A composite hemostatic material and method of making same

Abstract

The application discloses a composite hemostatic material and a preparation method thereof. The composite hemostatic material comprises a crosslinked product of double bond modified collagen and double bond modified sodium alginate and vascular endothelial growth factor loaded on the crosslinked product. The double bond modified collagen is made to have unsaturated double bonds, the double bond modified sodium alginate is made to have unsaturated double bonds, then the crosslinked product is reacted to obtain a hydrogel, and finally the hydrogel is immersed in a VEGF solution to obtain the composite hemostatic material. The three-component composite hemostatic material can accelerate the wound healing ability, and the hemostatic performance and the ability to promote wound healing are obviously improved.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to a composite hemostatic material and its preparation method. Background Technology

[0002] In general surgery, neurosurgery, cardiovascular surgery, hepatology, and orthopedic surgeries, large-area bleeding is a frequent challenge for surgeons and a major cause of surgical failure. Furthermore, statistics show that if bleeding is controlled within 30 minutes of an accident, the survival rate of injured individuals can increase by more than 40%. Effective hemostasis within the golden timeframe after bleeding is crucial for improving patient survival and surgical success rates. On the other hand, improper handling after wound scabbing can lead to recurrence; accelerating wound healing can minimize the risk of recurrence.

[0003] In mild cases such as bleeding from capillaries or small veins, traditional methods like compression and suturing are sufficient to stop the bleeding. However, these methods are ineffective for severe bleeding, requiring the use of hemostatic materials. But materials like bandages, bone wax, and zeolite are not ideal for hemostasis, take a long time to stop, and their non-biodegradability can cause secondary injury to the patient. Therefore, absorbable hemostatic materials have emerged. Because of their absorbability, these materials do not need to be removed after hemostasis, making safe and efficient hemostasis the development direction of hemostatic materials in recent years.

[0004] Currently, mainstream absorbable materials include collagen, oxidized cellulose, gelatin, chitosan, and fibrin. However, each has its significant drawbacks: cellulose degrades into acid, easily triggering tissue inflammation; gelatin and sodium alginate require the body's clotting factors, making them unsuitable for patients with coagulation disorders; fibrin has poor mechanical strength and adhesion, making it unsuitable for arterial and large vein bleeding; chitosan has poor water solubility and hemostatic effects; in comparison, while collagen has advantages such as suitability for patients with coagulation disorders and better hemostasis and healing promotion than other materials, its single-component nature also results in poor mechanical properties, limited water absorption, and limited healing-promoting effects. Therefore, single-component hemostatic materials often have limitations and cannot meet the needs of various applications. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, one objective of this invention is to provide a composite hemostatic material with good mechanical properties, excellent water absorption, and the ability to accelerate wound healing; another objective of this invention is to provide a method for preparing the composite hemostatic material.

[0006] One of the objectives of this invention is achieved through the following technical solution:

[0007] A composite hemostatic material comprising a crosslinked compound of double-bond modified collagen and double-bond modified sodium alginate, and vascular endothelial growth factor loaded on the crosslinked compound.

[0008] Furthermore, the mass ratio of the double-bond modified collagen to the double-bond modified sodium alginate is 1:(0.5–1.5); the loading of the vascular endothelial growth factor is 0.1–0.5 μg / cm³. 2 .

[0009] Furthermore, the double-bond modified collagen is obtained by reacting collagen with a compound containing double bonds, and the specific preparation method includes the following steps:

[0010] If the compound containing a double bond is a compound containing a carboxyl group and / or a compound containing an amino group, the compound containing a double bond undergoes an amidation reaction with collagen to obtain collagen modified with double bonds.

[0011] If the compound containing a double bond is an aldehyde-containing compound containing a double bond, the compound containing the double bond undergoes an aldehyde-amine condensation reaction with collagen to obtain collagen modified with double bonds.

[0012] If the compound containing a double bond is a compound containing an epoxy group, the epoxy group reacts with the amino group of collagen to obtain collagen modified with a double bond.

[0013] If the compound containing a double bond is a compound containing an anhydride, the anhydride reacts with the amino group of collagen to undergo an acylation reaction, resulting in collagen modified with a double bond.

[0014] Furthermore, the double-bond modified sodium alginate is obtained by reacting sodium alginate with a compound containing a double bond. The specific preparation method includes the following steps:

[0015] If the compound containing a double bond is an amino group, sodium alginate undergoes an amidation reaction with the amino group in the compound containing the double bond.

[0016] If the compound containing a double bond is a compound containing an epoxy group, sodium alginate and the epoxy group in the compound containing a double bond undergo a cross-linking reaction to obtain sodium alginate modified with a double bond.

[0017] If the compound containing a double bond is a compound containing an anhydride, sodium alginate and the anhydride in the compound containing a double bond undergo a cross-linking reaction to obtain sodium alginate modified with double bonds.

[0018] Furthermore, the compound containing a carboxyl group and a double bond is one or more of acrylic acid, sodium acrylate, methacrylic acid, and sodium methacrylate; the compound containing an aldehyde group and a double bond is one or more of acrolein and methacrolein.

[0019] Furthermore, the compound containing an amino group and a double bond is one or more of acrylamide, methacrylamide, p-propenylaniline, acrylhydrazide derivatives, or p-methpropenylphenethylamine; the compound containing an epoxy group and a double bond is one or more of epoxide, glycidyl methacrylate, or allyl glycidyl ether; and the compound containing an anhydride and a double bond is one or more of maleic anhydride, methacrylic anhydride, or acrylic anhydride.

