A chitosan hemostatic material for rapid arterial hemostasis and a preparation process and application thereof

Abstract

This invention provides a chitosan hemostatic material for rapid arterial hemostasis, its preparation process, and its applications, relating to the field of medical technology. The chitosan hemostatic material prepared by this invention can significantly shorten hemostasis time, maintain stable adhesion to the wound surface even under the impact of arterial blood flow, and is not easily detached, exhibiting strong wet adhesion and erosion resistance. The chitosan hemostatic material prepared by this invention not only improves the mechanical strength of the material but also provides more bioactive sites, activating intrinsic coagulation pathways and platelet aggregation, thereby improving hemostasis efficiency and showing significant advantages in emergency applications for arterial bleeding. The chitosan hemostatic material prepared by this invention combines rapid coagulation and stable hemostasis, and has good biocompatibility, controllable degradation, reduced inflammatory response, and high safety. The porous structure of the chitosan hemostatic material prepared by this invention is beneficial for tissue repair, does not adhere to other tissues, promotes healing, and has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a chitosan hemostatic material for rapid arterial hemostasis, its preparation process, and its application. Background Technology

[0002] Rapid control of arterial bleeding is crucial in emergency and surgical care. Ideal hemostatic materials need to be fast-acting, biocompatible, and easy to use. Active hemostatic materials based on natural polymers are currently an important research direction. Chitosan (CS) is a positively charged natural polymeric polysaccharide with characteristics such as non-toxicity, biodegradability, good biocompatibility, and low immunogenicity, making it suitable for biomedical applications. Due to its positive charge, it can serve as a delivery material for negatively charged DNA, RNA, and other biomolecules. The hemostatic and antibacterial activity of CS is mainly due to the presence of -NH2 groups in its molecular chain. The positively charged structure of CS causes negatively charged red blood cells to aggregate, thereby accelerating the rate of thrombus formation. Furthermore, CS can alter bacterial permeability after binding to the negatively charged cell membranes of bacteria, thus accelerating bacterial apoptosis. In recent years, chitosan or its derivatives have been utilized to varying degrees and designed into hemostatic products such as hemostatic sponges and hydrogels.

[0003] However, when using single-component chitosan as a hemostatic material, in situations with rapid arterial bleeding and strong blood flow, existing chitosan hemostatic materials are easily washed away by the gushing blood, failing to form a stable blood clot at the rupture site. Their adhesion in a moist environment is also insufficient; the presence of a blood film on the arterial surface makes it difficult for the material to adhere firmly, and fluctuations in blood pressure can lead to the occluder detaching and rebleeding. Some studies have reported attempts to improve material stability through surface hydrophobic modifications, but these modifications often uncontrollably reduce the material's hydrophilicity, thus inhibiting the accumulation of clotting factors. Furthermore, in situations like high-flow, high-pressure arterial bleeding, some hemostatic materials degrade too quickly or too slowly, affecting wound healing and even causing inflammation or foreign body reactions. Summary of the Invention

[0004] In view of this, the present invention proposes a chitosan hemostatic material for rapid arterial hemostasis, its preparation process and application, thereby solving the above problems.

[0005] The technical solution of this invention is implemented as follows: A preparation process for a chitosan hemostatic material for rapid arterial hemostasis includes the following steps: S1. Add chitosan to an aqueous acetic acid solution to prepare a chitosan solution. Mix the amidated modified carboxymethyl cellulose and the chitosan solution by ultrasonication and adjust the pH to 4.5-5.5 to obtain component A. S2. Chitosan is added to an aqueous acetic acid solution to prepare a chitosan solution. Polylactic acid copolymer crosslinked polyaspartic acid is added to dichloromethane. Chitosan solution is added, ultrasonic emulsified, and dichloromethane is removed by rotary evaporation under reduced pressure to obtain component B. S3. Inject component A into the mold and pre-freeze it. Slowly add component B to the top layer of the pre-frozen component A, continue freezing, and finally freeze-dry to obtain the target chitosan hemostatic material.

[0006] Furthermore, in steps S1 and S2, the chitosan has a molecular weight of 20-30 kDa and a degree of deacetylation ≥85%; the chitosan solution has a mass concentration of 30%-50%.

[0007] Furthermore, in step S1, the method for preparing the amidated modified carboxymethyl cellulose includes: Carboxymethyl cellulose was added to anhydrous ethanol, and hexamethylenediamine was added to react the mixture. The mixture was filtered and washed until neutral, and then freeze-dried to obtain amidated modified carboxymethyl cellulose.

