oxidized quaternized starch and methacrylated gelatin.

[0009] In another preferred embodiment, the mass ratio of the oxidized quaternized starch to the methacrylated gelatin is 1:(2 - 20), preferably 1:(5 - 12), more preferably 1:(6 - 9).

[0010] In another preferred embodiment, the starch-based hemostatic material has a pore structure, and the pore diameter is 100 - 500 μm, preferably 150 - 300 μm.

[0011] In another preferred embodiment, the porosity of the starch-based hemostatic material is 50 - 80%, preferably 55 - 80%, more preferably 65 - 80%.

[0012] In another preferred embodiment, the starch-based hemostatic material is a freeze-dried cryogel in a fully solid form.

[0013] In another preferred embodiment, the starch-based hemostatic material is a compressible injectable material.

[0014] In another preferred embodiment, after the compressed starch-based hemostatic material comes into contact with water or blood, it can expand to its original shape within 5 - 20 seconds, preferably 10 - 15 seconds.

[0015] In another preferred embodiment, the starch-based hemostatic material is an injectable cryogel, which is pushed into the bleeding wound through a syringe.

[0016] In another preferred embodiment, the oxidized quaternized starch is formed by modifying starch with a quaternizing reagent and then oxidizing it.

[0017] In another preferred embodiment, the starch is selected from the group consisting of 75% high amylose starch, corn starch, pea starch, potato starch, tapioca starch, amylopectin, soluble starch, pregelatinized starch, crosslinked starch, enzyme-treated starch, or a combination thereof; preferably, the starch is 75% high amylose starch.

[0018] In another preferred embodiment, the quaternizing reagent is selected from the group consisting of 3-chloro-2-hydroxypropyl-trimethylammonium chloride, N-(2,3-epoxypropyl)trimethylammonium chloride, cetyltrimethylammonium bromide, alkyltrimethyl quaternary ammonium salt, dimethylbenzylammonium chloride, benzyl quaternary ammonium salt, alkyldimethylbenzylammonium chloride, tetramethylammonium hydroxide, trimethylphenylammonium hydroxide, choline chloride, tetrabutylammonium bromide, or a combination thereof.

[0019] In another preferred example, the oxidation is formed by reacting an oxidizing agent with a quaternized modified starch;

[0020] Among them, the oxidizing agent is selected from the group consisting of: sodium periodate, hydrogen peroxide, peracetic acid, peroxide, dichromate, potassium permanganate, hypochlorite, sodium percarbonate, nitric acid, or a combination thereof; preferably sodium periodate.

[0021] In another preferred example, the methacrylated gelatin is formed by modifying gelatin with methacrylic anhydride.

[0022] In another preferred example, the methacrylated gelatin is obtained by an acylation reaction of gelatin with methacrylic anhydride.

[0023] In a preferred example, in the oxidized quaternized starch, the grafting rate of the quaternary ammonium group is 10% - 50%, preferably 15% - 40%, more preferably 20% - 40%.

[0024] In another preferred example, in the oxidized quaternized starch, the grafting rate of the quaternary ammonium group is 10% - 40%, preferably 20% - 40%.

[0025] In a preferred example, the degree of oxidation of the oxidized quaternized starch is 30% - 60%, preferably 40% - 60%, more preferably 50% - 60%.

[0026] In a preferred example, the starch-based hemostatic material has one or more of the following characteristics:

[0027] 1) The blood absorption rate of the material within 5 - 60 s (preferably 5 - 15 s) is 750% - 2000%;

[0028] 2) The compression modulus of the material is 0.5 - 3.0 MPa, preferably 1.5 - 2.5 MPa;

[0029] 3) The shape recovery time of the material in blood is 5 - 20 s;

[0030] 4) The shape recovery rate of the material in blood is 90 - 100%;

[0031] 5) The coagulation index of the material in 4 min is 2 - 20%;

[0032] 6) The degradation rate of the material in the simulated degradation solution in 5 - 15 days is 100%;

[0033] 7) The antibacterial rate of the material against E. coli and S. aureus is 50 - 90%;

[0034] 8) The bleeding volume after the material stops bleeding and plugs non-compressible wounds in the body is 60-90% of that of the commercial type B gelatin hemostatic sponge of Xiangen Medical;

[0035] 10) The hemostasis time of the material for plugging non-compressible wounds in the body is 40-70% of that of the commercial type B gelatin hemostatic sponge of Xiangen Medical;

[0036] 11) The hemolysis rate of the material is 0.1-5%;

[0037] 12) The cytocompatibility of the material is 75-100%.

[0038] In another preferred example, the blood absorption rate of the material within 5-60 s (preferably 5-20 s, more preferably 5-15 s) is 1200%-2000%, preferably 1300%-1600%, more preferably 1300%-1800%.

[0039] In another preferred example, the compression modulus of the material is 0.5-3.0 MPa, preferably 1.0-3.0 MPa, more preferably 1.5-3.0 MPa, and most preferably 1.5-2.5 MPa.

[0040] In another preferred example, the shape recovery time of the material in blood is 10-30 s, preferably 10-20 s, more preferably 12-18 s, and most preferably 13-17 s.

[0041] In another preferred example, the shape recovery rate of the material in blood is 90-100%, preferably 92-100%, more preferably 95-100%.

[0042] In another preferred example, the coagulation index of the material in 4 min is 5-50%, preferably 5-40%, more preferably 5-30%, and most preferably 5-20%.

[0043] In another preferred example, the degradation rate of the material in the simulated degradation solution in 5-15 days is 100%, preferably 5-13 days, more preferably 5-12 days.

[0044] In another preferred example, the degradation rate of the material in the simulated degradation solution in 11-15 days is 100%, preferably 11-13 days, more preferably 11-12 days.

[0045] In another preferred example, the antibacterial rates of the material against E. coli and S. aureus are 60%-90%, preferably 65-80%, more preferably 65-80%.

[0046] In another preferred example, the bleeding volume after the material stops bleeding and seals an incompressible wound in the body is 60-90% of that of the commercial type B gelatin hemostatic sponge of Xiangen Medical, preferably 60-87%, more preferably 60-86%.

[0047] In another preferred example, the bleeding volume after the material stops bleeding and seals an incompressible wound in the body is 70-90% of that of the commercial type B gelatin hemostatic sponge of Xiangen Medical, preferably 75-87%, more preferably 80-86%.

[0048] In another preferred example, the hemostasis time of the material for stopping bleeding and sealing an incompressible wound in the body is 40-70% of that of the commercial type B gelatin hemostatic sponge of Xiangen Medical, preferably 40-62%.

[0049] In another preferred example, the hemostasis time of the material for stopping bleeding and sealing an incompressible wound in the body is 50-70% of that of the commercial type B gelatin hemostatic sponge of Xiangen Medical, preferably 55-65%.

[0050] In another preferred example, the hemolysis rate of the material is 0.1-5%, preferably 1-4%, more preferably 2-4%.

[0051] In another preferred example, the cytocompatibility of the material is 80-98%, preferably 85-95%.