[0020] Furthermore, a catalyst is added to the amidation reaction. The catalyst is one or more of the following: carbodiimide hydrochloride / 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azabenzotriazole), N-hydroxysuccinimide, 1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate, and N′,N″-carbonyldiimidazole.

[0021] The second objective of this invention is achieved by the following technical solution:

[0022] The preparation method of the above-mentioned composite hemostatic material includes the following steps:

[0023] 1) Double-bond modified collagen and double-bond modified sodium alginate were dissolved in a solvent, and a photoinitiator was added. The photocrosslinking reaction was carried out under ultraviolet light to obtain a photocurable hydrogel.

[0024] 2) The photocurable hydrogel was immersed in a vascular endothelial growth factor solution to react and obtain a composite hemostatic material.

[0025] Furthermore, the photoinitiator is one or more of the following: benzophenone, 4′,4″-bis(dimethylamino)benzophenone, Irgacure2959 (2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone), LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), 2′,2″-dimethoxy-2-phenylacetophenone, and riboflavin.

[0026] Furthermore, the mass ratio of the double-bond modified collagen, the double-bond modified sodium alginate, and the photoinitiator is 1:(0.5-1.5):(0.01-0.05); the concentration of the vascular endothelial growth factor solution is 1-5 μg / mL.

[0027] Furthermore, in step 1), the wavelength of the ultraviolet light is 254–400 nm, and the energy density is 150–1500 mW / cm². 2 In step 2), the photocurable hydrogel is immersed in the vascular endothelial growth factor solution for 0.5 to 2 hours.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) The composite hemostatic material of the present invention comprises a crosslinked product of double-bond modified collagen and double-bond modified sodium alginate, and vascular endothelial growth factor loaded on the crosslinked product. Double-bond modification of collagen is used to obtain unsaturated double bonds, and then double-bond modification of sodium alginate is used to obtain unsaturated double bonds. These are then crosslinked to obtain a hydrogel, which is finally immersed in a VEGF solution to obtain the composite hemostatic material. This three-component composite hemostatic material can accelerate wound healing, and its hemostatic performance and wound healing promotion ability are significantly improved.

[0030] (2) The preparation method of the composite hemostatic material of the present invention utilizes double-bond modified collagen and double-bond modified sodium alginate to obtain unsaturated double bonds, then places the two under light for cross-linking reaction to obtain a photocurable hydrogel, and finally immerses the photocurable hydrogel in VEGF solution to obtain the composite hemostatic material. This preparation method has mild reaction conditions, does not denature collagen, endows the collagen with a cross-linked network structure, and provides excellent mechanical properties and flexibility; it has good hydrophilicity, high liquid absorption capacity and absorption speed; the water absorption swelling degree can be controlled, and the water absorption swelling degree can be reduced by increasing the cross-linking density, allowing for the selection of gel materials with different degrees of cross-linking for different application areas; by loading growth factors, the material is endowed with excellent wound healing ability; at the same time, the raw materials used are biodegradable, gradually degrading and being excreted from the body after wound healing, making it safe and effective. This solves the problems of insufficient water absorption and poor mechanical properties of single-polymer raw materials. Attached Figure Description

[0031] Figure 1 This is a process flow diagram of the composite hemostatic material of the present invention. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more comprehensive description of the invention will be provided below in conjunction with specific embodiments. Preferred embodiments of the invention are given in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. It should be understood that these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms “and” and “or” as used herein include any and all combinations of one or more of the associated listed items.

[0034] A composite hemostatic material, comprising double-bond modified collagen, double-bond modified sodium alginate, and vascular endothelial growth factor (VEGF) as raw materials; wherein, the double-bond modified collagen and double-bond modified sodium alginate are cross-linked and cured to obtain a hydrogel, and the hydrogel is then mixed with VEGF to obtain the composite hemostatic material; the double-bond modified collagen is obtained by reacting collagen with a compound containing double bonds; the double-bond modified sodium alginate is obtained by reacting sodium alginate with a compound containing double bonds.

[0035] The following section will provide a detailed introduction to composite hemostatic materials, focusing on their preparation methods. For example... Figure 1 As shown, the method includes the following steps:

[0036] Step S1: React collagen with compounds containing double bonds to obtain double-bond modified collagen. Since collagen itself possesses many functional groups available for reaction—hydroxyl, carboxyl, and amino groups—these functional groups can be used to perform grafting reactions with other functional groups: esterification of hydroxyl groups with acid anhydrides, condensation of carboxyl groups with amino amides, ring-opening reactions of carboxyl groups with epoxy groups, and condensation reactions of amino groups with aldehydes. The amide condensation of carboxyl and amino groups requires specific conditions to form a covalent bond; the amino and carboxyl groups in collagen themselves can only be ion-adsorbed and are easily broken. Therefore, the following five methods can be used to modify the double bonds of collagen:

[0037] In some embodiments, collagen is amidated with a carboxyl-containing compound containing a double bond in a solution of a catalyst for a reaction time of 0.5–24 h, followed by dialyzing and lyophilization. The mass ratio of collagen, carboxyl-containing compound containing a double bond, and amidation catalyst is 1.0:0.2–0.5:0.2–0.5. For example, the mass ratio of collagen, carboxyl-containing compound containing a double bond, and amide catalyst is 1.0:0.2:0.2, 1.0:0.2:0.5, 1.0:0.5:0.2, 1.0:0.5:0.5, or any range of two of these values.