[0008] Furthermore, the solid-liquid ratio of carboxymethyl cellulose to anhydrous ethanol is 1:10-20 g / mL; the amount of hexamethylenediamine added is 1-1.5 times the mass of carboxymethyl cellulose; the reaction temperature is 50-70℃, and the reaction time is 2-4 h.

[0009] Furthermore, in step S1, the solid-liquid ratio of the amidated modified carboxymethyl cellulose and chitosan solution is 1:20-30 g / mL; the ultrasonic mixing is performed at 15-20 KHz and 30-40℃ for 1-2 hours.

[0010] Further, in step S2, the solid-liquid ratio of the polylactic acid copolymer crosslinked polyaspartic acid to dichloromethane is 1:5-7 g / mL; the solid-liquid ratio of the polylactic acid copolymer crosslinked polyaspartic acid to chitosan solution is 1:20-30 g / mL; the ultrasonic emulsification specifically involves adding surfactant Tween-80 and intermittently ultrasonicating at 25-30℃ and 50-80 rpm for 30-60 seconds each time, for a total of 5-10 minutes. The amount of surfactant added is 0.8-1.2 times the mass of the polylactic acid copolymer crosslinked polyaspartic acid.

[0011] Furthermore, in step S3, the mass ratio of component A to component B is 1:1-3.

[0012] Furthermore, in step S3, the pre-freezing temperature is -15℃ to -10℃, and the time is 2-3 hours; the continued freezing time is 4-5 hours.

[0013] A chitosan hemostatic material for rapid arterial hemostasis, prepared by any of the above-described preparation processes.

[0014] Application of a chitosan hemostatic material for rapid arterial hemostasis in the preparation of rapid arterial hemostatic materials.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) The chitosan hemostatic material prepared by the present invention can significantly shorten the hemostasis time, and can still stably adhere to the wound surface under the impact of arterial blood flow, and is not easy to fall off. It has strong wet adhesion and anti-erosion ability.

[0016] (2) The chitosan hemostatic material prepared by the present invention not only improves the mechanical strength of the material, but also provides more bioactive sites, which can activate the intrinsic coagulation pathway and platelet aggregation, improve hemostasis efficiency, and has significant advantages in emergency application of arterial bleeding.

[0017] (3) The chitosan hemostatic material prepared by the present invention has both rapid coagulation and stable hemostasis, and has good biocompatibility, controllable degradation, reduced inflammatory response, and high safety.

[0018] (4) The porous structure of the chitosan hemostatic material prepared by the present invention is beneficial to tissue repair and does not adhere to other tissues, thus promoting healing and showing good application prospects. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0020] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0022] The polylactic acid copolymer crosslinked polyaspartic acid of this invention was purchased from Guangzhou Carbon Technology Co., Ltd.

[0023] The chitosan of this invention has a molecular weight of 20-30 kDa and a degree of deacetylation ≥85%.

[0024] Preparation Example Methods for preparing amidated modified carboxymethyl cellulose include: Carboxymethyl cellulose was added to anhydrous ethanol at a solid-liquid ratio of 1:15 g / mL, and hexamethylenediamine with a mass of 1.5 times that of carboxymethyl cellulose was added. The mixture was reacted at 60°C for 3 h, filtered, washed until neutral, and freeze-dried to obtain amidated modified carboxymethyl cellulose.

[0025] Example 1 A preparation process for a chitosan hemostatic material for rapid arterial hemostasis includes the following steps: S1. Add chitosan to an aqueous acetic acid solution to prepare a 40wt% chitosan solution. According to the solid-liquid ratio of 1:25 g / mL, sonicate the amidated modified carboxymethyl cellulose and chitosan solution at 20 kHz and 35℃ for 1.5 h, and adjust the pH to 5.0±0.1 to obtain component A. S2. Chitosan was added to an aqueous acetic acid solution to prepare a 40wt% chitosan solution. The solid-liquid ratio was 1:6 g / mL. Polylactic acid copolymer cross-linked polyaspartic acid was added to dichloromethane, followed by the chitosan solution. The solid-liquid ratio of polylactic acid copolymer cross-linked polyaspartic acid to chitosan solution was 1:25 g / mL. Surfactant Tween-80 (added in an amount equal to the mass of polylactic acid copolymer cross-linked polyaspartic acid) was added. The mixture was then subjected to intermittent sonication at 28℃ and 70 rpm for 45 seconds each time, for a total sonication treatment of 7.5 min. Finally, dichloromethane was removed by rotary evaporation under reduced pressure to obtain component B. S3. Inject component A into the mold and pre-freeze at -15℃ for 2.5h. Slowly add component B to the pre-frozen component A. The mass ratio of component A to component B is 1:2. Continue freezing for 4.5h. Finally, freeze-dry to obtain the target chitosan hemostatic material.