[0052] In the second aspect of the present invention, there is provided a preparation method of the starch-based hemostatic material as described in the first aspect of the present invention, and the method includes the following steps:

[0053] (a) In the presence of a first alkali reagent, starch reacts with a quaternization reagent to obtain quaternized starch;

[0054] (b) In the presence of a first acid reagent, the quaternized starch obtained in step (a) reacts with an oxidant to obtain oxidized quaternized starch;

[0055] (c) In the presence of a buffer solution, gelatin reacts with methacrylic anhydride to obtain methacryloylated gelatin;

[0056] (d) Mix the oxidized quaternized starch obtained in step (b), the methacryloylated gelatin obtained in step (c) and a photoinitiator, first carry out a photocuring reaction under ultraviolet light, then carry out a Schiff base reaction at -10 to -60 °C, and freeze-dry to obtain the starch-based hemostatic material as described in the first aspect of the present invention.

[0057] In a preferred example, in step (a), the starch is selected from the group consisting of: 75% high amylose starch, corn starch, pea starch, potato starch, cassava starch, amylopectin, soluble starch, pregelatinized starch, cross-linked starch, enzyme-treated starch, or a combination thereof.

[0058] In another preferred example, the starch is 75% high amylose starch.

[0059] In a preferred example, in step (a), the quaternizing reagent is selected from the group consisting of: 3-chloro-2-hydroxypropyltrimethylammonium chloride, N-(2,3-epoxypropyl)trimethylammonium chloride, cetyltrimethylammonium bromide, alkyltrimethyl quaternary ammonium salt, dimethylbenzylammonium chloride, benzyl quaternary ammonium salt, alkyldimethylbenzylammonium chloride, tetramethylammonium hydroxide, trimethylphenylammonium hydroxide, choline chloride, tetrabutylammonium bromide, or a combination thereof.

[0060] In another preferred example, the quaternizing reagent is 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0061] In another preferred example, in step (a), the first base reagent is selected from the group consisting of: sodium hydroxide, potassium hydroxide, barium hydroxide, sodium carbonate, sodium bicarbonate, or a combination thereof; preferably sodium hydroxide.

[0062] In another preferred example, in step (a), the first base reagent exists in the form of an aqueous solution of the first base reagent, and the concentration of the aqueous solution of the first base reagent is 0.5 - 2M, preferably 1 - 1.5M.

[0063] In another preferred example, step (a) further includes the following step: the starch is dissolved in the aqueous solution of the first base reagent, and the dissolution temperature is 30 - 50°C, preferably 35 - 45°C.

[0064] In another preferred example, in step (a), the mass-volume ratio of the starch to the aqueous solution of the first base reagent is (10 - 100):1 g / L, preferably (50 - 100):1 g / L, more preferably (70 - 90):1 g / L.

[0065] In another preferred example, in step (a), the mass ratio of the starch to the quaternizing reagent is 1:(0.1 - 5), preferably 1:(0.2 - 2), more preferably 1:(0.5 - 1.5).

[0066] In another preferred example, in step (a), the reaction temperature is 50 - 70°C, preferably 60 - 70°C.

[0067] In another preferred example, in step (a), the reaction time is 3 - 10 h, preferably 5 - 7 h.

[0068] In another preferred example, step (a) further includes the following steps: after the reaction is completed, the obtained quaternized starch is added to anhydrous methanol, precipitated and filtered by suction. The suction filtration product is washed with an aqueous alcohol solution of 60-80%, until no precipitate is formed when silver nitrate is added dropwise to the washing solution, and then washed with anhydrous methanol and dried to obtain the quaternized starch.

[0069] In another preferred example, in step (b), the first acid reagent is selected from the group consisting of: sulfuric acid, hydrochloric acid, nitric acid, perchloric acid, acetic acid, formic acid, or a combination thereof; preferably sulfuric acid.

[0070] In another preferred example, in step (b), the first acid reagent exists in the form of an aqueous solution of the first acid reagent, and the pH of the aqueous solution of the first acid reagent is 3-6, preferably 5-6.

[0071] In another preferred example, in step (b), the mass-volume ratio of the quaternized starch to the aqueous solution of the first acid reagent is (5-50):1 g / L, preferably (5-30):1 g / L, more preferably (5-20):1 g / L, and most preferably (8-12):1 g / L.

[0072] In another preferred example, in step (b), the oxidant is selected from the group consisting of: sodium periodate, hydrogen peroxide, peracetic acid, peroxides, dichromates, potassium permanganate, hypochlorites, sodium percarbonate, nitric acid, or a combination thereof; preferably sodium periodate.

[0073] In another preferred example, in step (b), the mass ratio of the quaternized starch to the oxidant is 1:(0.1-5), preferably 1:(0.5-4), more preferably 1:(0.5-2), and most preferably 1:(1-1.5).

[0074] In another preferred example, in step (b), the reaction is carried out under light-shielded conditions.

[0075] In another preferred example, in step (b), the reaction temperature is 30-60 °C, preferably 35-45 °C.

[0076] In another preferred example, in step (b), the reaction time is 2-8 h, preferably 3-5 h.

[0077] In another preferred example, step (b) further includes the following steps: after the reaction is completed, 1% (V / V) volume of ethylene glycol is added to the reaction solution, stirred for 10-20 min to terminate the reaction; the reaction solution is loaded into a dialysis bag with a molecular weight of 3500, dialyzed for 3-5 days, with the water changed 3-5 times a day, and freeze-dried to obtain oxidized quaternized starch.

[0078] In another preferred example, in step (c), the gelatin is selected from the group consisting of: alkali-processed gelatin, acid-processed gelatin, enzyme-processed gelatin, collagen, fibrin, keratin, fibroin, or a combination thereof; preferably alkali-processed gelatin.

[0079] In another preferred example, in step (c), the buffer solution is selected from the group consisting of: CB buffer solution, PBS buffer solution, Tris buffer solution, MES buffer solution, HEPES buffer solution, or a combination thereof; preferably CB buffer solution.

[0080] In another preferred example, in step (c), the pH of the buffer solution is 9 - 12, preferably 9 - 10.

[0081] In another preferred example, in step (c), the mass-volume ratio of the gelatin to the buffer solution is (10 - 50):1 g / L, preferably (10 - 40):1 g / L, more preferably (20 - 30):1 g / L, and most preferably (20 - 25):1 g / L.

[0082] In another preferred example, in step (c), the volume-mass ratio of methacrylic anhydride to gelatin is (10 - 100):1 μL / g, preferably (20 - 90):1 μL / g, more preferably (30 - 70):1 μL / g, and most preferably (35 - 65):1 μL / g.

[0083] In another preferred example, in step (c), the reaction temperature is 30 - 60 °C, preferably 40 - 60 °C.

[0084] In another preferred example, in step (c), the reaction time is 1 - 5 h, preferably 2 - 4 h.

[0085] In another preferred example, step (c) further includes the following steps: after the reaction is completed, the solution is filled into a dialysis bag with a molecular weight cut-off of 3500, dialyzed for 3 - 5 days, with the water changed 3 - 5 times a day, and then freeze-dried to obtain methacrylated gelatin.

[0086] In another preferred example, in step (d), both the oxidized quaternized starch and the methacrylated gelatin exist in the form of an aqueous solution.