[0038] Furthermore, the amidation catalyst is selected from any one or a combination of several of EDC (carbodiimide hydrochloride) / HOBT (1-hydroxybenzotriazole), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), HATU (2-(7-azabenzotriazole), EDC / NHS (N-hydroxysuccinimide), BOP (1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate), and CDI (N′,N″-carbonyldiimidazole).

[0039] Furthermore, the carboxyl-containing compound containing a double bond is selected from any one or a combination of several of acrylic acid, sodium acrylate, methacrylic acid, and methacrylic acid.

[0040] In some embodiments, collagen and an amino-containing compound with a double bond are added to an amidation catalyst for an amidation reaction for 0.5–24 h, followed by dialyzing and lyophilization. The mass ratio of collagen, the amino-containing compound with a double bond, and the amidation catalyst is 1.0:0.2–0.5:0.2–0.5. For example, the mass ratio of collagen, the amino-containing compound with a double bond, and the amidation catalyst is 1.0:0.2:0.2, 1.0:0.2:0.2, 1.0:0.5:0.2, 1.0:0.5:0.5, or any range of two of these values.

[0041] Furthermore, the amidation catalyst is selected from any one or a combination of several of EDC (carbodiimide hydrochloride) / HOBT (1-hydroxybenzotriazole), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), HATU (2-(7-azabenzotriazole), EDC / NHS (N-hydroxysuccinimide), BOP (1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate), and CDI (NN-carbonyldiimidazole).

[0042] Furthermore, the compound containing an amino group and a double bond is selected from any one or a combination of several of acrylamide, methacrylamide, p-propenylaniline, acrylhydrazide derivatives, or p-methpropenylphenethylamine.

[0043] In some embodiments, collagen reacts with a compound containing an aldehyde group and a double bond in solution for 0.5–2.5 h, followed by reduction with sodium cyanoborohydride. After the reaction is complete, the mixture is dialyzed and lyophilized. The pH of the solution should be 3.5. The mass ratio of collagen, the compound containing an aldehyde group and a double bond, and sodium cyanoborohydride is 1.0:0.2–0.5:0.02–0.05. For example, the mass ratio of collagen, the compound containing an aldehyde group and a double bond, and sodium cyanoborohydride is 1.0:0.2:0.02, 1.0:0.2:0.05, 1.0:0.5:0.02, 1.0:0.5:0.05, or any range of two of these values.

[0044] Furthermore, the compound containing an aldehyde group and a double bond is selected from any one or a combination of acrolein and methacrolein.

[0045] In some embodiments, collagen is reacted with an epoxy-containing compound containing double bonds in solution for 2–10 days, followed by dialyzing and lyophilization. The pH of the solution should be 6.5–8.5, and the mass ratio of collagen to the epoxy-containing compound containing double bonds is 1.0:0.2–0.5.

[0046] Furthermore, the compound containing an epoxy group and a double bond is selected from any one or a combination of several of glycidyl methacrylate, allyl glycidyl ether, and epoxybutene.

[0047] In some embodiments, collagen is reacted with an anhydride-containing compound containing double bonds in solution for 3–24 h, followed by dialyzing and lyophilization. The solution pH should be 8.0, and the mass ratio of collagen to the anhydride-containing compound containing double bonds is 1.0:0.2–0.5.

[0048] Furthermore, the compound containing anhydride and containing a double bond is selected from any one or a combination of maleic anhydride, methacrylic anhydride, and acrylic anhydride.

[0049] Step S2: React sodium alginate with a compound containing a double bond to obtain double-bond modified sodium alginate. Sodium alginate also has functional groups available for reaction: carboxyl and hydroxyl groups. Therefore, the double bond can be further modified using covalent reactions between functional groups, such as the amide condensation reaction of carboxyl and amino groups, the esterification reaction of hydroxyl groups with acid anhydrides, and the ring-opening reaction of carboxyl and epoxy groups. Therefore, double-bond modified sodium alginate can be achieved using the following three methods:

[0050] In some embodiments, sodium alginate is subjected to an amidation reaction with an amino-containing compound containing a double bond in the presence of an amidation catalyst for 0.5–24 h, followed by dialyzing and lyophilization. The mass ratio of sodium alginate, the amino-containing compound containing a double bond, and the amidation catalyst is 1.0:0.2–0.5:0.2–0.5. For example, the mass ratio of sodium alginate, the amino-containing compound containing a double bond, and the amidation catalyst is 1.0:0.2:0.2, 1.0:0.2:0.5, 1.0:0.5:0.2, 1.0:0.5:0.5, or any range thereof.

[0051] Furthermore, the amidation catalyst is selected from any one or a combination of several of EDC (carbodiimide hydrochloride) / HOBT (1-hydroxybenzotriazole), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), HATU (2-(7-azabenzotriazole), EDC / NHS (N-hydroxysuccinimide), BOP (1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate), and CDI (N′,N″-carbonyldiimidazole).