[0026] Example 2 A preparation process for a chitosan hemostatic material for rapid arterial hemostasis includes the following steps: S1. Add chitosan to an aqueous acetic acid solution to prepare a 30wt% chitosan solution. According to the solid-liquid ratio of 1:20 g / mL, sonicate the amidated modified carboxymethyl cellulose and chitosan solution at 15 kHz and 30℃ for 1 h, and adjust the pH to 5.0±0.1 to obtain component A. S2. Chitosan was added to an aqueous acetic acid solution to prepare a 30wt% chitosan solution. The solid-liquid ratio was 1:5 g / mL. Polylactic acid copolymer cross-linked polyaspartic acid was added to dichloromethane, followed by the chitosan solution. The solid-liquid ratio of polylactic acid copolymer cross-linked polyaspartic acid to chitosan solution was 1:20 g / mL. Surfactant Tween-80 (0.8 times the mass of polylactic acid copolymer cross-linked polyaspartic acid) was added. The mixture was then subjected to intermittent sonication at 25°C and 50 rpm for 30 seconds each time, for a total of 5 minutes. Finally, the dichloromethane was removed by rotary evaporation under reduced pressure to obtain component B. S3. Inject component A into the mold and pre-freeze at -15℃ for 2 hours. Slowly add component B to the top layer of the pre-frozen component A. The mass ratio of component A to component B is 1:1. Continue freezing for 4 hours. Finally, freeze-dry to obtain the target chitosan hemostatic material.

[0027] Example 3 A preparation process for a chitosan hemostatic material for rapid arterial hemostasis includes the following steps: S1. Add chitosan to an aqueous acetic acid solution to prepare a 50wt% chitosan solution. According to the solid-liquid ratio of 1:30 g / mL, sonicate the amidated modified carboxymethyl cellulose and chitosan solution at 20 kHz and 40℃ for 2 h, and adjust the pH to 5.0±0.1 to obtain component A. S2. Chitosan was added to an aqueous acetic acid solution to prepare a 50wt% chitosan solution. The solid-liquid ratio was 1:7 g / mL. Polylactic acid copolymer cross-linked polyaspartic acid was added to dichloromethane, followed by the chitosan solution. The solid-liquid ratio of polylactic acid copolymer cross-linked polyaspartic acid to chitosan solution was 1:30 g / mL. Surfactant Tween-80 (1.2 times the mass of polylactic acid copolymer cross-linked polyaspartic acid) was added. The mixture was then subjected to intermittent ultrasonic treatment at 30℃ and 80 rpm for 60 seconds each time, for a total of 10 minutes. Finally, dichloromethane was removed by rotary evaporation under reduced pressure to obtain component B. S3. Inject component A into the mold and pre-freeze at -10℃ for 3 hours. Slowly add component B to the top layer of the pre-frozen component A. The mass ratio of component A to component B is 1:3. Continue freezing for 5 hours. Finally, freeze-dry to obtain the target chitosan hemostatic material.

[0028] Comparative Example 1 The difference between this comparative example and Example 1 is that step S2 is missing; otherwise, they are the same as Example 1.

[0029] The preparation process of a chitosan hemostatic material for rapid arterial hemostasis, as described in this comparative example, includes the following specific steps: Chitosan was added to an aqueous acetic acid solution to prepare a 40wt% chitosan solution. The solid-liquid ratio was 1:25 g / mL. The amidated modified carboxymethyl cellulose and chitosan solution were ultrasonically treated at 20 kHz and 35℃ for 1.5 h. The pH was adjusted to 5.0±0.1. The solution was injected into a mold and pre-frozen at -15℃ for 2.5 h. After freeze-drying, chitosan hemostatic material was obtained.

[0030] Comparative Example 2 The difference between this comparative example and Example 1 is that in step S1, the amidated modified carboxymethyl cellulose is replaced with carboxymethyl cellulose, while the rest is the same as in Example 1.

[0031] Comparative Example 3 The difference between this comparative example and Example 1 is that in step S2, the polylactic acid copolymer crosslinked polyaspartic acid is replaced with polyethylene glycol-polyglutamic acid block copolymer, while the rest is the same as in Example 1.

[0032] Comparative Example 4 The difference between this comparative example and Example 1 is that in step S3, component A and component B are mixed and injected into a mold and pre-frozen at -10°C for 3 hours, and finally freeze-dried to obtain chitosan hemostatic material. The rest is the same as in Example 1.