[0087] In another preferred example, in step (d), the concentration of the oxidized quaternized starch is 0.5 - 5 wt%, preferably 0.5 - 4 wt%, more preferably 1 - 3 wt%, and most preferably 1 - 2 wt%.

[0088] In another preferred example, in step (d), the concentration of the methacrylated gelatin is 1 - 20 wt%, preferably 2 - 15 wt%, more preferably 5 - 12 wt%.

[0089] In another preferred example, in step (d), the concentration of the methacrylated gelatin is 5-9.5 wt%, preferably 6-9 wt%, and most preferably 6.5-8.5 wt%.

[0090] In another preferred example, in step (d), the volume ratio of the oxidized quaternized starch aqueous solution to the methacrylated gelatin aqueous solution is 1:(0.5-5), preferably 1:(0.5-2), and more preferably 1:(0.5-1.5).

[0091] In another preferred example, step (d) further includes the following steps: mixing the oxidized quaternized starch obtained in step (b) and the methacrylated gelatin obtained in step (c) to obtain a mixed solution; adding a photoinitiator to the mixed solution.

[0092] In another preferred example, in step (d), the photocrosslinking and curing reaction is carried out in the presence of a photoinitiator.

[0093] In another preferred example, the photoinitiator is selected from the group consisting of: I2959, LAP, TPO, ITX, BDK, OMBB, or a combination thereof; preferably I2959.

[0094] In another preferred example, in step (d), the mass-to-volume ratio of the photoinitiator to the mixed solution is (1-10):1 mg / mL, preferably (1-5):1 mg / mL, and more preferably (1-3):1 mg / mL.

[0095] In another preferred example, in step (d), the time of the photocuring reaction is 2-15 min, preferably 3-10 min, and more preferably 3-8 min.

[0096] In another preferred example, in step (d), the temperature of the photocuring reaction is 10-40 °C, preferably 20-30 °C.

[0097] In another preferred example, in step (d), the temperature of the Schiff base reaction is -10 to -40 °C, preferably -10 to -30 °C, and more preferably -15 to -25 °C.

[0098] In another preferred example, in step (d), the time of the Schiff base reaction is 6-48 h, preferably 12-36 h, and more preferably 22-28 h.

[0099] In a preferred example, in step (d), the mass ratio of the oxidized quaternized starch to the methacrylated gelatin is 1:(1-15), preferably 1:(2-12), and more preferably 1:(5-10).

[0100] The third aspect of the present invention provides a use of the starch-based hemostatic material as described in the first aspect of the present invention for preparing hemostatic products.

[0101] In another preferred embodiment, the hemostatic product is a hemostatic product for emergency bleeding or a hemostatic product for controlling massive bleeding;

[0102] The hemostatic products for emergency bleeding are selected from the group consisting of: hemostatic products for traumatic bleeding, hemostatic products for penetrating wound bleeding, hemostatic products for nasal bleeding, hemostatic products for osseous bleeding, and hemostatic products for gynecological bleeding;

[0103] The hemostatic products for controlling massive bleeding are selected from the group consisting of: hemostatic products for tissue penetrating wounds, hemostatic products for tissue defect wounds, and hemostatic products for narrow wounds.

[0104] The fourth aspect of the present invention provides a hemostatic product, a tissue engineering product or a wound repair product, comprising the starch-based hemostatic material as described in the first aspect of the present invention.

[0105] In another preferred embodiment, the hemostatic product, tissue engineering product or wound repair product further comprises a pharmaceutically or materially acceptable carrier.

[0106] The fifth aspect of the present invention provides a hemostatic material obtained by the preparation method as described in the second aspect of the present invention.

[0107] The sixth aspect of the present invention provides a method for rapid hemostasis, which comprises applying the starch-based hemostatic material as described in the first aspect or the fifth aspect of the present invention, or the hemostatic product, tissue engineering product or wound repair product as described in the fourth aspect of the present invention to an object in need thereof.

[0108] In another preferred embodiment, the object is a human or a non-human mammal;

[0109] The non-human mammals are selected from the group consisting of: rats, mice, rabbits, dogs, sheep, pigs, and cows.

[0110] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 1H NMR spectra of the products of Example 2 and Example 5 of the present invention.

[0112] Figure 2 Degree of oxidation test chart of the oxidized quaternized starch prepared in Example 5 of the present invention.

[0113] Figure 3 SEM images of the internal structures of the starch-based hemostatic cryogels prepared in Examples 10, 11, and 12 of the present invention.

[0114] Figure 4 Morphologies of the starch-based hemostatic cryogels prepared in Examples 10, 11, and 12 of the present invention before and after blood absorption.

[0115] Figure 5 Diagram showing the degradation of the starch-based hemostatic cryogels prepared in Examples 10, 11, and 12 of the present invention.

[0116] Figure 6 Antibacterial effects of the starch-based hemostatic cryogels prepared in Examples 10, 11, and 12 of the present invention against E. coli and S. aureus after 6 h.

[0117] Figure 7 BCI results of the starch-based hemostatic cryogels prepared in Examples 10, 11, and 12 of the present invention.

[0118] Figure 8 In vivo hemostasis effect diagrams of the starch-based hemostatic cryogels prepared in Examples 10, 11, and 12 of the present invention. Detailed implementation manners

[0119] After extensive and in-depth research, the inventor of the present invention unexpectedly discovered a starch-based hemostatic material. Specifically, the inventor optimized the starch modification route to prepare oxidized quaternized starch with excellent comprehensive properties. After cross-linking and curing oxidized quaternized starch and methacrylated gelatin under ultraviolet light, Schiff base cross-linking was continued, and then freeze-drying was carried out to obtain a super-elastic starch-based highly efficient hemostatic cryogel. The super-elastic starch-based hemostatic cryogel of the present invention has advantages such as strong liquid absorption performance, high mechanical strength, rapid expansion and injectability, and active blood coagulation promotion. It can achieve rapid hemostasis and avoid secondary bleeding, and the hemostasis effect is better than that of commercially available sponge-like hemostatic materials. In addition, the starch-based hemostatic cryogel is prepared from natural polymer derivatives, has good biocompatibility and is biodegradable in vivo. Based on this, the present invention was completed.

[0120] Terms

[0121] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0122] As used herein, the terms "starch-based hemostatic cryogel", "starch-based hemostatic material", and "starch-based cryogel" are used interchangeably and refer to the hemostatic cryogel obtained by photocrosslinking and freeze-drying oxidized quaternary ammonium starch (formed by quaternary ammonium starch and an oxidant) and methacrylated gelatin of the present invention.

[0123] As used herein, the terms "photocuring reaction", "photocrosslinking reaction", and "photocrosslinking and curing reaction" are used interchangeably and refer to the process in which covalent bonds (such as C-C and C-O bonds) are formed between molecules induced by light, connecting linear or branched molecular chains into a network structure.

[0124] As used herein, the terms "Schiff base reaction" and "Schiff base crosslinking" are used interchangeably and refer to the process in which an amine compound and an aldehyde / ketone compound form an imine (C=N bond) through a nucleophilic addition-elimination reaction.

[0125] As used herein, when used in reference to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0126] As used herein, the terms "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the terms also include "consisting essentially of" or "consisting of".

[0127] As used herein, the term "room temperature" or "ambient temperature" refers to a temperature of 4 to 40 °C, preferably 25 ± 5 °C.