[0052] Furthermore, the compound containing an amino group and a double bond is selected from any one or a combination of several of acrylamide, methacrylamide, p-propenylaniline, acrylhydrazide derivatives, or p-methpropenylphenethylamine.

[0053] In some embodiments, sodium alginate is cross-linked with a compound containing an anhydride double bond in solution for 3–24 h, followed by dialyzing and lyophilization. The solution pH should be 8.0, and the mass ratio of sodium alginate to the anhydride-containing compound containing a double bond is 1.0:0.2–0.5.

[0054] Furthermore, the compound containing anhydride and containing a double bond is selected from any one or a combination of maleic anhydride, methacrylic anhydride, and acrylic anhydride.

[0055] In some embodiments, sodium alginate is cross-linked with an epoxy-containing compound containing a double bond in solution for 2–10 days, followed by dialysis and lyophilization after the reaction is complete. The solution pH should be 6.5–8.5, and the mass ratio of sodium alginate to the epoxy-containing compound containing a double bond is 1.0:0.2–0.5.

[0056] Furthermore, the compound containing an epoxy group and a double bond is selected from any one or a combination of several of glycidyl methacrylate, allyl glycidyl ether, and epoxybutene.

[0057] Step S3: Dissolve the double-bond modified collagen and double-bond modified sodium alginate in a solvent, add a photoinitiator, and carry out a photocrosslinking reaction under ultraviolet light for a certain period of time to obtain a photocurable hydrogel.

[0058] Specifically, photocrosslinking refers to the crosslinking of unsaturated double bonds under light conditions, forming a network structure on the polymer chain.

[0059] Furthermore, the photoinitiator is selected from any one or a combination of several of the following: benzophenone, 4,4-bis(dimethylamino)benzophenone, Irgacure2959 (2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone), LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), 2′,2″-dimethoxy-2-phenylacetophenone, and riboflavin.

[0060] In some embodiments, the mass ratio of modified collagen to modified sodium alginate and photoinitiator is 1:0.5–1.5:0.01–0.05. In a specific embodiment, the mass ratio of modified collagen to modified sodium alginate is 1:0.5:0.01, 1:0.5:0.05, 1:1.5:0.01, 1:1.5:0.05, 1:1:0.02, or any range of two of these values.

[0061] In some embodiments, the wavelength of the ultraviolet light is 254 nm to 400 nm, the energy density is 150 to 1500 mw / cm2, and the illumination time is 10 min to 120 min.

[0062] Step S4: Immerse the photocurable hydrogel in a VEGF solution and react for a certain period of time to obtain a composite hemostatic material. VEGF is a positively charged growth factor. By loading VEGF onto sodium alginate, which carries a large number of negative charges, it can be slowly released during use due to ion adsorption, thereby promoting wound healing.

[0063] In some embodiments, the VEGF concentration is 0.1–0.5 μg / cm³. 2 The immersion reaction time is 0.5h to 2h.

[0064] The aforementioned method for preparing the composite hemostatic material involves modifying the host material with double bonds, cross-linking these double bonds to form a network structure, resulting in a hydrogel with strong water absorption properties. Further loading with VEGF growth factor imparts wound-healing promotion capabilities, yielding the composite hemostatic material. This method addresses the shortcomings of current marketed hemostatic materials by providing a certain degree of cross-linking, resulting in excellent mechanical properties and flexibility; good hydrophilicity, high liquid absorption capacity and speed; and adjustable water absorption swelling. Increasing the cross-linking density reduces water absorption swelling, allowing for the selection of gel materials with different degrees of cross-linking for different application areas. Loading with growth factor imparts excellent wound-healing promotion capabilities. Furthermore, the raw materials used are biodegradable, gradually degrading and being eliminated from the body after wound healing, ensuring safety and effectiveness.

[0065] The aforementioned composite hemostatic material demonstrates good safety and effectiveness in stopping bleeding and promoting wound healing, and does not require removal, thus avoiding secondary harm to the patient. The preparation process of this composite hemostatic material is mild, which helps maintain the material's inherent properties.

[0066] The present invention also provides a composite hemostatic material according to an embodiment, comprising polymer molecules having a network structure, wherein the polymer molecules include double-bond modified collagen and double-bond modified sodium alginate, which are cross-linked by light and then introduced into a VEGF solution.

[0067] This invention provides a composite hemostatic material, which is prepared by any of the above-described preparation methods.

[0068] To make the objectives, technical solutions, and advantages of this invention clearer and more concise, the preparation method and effects of the composite hemostatic material of this invention are further described in detail below with reference to specific embodiments. It should be understood that the embodiments described below are merely preferred embodiments of this invention and can be used to describe this invention, but should not be used to limit this invention. It should be noted that any modifications, improvements, or substitutions made within the principles of this invention should be included within the scope of protection of this invention. Unless otherwise specified, the drugs and instruments used in the following embodiments are conventional choices in the art. Experimental methods not specified in the embodiments are implemented according to conventional conditions, such as those described in books or literature, or methods recommended by the manufacturer.

[0069] To better illustrate the present invention, the following embodiments are provided for further explanation. The specific embodiments are as follows.

[0070] Examples 1-5

[0071] A method for preparing a composite hemostatic material comprises the following steps:

[0072] (1) Double bond modification of collagen (this step differs in Examples 1-5)

[0073] Example 1:

[0074] Dissolve 1g of collagen in acetic acid solution and set aside. Take 0.2g of acrylic acid solution, add 0.2g of EDC / NHS to activate it, add collagen solution after 0.5h, stir and react at 0℃ for 6h, graft dialyze and freeze dry to obtain double bond modified collagen.