[0128] As used herein, the term "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0129] Starch-based hemostatic cryogel

[0130] The hemostatic material of the present invention is a starch-based hemostatic cryogel with superelasticity, obtained by photocrosslinking and curing reaction, Schiff base crosslinking, and freeze-drying of oxidized quaternary ammonium starch and methacrylated gelatin.

[0131] In a preferred embodiment, the oxidized quaternary ammonium starch is obtained by reacting starch with a quaternary ammonium reagent and an oxidant in sequence.

[0132] In a preferred embodiment, the oxidized quaternary ammonium starch is obtained by reacting quaternary ammonium starch with an oxidant.

[0133] In a preferred embodiment, the quaternary ammonium starch is obtained by reacting starch with a quaternary ammonium reagent.

[0134] In a preferred embodiment, the methacrylated gelatin is obtained by the amide reaction of gelatin and methacrylic anhydride.

[0135] In a preferred embodiment, the gelatin is selected from the group consisting of: alkali-processed gelatin, acid-processed gelatin, enzyme-processed gelatin, collagen, fibrin, keratin, silk fibroin, or a combination thereof; preferably alkali-processed gelatin.

[0136] The hemostatic material of the present invention is based on starch and gelatin;

[0137] Among them, starch is reacted with a quaternizing reagent and an oxidizing agent in sequence to obtain oxidized quaternized starch;

[0138] Gelatin reacts with methacrylic anhydride to obtain methacrylated gelatin.

[0139] The hemostatic material of the present invention is a multi-crosslinked network hemostatic cryogel obtained by Schiff base crosslinking and photocrosslinking reactions.

[0140] The starch-based hemostatic material of the present invention has one or more (1, 2, 3, 4, 5, 6, 7, 8, or 9) of the following characteristics:

[0141] 1) The blood absorption rate of the material is 750% - 2000% within 5 - 60 s (preferably 5 - 15 s), more preferably 1200% - 2000%;

[0142] 2) The compression modulus of the material is 0.5 - 2.5 MPa, more preferably 1.5 - 2.5 MPa;

[0143] 3) The shape recovery time of the material in blood is less than 30 s, more preferably less than 20 s;

[0144] 4) The shape recovery rate of the material in blood is more than 90%, more preferably more than 95%;

[0145] 5) The coagulation BCI index of the material in 4 minutes is less than 30%, more preferably less than 20%;

[0146] 6) The material is biocompatible and biodegradable;

[0147] 7) The antibacterial rate of the material against E. coli and S. aureus after 6 hours is more than 60%, more preferably more than 70%;

[0148] 8) The blood loss of the material for hemostatically plugging non-compressible wounds in the body is 70% of that of commercial gelatin hemostatic sponges, more preferably 50%;

[0149] 9) The hemostasis time of the material for hemostatically plugging non-compressible wounds in the body is 90% of that of commercial gelatin hemostatic sponges, more preferably 88%.

[0150] The oxidized quaternary ammonium starch in the present invention has both blood coagulation promoting and cross-linking functions, effectively solving the technical problems of traditional starch being difficult to load, difficult to form, and having no blood coagulation promoting activity.

[0151] The preparation reaction of the oxidized quaternary ammonium starch of the present invention is stable and controllable. The grafting rate of the quaternary ammonium group exceeds 10%, preferably exceeds 20%, more preferably exceeds 30% (such as 30%, 35%, 40%), and can reach 30.9%. The degree of oxidation is greater than 30% and does not exceed 80%. For example, the degree of oxidation can be increased to 58%.

[0152] The starch-based hemostatic cryogel of the present invention has superelasticity and rapid active blood coagulation promoting function, and can achieve efficient hemostasis in emergency scenarios.

[0153] The hemostatic material of the present invention can be directly placed after simple compression or filled into the bleeding wound using a syringe. It can quickly absorb and concentrate blood, restore its shape and expand, and achieve preliminary hemostasis by plugging. At the same time, red blood cells and platelets can adhere and aggregate inside the hemostatic material of the present invention. Under the action of the quaternary ammonium group, platelets are further activated, the coagulation cascade reaction is activated, the formation of blood clots is accelerated, and effective hemostasis is achieved and secondary bleeding is prevented.

[0154] First, the superelasticity of the starch-based hemostatic cryogel of the present invention comes from the dual cross-linking network system formed by the photocrosslinking reaction and the Schiff base reaction. Compared with the single photocrosslinking network of methacrylated gelatin, the Schiff base bonds formed by the oxidized quaternary ammonium starch increase the overall cross-linking density, the molecular chains are more tightly bound, making the pore structure more orderly and regular, and maintaining high mechanical strength. The long-chain molecules of the oxidized quaternary ammonium starch store and release energy during the compression and shape recovery processes through the folding and unfolding of the chain segments in the material. After contacting with blood, due to the regular pore structure and high hydrophilicity of the cryogel, the compressed dry cryogel will quickly absorb blood, release the energy of the internal molecular chains, and produce a shape memory function.

[0155] Secondly, the oxidized quaternary ammonium starch component in the starch-based hemostatic material of the present invention not only realizes active blood coagulation promotion, but also can change the membrane permeability of bacteria and play an antibacterial role. The hemostatic cryogel has good water retention and biocompatibility, and the degradation products can accelerate wound healing, showing broad prospects in the fields of hemostasis, wound repair, etc.

[0156] Preparation method

[0157] The reaction mechanism of the oxidized quaternary ammonium starch and methacrylated gelatin is as follows:

[0158]

[0159] The present invention provides a method for preparing a starch-based hemostatic cryogel, comprising the following steps:

[0160] (a) Mix starch with sodium hydroxide solution, stir at 40 °C until completely dissolved, add 3-chloro-2-hydroxypropyltrimethylammonium chloride, raise the temperature to 60 °C and react for 6 h. After the reaction, filter with anhydrous methanol, wash, and dry to obtain the quaternized starch.

[0161] (b) Dissolve the quaternized starch in sulfuric acid solution, raise the temperature to 40 °C and react with an oxidant in the dark for 4 h. After the reaction, terminate the reaction with ethylene glycol and dialyze with ultrapure water to obtain oxidized quaternized starch.

[0162] (c) Dissolve gelatin in CB buffer at 50 °C, add methacrylic anhydride and react for 3 h, dialyze with ultrapure water to obtain methacrylated gelatin.

[0163] (d) Mix the oxidized quaternized starch solution and the methacrylated gelatin solution, under the action of I2959 photoinitiator, after crosslinking and curing, place it in an environment at -20 °C and freeze for 24 h to complete the Schiff base reaction, and freeze-dry to obtain the starch-based hemostatic cryogel.

[0164] According to the above method, in step (a), the starch is selected from the following group: 75% high amylose starch, corn starch, pea starch, potato starch, cassava starch, amylopectin, soluble starch, pregelatinized starch, crosslinked starch, enzyme-treated starch, or a combination thereof.

[0165] According to the above method, in step (a), the concentration of starch in the sodium hydroxide solution is 10-100 g / L.

[0166] According to the above method, in step (a), the mass ratio of starch to 3-chloro-2-hydroxypropyltrimethylammonium chloride is 1:0.5-5.

[0167] According to the above method, in step (b), the concentration of quaternized starch is 5-20 g / L.

[0168] According to the above method, in step (b), the mass ratio of quaternized starch to the oxidant is 1:0.5-5.

[0169] According to the above method, in step (c), the concentration of gelatin in the CB buffer is 10-50 g / L.

[0170] According to the above method, in step (c), the volume-to-mass ratio (μL / g) of methacrylic anhydride to gelatin is 10-100:1.

[0171] According to the above method, in step (d), the concentration of oxidized quaternized starch is 0.5-5 wt%.

[0172] According to the above method, in step (d), the concentration of methacrylated gelatin is 1-10 wt%.

[0173] According to the above method, in step (d), the volume ratio of the oxidized quaternized starch solution to the methacrylated gelatin solution is 1:0.5-2.

[0174] Application

[0175] The starch-based hemostatic cryogel of the present invention has super elasticity and active blood coagulation promotion efficacy, and can be used for massive bleeding in penetrating tissue wounds, tissue defect wounds or narrow wounds. It can be directly placed after simple compression or filled into the bleeding wound site by using a syringe injection, solving the defects that the existing starch powder hemostatic material is easily washed away by blood flow, and the starch sponge hemostatic material cannot expand after compression, is easily broken during use, and has no procoagulant activity.

[0176] It can be seen from the experimental results of the present invention that the starch-based cryogel obtained by the present invention also has functions in the biomedical field (such as bleeding control, antibacterial, tissue repair, wound healing, drug release, etc.), and thus has a wide range of uses.

[0177] The main advantages of the present invention include:

[0178] 1. The starch-based hemostatic material of the present invention has super elasticity, strong liquid absorption ability, can efficiently concentrate blood, has a shape memory function, can achieve rapid expansion and plugging for hemostasis, and does not break or disperse during in vivo circulation compression. It is light in weight, high in strength, portable and resistant to storage.

[0179] 2. The starch-based hemostatic material of the present invention can promote blood cell adhesion and activation, activate the coagulation cascade reaction, actively coagulate blood, and has a better hemostatic effect than commercially available sponge-like hemostatic materials and related starch sponge hemostatic material inventions, and has great clinical application value.

[0180] 3. The preparation of the product adopts a homogeneous reaction, with high production efficiency and controllable process, reducing the time cost and energy consumption. In particular, the quaternary ammonium group substitution degree and oxidation degree of the oxidized quaternized starch can reach 30.9% and 58.3% respectively.

[0181] 4. The method and the starch-based hemostatic cryogel of the present invention greatly expand the application of starch substances and contribute to the development of high-performance hemostatic materials.

[0182] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0183] Synthesis of Quaternary Ammonium Starch in Example 1

[0184] Weigh 6.4 g of dry 75% high amylose starch and add it to 80 ml of sodium hydroxide solution (1.25 M). Heat to 40 °C and stir until dissolved.

[0185] Dropwise add 4 g of 3-chloro-2-hydroxypropyl-trimethyl ammonium chloride, continue to heat to 60 °C, and stir for 6 h.

[0186] After the reaction, drop the solution into anhydrous methanol for dispersion, precipitate and filter by suction. Wash the suction-filtered product with 80% methanol aqueous solution until no precipitate forms when silver nitrate is added dropwise to the washing solution, then wash with anhydrous methanol and dry to obtain quaternary ammonium starch.

[0187] Synthesis of Quaternary Ammonium Starch in Example 2

[0188] Weigh 6.4 g of dry 75% high amylose starch and add it to 80 ml of sodium hydroxide solution (1.25 M). Heat to 40 °C and stir until dissolved.

[0189] Dropwise add 8 g of 3-chloro-2-hydroxypropyl-trimethyl ammonium chloride, continue to heat to 60 °C, and stir for 6 h.

[0190] After the reaction, drop the solution into anhydrous methanol for dispersion, precipitate and filter by suction. Wash the suction-filtered product with 80% methanol aqueous solution until no precipitate forms when silver nitrate is added dropwise to the washing solution, then wash with anhydrous methanol and dry to obtain quaternary ammonium starch.

[0191] Compared with Example 1, the difference is that the amount of quaternary ammonium salt 3-chloro-2-hydroxypropyl-trimethyl ammonium chloride used in this example is increased.

[0192] Synthesis of Oxidized Quaternary Ammonium Starch in Example 3

[0193] Dissolve 2 g of the quaternary ammonium starch in Example 1 in 200 mL of sulfuric acid solution (pH = 5), add 2 g of sodium periodate, and react in the dark at 40 °C for 4 h.

[0194] After the reaction, add 2 mL of ethylene glycol and stir for 10 min to terminate the reaction. Put the reaction solution into a dialysis bag with a molecular weight cut-off of 3500 and dialyze for 3 days, changing the water 3 times a day, and then freeze-dry to obtain oxidized quaternary ammonium starch.

[0195] Synthesis of Oxidized Quaternary Ammonium Starch in Example 4

[0196] Dissolve 2 g of the quaternary ammonium starch in Example 2 in 200 mL of sulfuric acid solution (pH = 5), add 1 g of sodium periodate, and react in the dark at 40 °C for 4 h.

[0197] After the reaction was completed, 2 mL of ethylene glycol was added and stirred for 10 min to terminate the reaction. The reaction solution was placed in a dialysis bag with a molecular weight cut-off of 3500 and dialyzed for 3 days, with the water changed 3 times a day. Then it was freeze-dried to obtain oxidized quaternized starch.

[0198] Example 5 Synthesis of Oxidized Quaternized Starch

[0199] Dissolve 2 g of the quaternized starch in Example 2 in 200 mL of sulfuric acid solution (pH = 5), add 2 g of sodium periodate, and react at 40 °C in the dark for 4 h.

[0200] After the reaction was completed, 2 mL of ethylene glycol was added and stirred for 10 min to terminate the reaction. The reaction solution was placed in a dialysis bag with a molecular weight cut-off of 3500 and dialyzed for 3 days, with the water changed 3 times a day. Then it was freeze-dried to obtain oxidized quaternized starch.

[0201] Example 6 Synthesis of Methacryloylated Gelatin

[0202] Add 2.25 g of alkali-processed gelatin to 100 mL of CB buffer solution (pH = 9), heat to 50 °C to dissolve, dropwise add 88 μL of methacrylic anhydride and maintain the solution pH = 9, and react at 50 °C for 3 h. After the reaction was completed, it was dialyzed and freeze-dried to obtain methacryloylated gelatin.

[0203] Example 7 Synthesis of Methacryloylated Gelatin

[0204] Add 2.25 g of alkali-processed gelatin to 100 mL of carbonate (CB) buffer solution (pH = 9), heat to 50 °C to dissolve, dropwise add 135 μL of methacrylic anhydride and maintain the solution pH = 9, and react at 50 °C for 3 h. After the reaction was completed, it was dialyzed and freeze-dried to obtain methacryloylated gelatin.