[0075] Example 2:

[0076] 1g of collagen was dissolved in acetic acid solution, 0.2g of EDC / NHS was added for activation, and 0.2g of methacrylamide was added after 0.5h. The mixture was stirred at 0℃ for 6h, and the double bond modified collagen was obtained by grafting, dialysis and freeze drying.

[0077] Example 3:

[0078] 1g of collagen and 0.2g of methacrolein were dissolved in acetic acid solution, the pH of the solution was adjusted to 3.5, and the reaction was carried out at 53℃ for 2h. Then, 0.02g of sodium cyanoborohydride was added for reduction, followed by dialyzing and freeze drying to obtain double bond modified collagen.

[0079] Example 4:

[0080] 1g of collagen and 0.2g of glycidyl methacrylate were dissolved in acetic acid solution. The pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. The reaction was carried out for 4 days, followed by dialyzing and freeze-drying to obtain double bond modified collagen.

[0081] Example 5:

[0082] 1g of collagen and 0.2g of methacrylic anhydride were dissolved in acetic acid solution, and the pH of the solution was adjusted to 8.0 with 0.2mol / L NaOH solution. The reaction was carried out for 12h, followed by dialyzing and freeze drying to obtain double bond modified collagen.

[0083] (2) Double bond modification of sodium alginate

[0084] 1g of sodium alginate was reacted with 0.2g of methacrylic anhydride in a NaOH solution with a pH of 8.0 for 8 hours, followed by dialyzing and freeze-drying to obtain double-bond modified sodium alginate.

[0085] (3) Preparation of collagen-sodium alginate composite material

[0086] 1 g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 0.2 mol / L NaOH solution.

[0087] 7.0, then add 0.5g sodium alginate and 0.02g benzophenone, stir for 5 minutes, and then irradiate the solution under ultraviolet light with a wavelength of 254nm and a light intensity of 500mw / cm2 for 30 minutes to obtain collagen-sodium alginate composite material.

[0088] (4) Preparation of collagen-sodium alginate-VEGF composite hemostatic material

[0089] The above collagen-sodium alginate composite material was immersed in a 5 μg / ml VEGF solution and reacted for 2 hours. After the reaction was completed, it was taken out and dried for 30 minutes to obtain the collagen-sodium alginate-VEGF composite hemostatic material.

[0090] Examples 6-7

[0091] A method for preparing a composite hemostatic material based on collagen / sodium alginate / VEGF, comprising the following steps:

[0092] (1) Double bond modification of collagen

[0093] Dissolve 1g of collagen in acetic acid solution and set aside. Take 0.2g of acrylic acid solution, add 0.2g of EDC / NHS to activate it, add collagen solution after 0.5h, stir and react at 0℃ for 6h, graft dialyze and freeze dry to obtain double bond modified collagen.

[0094] (2) Double bond modification of sodium alginate (this step differs slightly in Examples 6 and 7)

[0095] Example 6:

[0096] 1g of sodium alginate was dissolved in an aqueous solution, 0.2g of EDC / NHS was added for activation for 0.5h, and then 0.5g of methacrylamide was added. The mixture was stirred at 0℃ for 6h, followed by dialyzing and freeze-drying to obtain sodium alginate with double bond modification.

[0097] Example 7:

[0098] 1g of sodium alginate and 0.5g of glycidyl methacrylate were dissolved in an aqueous solution. The pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. The reaction was carried out for 4 days. After the reaction was completed, the double-bond modified sodium alginate was obtained by dialyzing and freeze-drying.

[0099] (3) Preparation of collagen-sodium alginate composite material

[0100] 1 g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 0.2 mol / L NaOH solution.

[0101] 7.0, then add 0.5g sodium alginate and 0.02g benzophenone, stir for 5 minutes, and then irradiate the solution under ultraviolet light with a wavelength of 254nm and a light intensity of 500mw / cm2 for 30 minutes to obtain collagen-sodium alginate composite material.

[0102] (4) Preparation of collagen-sodium alginate-VEGF composite hemostatic material

[0103] The above-mentioned collagen-sodium alginate composite material was immersed in a 5 μg / ml VEGF solution and reacted for 2 hours. After the reaction was completed, it was removed and dried for 30 minutes to obtain the collagen-sodium alginate-VEGF composite hemostatic material. Among them, the composite hemostatic materials prepared by methods a, b, and c correspond to Examples 1, 6, and 7, respectively.

[0104] Example 8

[0105] A method for preparing a composite hemostatic material based on collagen / sodium alginate / VEGF, comprising the following steps:

[0106] (1) Double bond modification of collagen

[0107] Dissolve 1g of collagen in acetic acid solution and set aside. Take 0.2g of methacrylic acid solution, add 0.2g of HATU to activate it, and add the collagen solution after 0.5h. Stir the reaction for 12h, then dialyze and freeze dry to obtain double bond modified collagen.

[0108] (2) Double bond modification of sodium alginate

[0109] Dissolve 1g of sodium alginate in an aqueous solution, add 0.2g of BOP, then add 0.5g of allylamine, stir for 6 hours, then dialyze and freeze dry to obtain double-bond modified sodium alginate.