[0205] Example 8 Synthesis of Starch-Based Hemostatic Freezing Gel

[0206] Prepare 500 μL of 1 wt% aqueous solution of the oxidized quaternized starch obtained in Example 3 and 500 μL of 8 wt% aqueous solution of the methacryloylated gelatin obtained in Example 6, mix them in a mold at a volume ratio of 1:1, add 2 mg of I2959 photoinitiator and mix evenly. Crosslink and cure with ultraviolet light at 25 °C for 5 min. After the photo-crosslinking was completed, place it in an environment at -20 °C for 24 h for Schiff base crosslinking, and then freeze-dry to obtain the starch-based hemostatic freezing gel.

[0207] Example 9 Synthesis of Starch-Based Hemostatic Freezing Gel

[0208] 500 μL of 1 wt% aqueous solution of the oxidized quaternized starch obtained in Example 4 and 500 μL of 8 wt% aqueous solution of the methacryloylated gelatin obtained in Example 6 were mixed in a mold at a volume ratio of 1:1. 2 mg of photoinitiator I2959 was added and mixed evenly. Then, it was crosslinked and cured by ultraviolet light at 25 °C for 5 min. After the photo-crosslinking was completed, it was placed in an environment at -20 °C for 24 h for Schiff base crosslinking, and then freeze-dried to obtain a starch-based hemostatic cryogel.

[0209] Synthesis of the starch-based hemostatic cryogel in Example 10

[0210] 500 μL of 0.5 wt% aqueous solution of the oxidized quaternized starch obtained in Example 5 and 500 μL of 8 wt% aqueous solution of the methacryloylated gelatin obtained in Example 6 were mixed in a mold at a volume ratio of 1:1. 2 mg of photoinitiator I2959 was added and mixed evenly. Then, it was crosslinked and cured by ultraviolet light at 25 °C for 5 min. After the photo-crosslinking was completed, it was placed in an environment at -20 °C for 24 h for Schiff base crosslinking, and then freeze-dried to obtain a starch-based hemostatic cryogel.

[0211] Synthesis of the starch-based hemostatic cryogel in Example 11

[0212] 500 μL of 1 wt% aqueous solution of the oxidized quaternized starch obtained in Example 5 and 500 μL of 8 wt% aqueous solution of the methacryloylated gelatin obtained in Example 6 were mixed in a mold at a volume ratio of 1:1. 2 mg of photoinitiator I2959 was added and mixed evenly. Then, it was crosslinked and cured by ultraviolet light at 25 °C for 5 min. After the photo-crosslinking was completed, it was placed in an environment at -20 °C for 24 h for Schiff base crosslinking, and then freeze-dried to obtain a starch-based hemostatic cryogel.

[0213] Compared with Example 10, the difference is that the dosage of the oxidized quaternized starch in this example is further increased.

[0214] Synthesis of the starch-based hemostatic cryogel in Example 12

[0215] 500 μL of 1 wt% aqueous solution of the oxidized quaternized starch obtained in Example 5 and 500 μL of 10 wt% aqueous solution of the methacryloylated gelatin obtained in Example 6 were mixed in a mold at a volume ratio of 1:1. 2 mg of photoinitiator I2959 was added and mixed evenly. Then, it was crosslinked and cured by ultraviolet light at 25 °C for 5 min. After the photo-crosslinking was completed, it was placed in an environment at -20 °C for 24 h for Schiff base crosslinking, and then freeze-dried to obtain a starch-based hemostatic cryogel.

[0216] Compared with Example 11, the difference is that the dosage of the methacryloylated gelatin in this example is further increased.

[0217] Example 13 Synthesis of Starch-based Hemostatic Freezing Gel

[0218] 500 μL of 1 wt% aqueous solution of the oxidized quaternized starch obtained in Example 5 and 500 μL of 8 wt% aqueous solution of the methacrylated gelatin obtained in Example 7 were mixed in a mold at a volume ratio of 1:1. 2 mg of photoinitiator I2959 was added and mixed evenly. Then, it was crosslinked and cured by ultraviolet light at 25 °C for 5 min. After the photo-crosslinking was completed, it was placed in an environment of -20 °C for 24 h for Schiff base crosslinking, and then freeze-dried to obtain the starch-based hemostatic freezing gel.

[0219] Example 14 1H NMR Analysis of Starch Modified Products

[0220] The chemical structure of 75% high amylose starch was characterized by a 400 MHz superconducting Fourier transform nuclear magnetic resonance spectrometer. The solvent was DMSO-d6. Under the same conditions, the quaternized starch prepared in Example 2 and the oxidized quaternized starch prepared in Example 5 were analyzed. The results are as Figure 1 shown.

[0221] In the 1H NMR spectrum, the - + N(CH3)3 methyl proton peak that did not appear in 75% high amylose starch appeared at 3.14 ppm, indicating that the quaternary ammonium salt had been successfully grafted onto the 75% high amylose starch molecule. The proton characteristic peak of the sugar ring (O-CH-O) in 75% high amylose starch was at 5.11 ppm. By integrating the peaks, the grafting rate of the quaternary ammonium group in the oxidized quaternized starch prepared in Example 5 was calculated to be 30.9%.

[0222] Example 15 Determination of the Degree of Oxidation of Oxidized Quaternized Starch

[0223] The degree of oxidation of the oxidized quaternized starch prepared in Example 5 was determined by the hydroxylamine hydrochloride-potentiometric titration method. 100 mg of the oxidized quaternized starch was dissolved in the hydroxylamine hydrochloride solution, and the pH of the solution was adjusted to 5.0 with 0.1 mol / L NaOH solution. The degree of oxidation could be calculated based on the relationship between pH and the volume of NaOH consumed. The results are as Figure 2 shown.

[0224] It was calculated that the degree of oxidation of the oxidized quaternized starch prepared in Example 5 was 58.3%.

[0225] Example 16 Microscopic Morphology of Starch-based Hemostatic Freezing Gel

[0226] The starch-based hemostatic freezing gels prepared in Example 10, Example 11, and Example 12 were observed by scanning electron microscopy. The results are as Figure 3 shown.

[0227] As can be seen from the figure, the starch-based hemostatic cryogel prepared by the present invention has a uniform and complete pore structure with a relatively small pore size (about 150 - 300 μm), and the starch-based hemostatic cryogel has a relatively high porosity (up to 69%), indicating that the hemostatic material of the present invention has better liquid absorption ability, can efficiently concentrate blood components, and improve the hemostatic efficiency.

[0228] Blood absorption performance of the starch-based hemostatic cryogel in Example 17

[0229] The blood absorption ability of the starch-based hemostatic cryogels prepared in Example 10, Example 11, and Example 12 was characterized by the mass ratio before and after blood absorption.

[0230] Weigh the mass of the starch-based hemostatic cryogel before liquid absorption as m0, and then immerse the compressed starch-based hemostatic cryogel in anticoagulated rabbit blood. Measure the time required to fully absorb the anticoagulated rabbit blood and the weight m1 of the starch-based hemostatic cryogel after full absorption. The blood absorption ability and blood absorption rate of the starch-based hemostatic cryogel are calculated according to the following formulas:

[0231]

[0232] Blood absorption rate (%) = Blood absorption ability * 100%

[0233] Take the average value of 3 test data, and the results are shown in Table 1 below:

[0234] Table 1. Blood absorption performance of the starch-based hemostatic cryogel of the present invention

[0235]

[0236] The experimental results show that the prepared starch-based hemostatic cryogel has excellent blood absorption ability. In addition, the liquid absorption performance of Example 11 is better than that of Example 8 and Example 9, indicating that increasing the usage amounts of the oxidizing agent and the quaternization reagent can improve the liquid absorption ability of the material;

[0237] The liquid absorption performance of Example 11 and Example 12 is better than that of Example 10, indicating that increasing the content of oxidized quaternized starch can improve the liquid absorption ability of the material;

[0238] The liquid absorption performance of Example 11 and Example 12 is better than that of Example 13, indicating that the usage amount of methacrylic anhydride should not be too high.