[0110] (3) Preparation of collagen-sodium alginate composite material

[0111] 1g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. Then, 1g of sodium alginate and 0.05g of LAP were added, and the mixture was stirred for 5min. The solution was then irradiated under ultraviolet light with a wavelength of 365nm and a light intensity of 1000mw / cm2 for 30min to obtain collagen-sodium alginate composite material.

[0112] (4) Preparation of collagen-sodium alginate-VEGF composite hemostatic material

[0113] The above collagen-sodium alginate composite material was immersed in a 5 μg / ml VEGF solution and reacted for 2 hours. After the reaction was completed, it was taken out and dried for 30 minutes to obtain the collagen-sodium alginate-VEGF composite hemostatic material.

[0114] Example 9

[0115] A method for preparing a composite hemostatic material based on collagen / sodium alginate / VEGF, comprising the following steps:

[0116] (1) Double bond modification of collagen

[0117] 1g of collagen and 0.2g of glycidyl methacrylate were dissolved in acetic acid solution. The pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. The reaction was carried out for 4 days, followed by dialyzing and freeze-drying to obtain double bond modified collagen.

[0118] (2) Double bond modification of sodium alginate

[0119] 1g of sodium alginate and 0.5g of allyl glycidyl ether were dissolved in an aqueous solution and reacted for 4 days. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain sodium alginate with double bond modification.

[0120] (3) Preparation of collagen-sodium alginate composite material

[0121] 1 g of double-bond modified collagen was dissolved in acetic acid solution. The pH of the solution was adjusted to 7.0 with 0.2 mol / L NaOH solution. Then, 1.5 g of sodium alginate and 0.05 g of 2959 initiator were added. After stirring for 5 min, the solution was placed in an atmosphere with a wavelength of 365 nm and an intensity of 1000 mw / cm². 2 Collagen-sodium alginate composite material was obtained by irradiating with ultraviolet light for 30 minutes.

[0122] (4) Preparation of collagen-sodium alginate-VEGF composite hemostatic material

[0123] The above collagen-sodium alginate composite material was immersed in a 5 μg / ml VEGF solution and reacted for 2 hours. After the reaction was completed, it was taken out and dried for 30 minutes to obtain the collagen-sodium alginate-VEGF composite hemostatic material.

[0124] Example 10

[0125] A method for preparing a composite hemostatic material based on collagen / sodium alginate / VEGF, the specific steps of which are similar to those in Example 1, except that the concentration of the VEGF solution in step (4) is 1 μg / ml.

[0126] Examples 11-12

[0127] A method for preparing a composite hemostatic material based on collagen / sodium alginate / VEGF, the specific steps of which are similar to those in Example 1, except that the light irradiation time in step (3) is 10 min or 120 min, corresponding to Example 11 and Example 12 respectively.

[0128] Example 13

[0129] A method for preparing a composite hemostatic material based on collagen / sodium alginate / VEGF, the specific steps of which are similar to those in Example 1, except that the mass of acrylic acid used in step (1) is 0.5g and the mass of methacrylic anhydride used in step (2) is 0.5g.

[0130] Comparative Example 1

[0131] Comparative Example 1: Collagen sponge hemostatic material was prepared using unmodified collagen. The specific steps are as follows:

[0132] (1) Preparation of collagen solution

[0133] Collagen was dissolved in 0.6 mol / L acetic acid solution, and then dialyzed in 0.6 mol / L acetic acid solution for 20 h to obtain collagen solution;

[0134] (2) Preparation of collagen sponge

[0135] Collagen sponge is obtained by freezing and drying the collagen solution for 24–36 hours.

[0136] Comparative Example 2

[0137] Comparative Example 2 prepared a hemostatic material using unmodified collagen and unmodified sodium alginate. The specific steps are as follows:

[0138] 1) Dissolve collagen in acetic acid solution to prepare a collagen solution with a mass fraction of 0.5%.

[0139] 2) Dissolve sodium alginate powder in water to prepare a sodium alginate solution with a mass fraction of 0.2%.

[0140] 3) Neutralize the collagen solution to pH 7.0 with 0.2M NaOH, add sodium alginate solution to mix, pump the mixture into a casting film casting machine, and cast the film.

[0141] Comparative Example 3

[0142] Comparative Example 3 provides a composite hemostatic material, the specific steps of which are similar to those of Example 1, except that in step (1), the collagen is not modified with double bonds.

[0143] The following is the specific test section:

[0144] (1) Liquid absorbency test:

[0145] Take approximately 20 mg of sample, weigh accurately, and record the weight as m1. Immerse the sample in a beaker of water at 20℃±1℃, gently rub with your fingers until completely wetted and all air is removed, taking care not to break the beaker. After it has absorbed enough water, gently hold one corner with small tweezers and remove it from the water. Gently drain the water from the surface with the tweezers for 1 minute, then weigh it again and record the weight as m2. Calculate the water absorption ratio using the following formula. Randomly select 5 samples and report the average value as the water absorption ratio:

[0146] A=(m2-m1) / m1

[0147] In the formula:

[0148] A—Sample water absorption ratio

[0149] m1—Mass of the sample before wetting, in grams (g)

[0150] m2 — The mass of the sample after wetting, in grams (g).