[0239] Shape recovery ability of the starch-based hemostatic cryogel in Example 18

[0240] The starch-based hemostatic cryogels prepared in Example 10, Example 11, and Example 12 were made into cylinders of the same size through a mold. The original height was recorded as h0, and then it was longitudinally compressed to the minimum height denoted as h1. Next, it was placed in deionized water to restore its shape (i.e., expand to the original shape), and the height h2 after shape restoration was recorded. The shape recovery rate was calculated according to the following formula:

[0241]

[0242] The average value of 3 test data was taken, and the results are shown in Table 2 below and Figure 4 as follows:

[0243] Table 2. Shape recovery ability of the starch-based hemostatic cryogel of the present invention

[0244]

[0245] The experimental results showed that the shape recovery rate of the starch-based hemostatic cryogel was above 95% and was completed within 16 s.

[0246] Therefore, the starch-based hemostatic cryogel of the present invention can rapidly absorb blood, can be filled into the wound site, rapidly undergoes shape recovery, and achieves preliminary plugging and hemostasis, having the potential for clinical application.

[0247] Compression modulus of the starch-based hemostatic cryogel of Example 19

[0248] The compression moduli of the starch-based hemostatic cryogels prepared in Example 10, Example 11, and Example 12 were respectively tested by a universal material testing machine. The starch-based hemostatic cryogel was compressed to 80% of the original height at a constant rate of 5 mm / min, and the compression modulus was recorded.

[0249] The average value of 3 test data was taken, and the results are shown in Table 3 below:

[0250] Table 3. Compression modulus of the starch-based hemostatic cryogel of the present invention

[0251] Sample Example 10 Example 11 Example 12 Compression modulus (MPa) 1.50±0.09 1.57±0.05 2.49±0.1

[0252] The experimental results showed that the starch-based hemostatic cryogels prepared in Example 10, Example 11, and Example 12 had a high compression modulus. The modulus of the starch-based hemostatic cryogel exceeded 1.5 MPa, which could effectively prevent the material from breaking before use and was convenient for storage. However, the increase in the content of methacrylated gelatin (GelMA) in Example 12 caused the compression modulus of the material to increase too high, which had an adverse effect on the compressible injectability. Therefore, the content of methacrylated gelatin should not be too high.

[0253] Degradation performance of the starch-based hemostatic cryogel of Example 20

[0254] Using PBS buffer as the simulated degradation solution, the starch-based hemostatic cryogel prepared in Example 10, Example 11 and Example 12 was immersed in the simulated degradation solution, incubated at room temperature. Samples were taken out every other day, washed with ultrapure water, freeze-dried and weighed. The degradation situation was evaluated by the ratio of the remaining mass to the original weight.

[0255] As Figure 5 shown, the degradation rate of the starch-based hemostatic cryogel prepared in Example 11 was less than 40% (31.1%) in the first 5 days, and the sponge degradation rate was greater than 80% (88.9%) after 7 days, and it was completely degraded after 11 days, indicating that the starch-based hemostatic cryogel of the present invention has good degradation performance.

[0256] Antibacterial property of the starch-based hemostatic cryogel of Example 21

[0257] 10 mg of the starch-based hemostatic cryogel prepared in Example 10, Example 11 and Example 12 were respectively co-incubated with 10 6 CFU / mL of E. coli and S. aureus bacterial solutions for 6 h, and the plate coating method was used to evaluate the antibacterial property of the starch-based hemostatic cryogel.

[0258] The results are as Figure 6 shown, the antibacterial rates of the starch-based hemostatic cryogel against E. coli and S. aureus were 66.6% and 73.2% respectively, both exceeding 65%, indicating that the starch-based hemostatic cryogel of the present invention has excellent antibacterial properties, and the antibacterial properties of Example 11 and Example 12 are better than those of Example 10, indicating that increasing the content of oxidized starch can improve the antibacterial property of the starch-based hemostatic cryogel.

[0259] In vitro hemostatic effect of the starch-based hemostatic cryogel of Example 22

[0260] The hemostatic effect was evaluated by testing the blood coagulation index (BCI) of the starch-based hemostatic cryogel prepared in Example 10, Example 11 and Example 12.

[0261] Weigh 5 mg of the starch-based hemostatic cryogel and place it in a 24-well plate, add 50 μL of fresh anticoagulated rabbit blood and 5 μL of 0.2 M calcium chloride solution, and incubate at 37 °C for 4 min. After the incubation is over, add 2 mL of deionized water to the well plate, continue to incubate for 10 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance OD1 of the supernatant at 540 nm. The absorbance of the blank group without the material is OD2, and the same mass of commercial gelatin hemostatic sponge is used as the commercial control group. Calculate the BCI value of the material according to the formula. The smaller the BCI value, the stronger the hemostatic ability.

[0262]

[0263] The results are as Figure 7 shown. The BCI values of Example 10, Example 11, and Example 12 are specifically 32.3%, 6.03%, and 15.48% respectively, which are much lower than the BCI value of the commercial gelatin hemostatic sponge (76.24%), indicating that the starch-based hemostatic cryogel of the present invention has excellent hemostatic effects. Moreover, the procoagulant properties of Example 11 and Example 12 are superior to those of Example 10, indicating that increasing the content of oxidized starch can improve the procoagulant properties of the starch-based hemostatic cryogel.

[0264] In vivo hemostatic effect of the starch-based hemostatic cryogel of Example 23

[0265] Before the experiment, SD rats were anesthetized and the abdominal hair was removed. The abdomen was incised to expose the middle lobe of the liver of the SD rats. The tissue exudate of the liver was removed, and a pre-weighed dry filter paper was placed below. A punch with a diameter of 2 mm was used to punch a hole at the center of the liver to create a circular penetrating wound in the liver. Immediately after the liver started bleeding, a commercial gelatin hemostatic sponge (Xiangen Medical, Type B) or the starch-based hemostatic cryogel prepared in Example 11 was placed at the wound. The blank group did not use any hemostatic material. The hemostatic process of the liver was observed, and the blood loss and hemostasis time of each SD rat were recorded.

[0266] The experimental results are as shown in Table 4 below and Figure 8 shown. The blood loss and hemostasis time of the starch-based hemostatic cryogel of the present invention are 85% and 60% of those of the commercial gelatin hemostatic sponge respectively, indicating that compared with the commercial gelatin hemostatic sponge, the starch-based hemostatic cryogel of the present invention significantly reduces the blood loss and hemostasis time of SD rats, further proving that the starch-based hemostatic cryogel of the present invention has excellent hemostatic effects.