[0151] The results are shown in Table 1:

[0152] Table 1

[0153] Example 1 60.5 Example 2 62.1 Example 3 61.3 Example 4 60.9 Example 5 61.2 Example 6 62.4 Example 7 63.2 Example 8 65.3 Example 9 70.1 Example 10 60.1 Example 11 69.0 Example 12 55.3 Example 13 71.2 Comparative Example 1 25 Comparative Example 2 30 Comparative Example 3 37

[0154] As shown in Table 1, the composite hemostatic material prepared by the present invention exhibits better water absorption performance compared with comparative examples 1, 2 and 3. Different double bond modification methods do not have significant differences in the water absorption performance of the samples. In addition, with the extension of light exposure time, excessive crosslinking density will lead to a slight decrease in water absorption. Increasing the proportion of sodium alginate and deepening the degree of modification will improve the water absorption performance of the samples.

[0155] (2) Elongation at break:

[0156] Cut samples of a fixed size, immerse them in purified water for 10 minutes, then remove them and measure the size of the swollen samples. Record the length as L1. Then, stretch the samples uniformly along their length at a set rate and record the length of the material at break as L2. The elongation at break is calculated as (L2 - L1) / L1 * 100%.

[0157] The results are shown in Table 2:

[0158] Table 2

[0159] Example 1 150％ Example 2 152％ Example 3 149％ Example 4 150％ Example 5 148％ Example 6 153％ Example 7 152％ Example 8 156％ Example 9 160％ Example 10 151％ Example 11 85％ Example 12 90％ Example 13 170％ Comparative Example 1 20％ Comparative Example 2 25％ Comparative Example 3 33％

[0160] As shown in Table 2, compared with the comparative examples, the composite hemostatic material prepared by the present invention exhibits excellent toughness, and different double bond modification methods have little effect on mechanical properties. However, the duration of light exposure, the amount of double bond monomers added, and the ratio of collagen to sodium alginate will affect the performance. Too long or too short light exposure time will reduce toughness, while increasing the amount of double bond monomers will enhance toughness, and increasing the proportion of sodium alginate will slightly improve toughness.

[0161] (3) Animal experiments

[0162] New Zealand rabbits were anesthetized with 10% chloral hydrate solution. Skin layers (approximately 1 cm in diameter) were removed from both sides of the midline of the rabbit's back, with six removals per rabbit. Samples were applied to each wound, and the wounds were bandaged with sterile gauze. Hemostasis time was recorded. The results are shown in Table 3.

[0163] Table 3

[0164] control group 7min Example 1 58s Example 8 1min Example 9 1 minute 30 seconds Example 10 1 minute 20 seconds Example 11 2min Example 12 1 minute 45 seconds Example 13 40s Comparative Example 1 5 min 50 s Comparative Example 2 5 min 30 s Comparative Example 3 5min

[0165] As shown in Table 3, the composite hemostatic material prepared in this invention exhibits significantly improved in vivo hemostatic performance compared to the control group and comparative examples. The duration of light exposure has a slight impact on hemostatic performance; both excessively long and short exposure times result in a slight decrease. The ratio of collagen to sodium alginate also affects hemostatic performance; a suitable ratio further enhances the hemostatic properties of the composite material. Since collagen can initiate intrinsic coagulation mechanisms, an excessively high sodium alginate content reduces the collagen content, leading to a slight decrease in hemostatic performance.

[0166] (4) Wound healing speed

[0167] Based on the specific test protocol (3), the gauze was removed and the wound was exposed three days after treatment, and the time taken for the wound to fully heal was observed daily. The results are shown in Table 4:

[0168] Table 4

[0169] control group 23 Example 1 8 Example 8 10 Example 9 11 Example 10 15 Comparative Example 1 20 Comparative Example 2 21 Comparative Example 3 20

[0170] As shown in Table 3, the hemostatic material prepared in this invention significantly improves wound healing speed compared to the control group and the comparative examples. The concentration of VEGF used has a significant impact on promoting healing speed; when the VEGF concentration is too low, its healing-promoting ability is limited, and the healing time is prolonged. The comparative examples, however, did not introduce any corresponding growth factors, resulting in no significant improvement in wound healing performance.

[0171] In summary, the composite hemostatic material of the present invention has excellent hemostatic properties, toughness, and the ability to promote wound healing.

[0172] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0173] The embodiments described above are merely illustrative of several implementations of the present invention, intended for detailed and specific description, but should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A composite hemostatic material, characterized in that, The composite hemostatic material includes a crosslink of double-bond modified collagen and double-bond modified sodium alginate, and vascular endothelial growth factor loaded on the crosslink. The composite hemostatic material is prepared by any of the following methods: (1) Double bond modification of collagen: 1g of collagen was dissolved in acetic acid solution and left to stand; 0.2g of acrylic acid solution was taken, 0.2g of EDC / NHS was added for activation, and after 0.5h, collagen solution was added. The reaction was stirred at 0℃ for 6h, and double bond modified collagen was obtained by grafting dialysis and freeze drying. Double bond modification of sodium alginate: 1g of sodium alginate and 0.2g of methacrylic anhydride were reacted in NaOH solution with a pH of 8.0 for 8h, followed by dialyzing and freeze drying to obtain double bond modified sodium alginate; Preparation of collagen-sodium alginate composite material: 1g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. Then, 0.5g of double-bond modified sodium alginate and 0.02g of benzophenone were added. After stirring for 5min, the solution was placed in an atmosphere with a wavelength of 254nm and an intensity of 500mw / cm². 2 Collagen-sodium alginate composite material was obtained by irradiating with ultraviolet light for 30 minutes; Preparation of collagen-sodium alginate-VEGF composite hemostatic material: The above collagen-sodium alginate composite material was immersed in 5μg / ml VEGF solution and reacted for 2h. After the reaction was completed, it was taken out and dried for 30min to obtain collagen-sodium alginate-VEGF composite hemostatic material. or, (2) Double bond modification of collagen: 1g of collagen and 0.2g of glycidyl methacrylate were dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. The reaction was carried out for 4 days, followed by dialysis and freeze drying to obtain double bond modified collagen. Double bond modification of sodium alginate: 1g of sodium alginate and 0.2g of methacrylic anhydride were reacted in NaOH solution with a pH of 8.0 for 8h, followed by dialyzing and freeze drying to obtain double bond modified sodium alginate; Preparation of collagen-sodium alginate composite material: 1g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. Then, 0.5g of double-bond modified sodium alginate and 0.02g of benzophenone were added. After stirring for 5min, the solution was placed in an atmosphere with a wavelength of 254nm and an intensity of 500mw / cm². 2 Collagen-sodium alginate composite material was obtained by irradiating with ultraviolet light for 30 minutes; Preparation of collagen-sodium alginate-VEGF composite hemostatic material: The above collagen-sodium alginate composite material was immersed in 5μg / ml VEGF solution and reacted for 2h. After the reaction was completed, it was taken out and dried for 30min to obtain collagen-sodium alginate-VEGF composite hemostatic material. or, (3) Double bond modification of collagen: Dissolve 1g of collagen in acetic acid solution and set aside; take 0.2g of methacrylic acid solution, add 0.2g of HATU to activate, add collagen solution after 0.5h, stir and react for 12h, then dialyze and freeze dry to obtain double bond modified collagen; Double bond modification of sodium alginate: 1g of double bond modified sodium alginate was dissolved in an aqueous solution, 0.2g of BOP was added, and then 0.5g of allylamine was added. The mixture was stirred for 6 hours, followed by dialysis and freeze drying to obtain double bond modified sodium alginate. Preparation of collagen-sodium alginate composite material: 1g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. Then, 1g of double-bond modified sodium alginate and 0.05g of LAP were added. After stirring for 5min, the solution was placed in a 365nm light source with an intensity of 1000mw / cm². 2 Collagen-sodium alginate composite material was obtained by irradiating with ultraviolet light for 30 minutes; Preparation of collagen-sodium alginate-VEGF composite hemostatic material: The above collagen-sodium alginate composite material was immersed in 5μg / ml VEGF solution and reacted for 2h. After the reaction was completed, it was taken out and dried for 30min to obtain collagen-sodium alginate-VEGF composite hemostatic material. or, (4) Double bond modification of collagen: 1g of collagen and 0.2g of glycidyl methacrylate were dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. The reaction was carried out for 4 days, followed by dialysis and freeze drying to obtain double bond modified collagen. Double bond modification of sodium alginate: 1g sodium alginate and 0.5g allyl glycidyl ether were dissolved in an aqueous solution and reacted for 4 days. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain double bond modified sodium alginate. Preparation of collagen-sodium alginate composite material: 1g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. Then, 1.5g of double-bond modified sodium alginate and 0.05g of 2959 initiator were added. After stirring for 5min, the solution was placed in a 365nm light source with an intensity of 1000mw / cm². 2 Collagen-sodium alginate composite material was obtained by irradiating with ultraviolet light for 30 minutes; Preparation of collagen-sodium alginate-VEGF composite hemostatic material: The above collagen-sodium alginate composite material was immersed in 5μg / ml VEGF solution and reacted for 2h. After the reaction was completed, it was taken out and dried for 30min to obtain collagen-sodium alginate-VEGF composite hemostatic material. or, (5) Double bond modification of collagen: Dissolve 1g of collagen in acetic acid solution and set aside; take 0.5g of acrylic acid solution, add 0.2g of EDC / NHS to activate, add collagen solution after 0.5h, stir at 0℃ for 6h, graft dialysis and freeze dry to obtain double bond modified collagen; Double bond modification of sodium alginate: 1g of sodium alginate and 0.5g of methacrylic anhydride were reacted in NaOH solution with a pH of 8.0 for 8h, followed by dialyzing and freeze drying to obtain double bond modified sodium alginate; Preparation of collagen-sodium alginate composite material: 1g of double-bond modified collagen was dissolved in acetic acid solution, and the pH of the solution was adjusted to 7.0 with 0.2mol / L NaOH solution. Then, 0.5g of double-bond modified sodium alginate and 0.02g of benzophenone were added. After stirring for 5min, the solution was placed in an atmosphere with a wavelength of 254nm and an intensity of 500mw / cm². 2 Collagen-sodium alginate composite material was obtained by irradiating with ultraviolet light for 30 minutes; Preparation of collagen-sodium alginate-VEGF composite hemostatic material: The above collagen-sodium alginate composite material was immersed in 5 μg / ml VEGF solution and reacted for 2 h. After the reaction was completed, it was taken out and dried for 30 min to obtain collagen-sodium alginate-VEGF composite hemostatic material.

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