[0267] Table 4: In vivo hemostatic performance of the starch-based hemostatic cryogel prepared in Example 11

[0268]

[0269] Blood compatibility of the starch-based hemostatic cryogel of Example 24

[0270] Fresh anticoagulated rabbit whole blood was centrifuged in a centrifuge, and the lower-layer red blood cells were taken. The red blood cell concentrate was uniformly mixed with PBS to obtain a red blood cell suspension. 10 mg of the starch-based hemostatic cryogel prepared in Example 10, Example 11, and Example 12 was incubated with 200 μL of the red blood cell suspension at 37 °C for 1 h. The incubated mixture was centrifuged at 3000 rpm for 5 min in a centrifuge, the supernatant was aspirated, and the absorbance at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate was calculated according to the formula. Deionized water was used as the positive control, and PBS buffer was used as the negative control.

[0271]

[0272] Among them, OD sample , OD negative and OD positive are the absorbances of the sample group, negative control group, and positive control group at 450 nm, respectively.

[0273] Take the average value of the three test data, and the results are shown in Table 5 below:

[0274] Table 5. Hemolysis rate of the starch-based hemostatic cryogel of the present invention

[0275] Sample Example 10 Example 11 Example 12 Hemolysis rate (%) 2.36±0.49 2.97±0.55 3.27±0.77

[0276] The experimental results show that the hemolysis rates of the starch-based hemostatic cryogels of Example 11 and Example 12 are less than the international standard (5%), indicating that the starch-based hemostatic cryogel of the present invention has good blood compatibility and is not likely to cause adverse reactions.

[0277] Cytotoxicity of the starch-based hemostatic cryogel of Example 25

[0278] Immerse the starch-based hemostatic cryogels prepared in Example 10, Example 11, and Example 12 in DMEM medium at a concentration of 5 mg / mL for 24 h. Test the cytocompatibility of the starch-based hemostatic cryogel through the extract, and use untreated DMEM as the blank control group.

[0279] Take the average value of the three test data, and the results are shown in Table 6 below:

[0280] Table 6. Cytotoxicity of the starch-based hemostatic cryogel of the present invention

[0281]

[0282] The experimental results show that the starch-based hemostatic cryogels prepared in Example 10, Example 11, and Example 12 all have qualified cytocompatibility (>75%), indicating that the starch-based hemostatic cryogel of the present invention does not have cytotoxicity and has high safety.

[0283] Conclusion

[0284] The inventors unexpectedly found that by first quaternizing and then oxidatively modifying conventional starch, excellent hemostatic materials can be obtained. Specifically, the quaternization reaction of the present invention endows starch with excellent hemostatic performance (positive charge of the quaternary ammonium group), good water solubility, and antibacterial properties. At the same time, after oxidative modification, it has chemical cross-linking ability (dialdehyde structure) and stable loading ability, and will not dissolve after being applied to the wound and affect the hemostatic effect.

[0285] The cryogel of the present invention not only has excellent physical, hemostatic, antibacterial and biodegradable properties, but also is not easy to cause adverse reactions and has high safety. For incompressible hemorrhage wounds, the cryogel of the present invention can be clinically compressed, injected and filled by virtue of its own superelasticity to achieve rapid physical blocking and hemostasis. At the same time, after absorbing blood, the cryogel can accelerate the coagulation cascade reaction, achieve rapid active coagulation, and minimize the impact of bleeding on the injured.

[0286] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A starch-based hemostatic material, characterized in that, The hemostatic material comprises oxidized quaternized starch and methacrylated gelatin.

2. The starch-based hemostatic material according to claim 1, wherein In the oxidized quaternized starch, the grafting rate of the quaternary ammonium group is 10% to 50%, preferably 15% to 40%, more preferably 20% to 40%.

3. The starch-based hemostatic material according to claim 1, wherein, The degree of oxidation of the oxidized quaternized starch is 30% to 60%, preferably 40% to 60%, more preferably 50% to 60%.

4. The starch-based hemostatic material according to claim 1, wherein The starch-based hemostatic material has one or more of the following characteristics: 1) The blood absorption rate of the material within 5 to 60 s (preferably 5 to 15 s) is 750% to 2000%; 2) The compression modulus of the material is 0.5 to 3.0 MPa, preferably 1.5 to 2.5 MPa; 3) The shape recovery time of the material in blood is 5 to 20 s; 4) The shape recovery rate of the material in blood is 90 to 100%; 5) The coagulation index of the material at 4 min is 2 to 20%; 6) The degradation rate of the material in the simulated degradation solution in 5 to 15 days is 100%; 7) The antibacterial rate of the material against E. coli and S. aureus is 50 to 90%; 8) The amount of bleeding after the material stops bleeding and plugs an incompressible wound in the body is 60 to 90% of that of the commercial gelatin hemostatic sponge of type B of Xiangen Medical; 10) The hemostasis time of the material for plugging an incompressible wound in the body is 40 to 70% of the hemostasis time of the commercial gelatin hemostatic sponge of type B of Xiangen Medical; 11) The hemolysis rate of the material is 0.1 to 5%; 12) The cell compatibility of the material is 75 to 100%.

5. A method for preparing the starch-based hemostatic material according to claim 1, characterized in that, The method comprises the following steps: (a) Reacting starch with a quaternizing reagent in the presence of a first base reagent to obtain quaternized starch; (b) Reacting the quaternized starch obtained in step (a) with an oxidant in the presence of a first acid reagent to obtain oxidized quaternized starch; (c) Reacting gelatin with methacrylic anhydride in the presence of a buffer solution to obtain methacrylated gelatin; (d) Mixing the oxidized quaternized starch obtained in step (b), the methacrylated gelatin obtained in step (c) and a photoinitiator, first carrying out a photocuring reaction under ultraviolet light, then carrying out a Schiff base reaction at -10 to -60 °C, and freeze-drying to obtain the starch-based hemostatic material as claimed in claim 1.

6. The preparation method according to claim 5, wherein, In step (a), the starch is selected from the group consisting of: 75% high amylose starch, corn starch, pea starch, potato starch, tapioca starch, amylopectin, soluble starch, pregelatinized starch, crosslinked starch, enzymatically treated starch, or a combination thereof.

7. The preparation method according to claim 5, wherein In step (a), the quaternizing reagent is selected from the group consisting of: 3-chloro-2-hydroxypropyl-trimethylammonium chloride, N-(2,3-epoxypropyl)trimethylammonium chloride, cetyltrimethylammonium bromide, alkyltrimethyl quaternary ammonium salts, dimethylbenzylammonium chloride, benzyl quaternary ammonium salts, alkyldimethylbenzylammonium chloride, tetramethylammonium hydroxide, trimethylphenylammonium hydroxide, choline chloride, tetrabutylammonium bromide, or a combination thereof.

8. The preparation method according to claim 5, wherein In step (d), the mass ratio of the oxidized quaternized starch to the methacrylated gelatin is 1: (1 to 15), preferably 1: (2 to 12), and more preferably 1: (5 to 10).

9. Use of a starch-based hemostatic material as described in claim 1, characterized in that, For the preparation of hemostatic products.

10. A hemostatic product, tissue engineering product or wound repair product, characterized in that, Comprising the starch-based hemostatic material according to claim 1.

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