[0033] Test Example 1 - Hemostatic Performance Test The hemostatic properties of the chitosan hemostatic materials prepared in Examples 1-3 and Comparative Examples 1-4, as well as the commercially available chitosan hemostatic sponge (purchased from Qingdao Boyite Biomaterials Co., Ltd.), were tested respectively.

[0034] Experimental Methods: Eighty healthy adult SD rats, weighing 200±10g, were randomly divided into 8 groups of 10 rats each: Examples 1-3, Comparative Examples 1-4, and a commercially available group. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (1.5%, 2mL / kg). After anesthesia, the rats were fixed supine on the operating table. The common carotid artery was dissected 2 cm, the proximal end was clamped, and the other end was tied with biodegradable sutures. A transverse incision was made along the artery, and a catheter was inserted and fixed through the incision. The proximal end hemostatic clamp was removed, and the catheter was connected to a physiological instrument. Heparin 300U / kg was injected intravenously. The groin area was shaved and disinfected. The femoral artery was dissected, the proximal end was clamped, and a transverse incision of 1 / 3 of the artery's diameter was made. The spurting blood was immediately wiped away, and gauze was applied to the wound. The gauze was then slowly loosened, and hemostatic material was immediately applied to the bleeding site with appropriate pressure. Timing was then started. After a certain period of time, remove the cotton ball and observe the hemostatic effect of different materials. Observe every 5 minutes thereafter until complete hemostasis is achieved, and record the hemostasis time and the amount of bleeding. The shorter the hemostasis time and the less bleeding, the better the hemostatic effect.

[0035] The test results are shown in Table 1.

[0036] Table 1

[0037] As can be seen from Table 1, the chitosan hemostatic materials prepared in Examples 1-3 of the present invention can significantly shorten the hemostasis time and reduce the amount of bleeding, and can achieve rapid hemostasis.

[0038] Test Example 2 - Wet Adhesion Strength Test The adhesion properties of the samples prepared in Examples 1-3 and Comparative Examples 1-4 were tested using an Instron machine 1185 (Instron, Boston, MA, USA) on a 100N load cell, according to the lap shear test (ASTM F2255-05) standard.

[0039] Fresh pigskin was selected and cut into rectangular slices measuring 4.0cm × 1.0cm, 0.2cm thick. Before use, the slices were moistened with PBS buffer. The slices were then removed, and the remaining PBS buffer was ground with filter paper. The test sample was then directly coated onto opposite sides of two pigskin slices, with a contact area of ​​1.0 × 1.0cm. 2 Two pigskin pieces were placed in contact with the surface for 40 minutes under a load of 250.0g, and then placed in a testing machine to test the adhesion strength of the samples at room temperature.

[0040] The results are shown in Table 2.

[0041] Table 2

[0042] As can be seen from Table 2, the chitosan hemostatic materials prepared in Examples 1-3 of the present invention have strong wet adhesion, can stably adhere to the wound surface, and are not easy to fall off.

[0043] Test Example 3 - Wound Healing Performance Test Healthy adult SD rats weighing 200±10g were selected and anesthetized by intraperitoneal injection of sodium pentobarbital (1.5%, 2mL / kg). A circular full-thickness skin defect model with a diameter of 1cm was then created on the back of the rats. The wound was then filled with chitosan hemostatic materials prepared in Examples 1-3 and Comparative Examples 1-4. The wound was photographed at regular intervals, the wound area was measured, and the wound healing rate was calculated on days 3, 7, and 14.

[0044] Wound healing rate (%) = (T0 – T t ) / T0×100%.

[0045] Where T0 is the initial area of ​​the wound, T t Let t be the area of ​​the wound at time t.

[0046] The test results are shown in Table 3.

[0047] Table 3

[0048] As can be seen from Table 3, the chitosan hemostatic materials prepared in Examples 1-3 of the present invention can promote wound healing. Compared with Comparative Examples 1-4, the chitosan hemostatic material of Example 1 can significantly shorten the healing time. On the 3rd day, the wound healing rate reaches 85%, and it can be completely healed on the 7th day.

[0049] Test Example 4 - Safety Performance Test The chitosan hemostatic materials prepared in Examples 1-3 were subjected to safety performance tests. The test items, test methods and test results are shown in Table 4.

[0050] Table 4

[0051] As can be seen from Table 4, the chitosan hemostatic materials prepared in Examples 1-3 of this invention are non-toxic, non-sensitizing, and highly safe.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A preparation process for a chitosan hemostatic material for rapid arterial hemostasis, characterized in that, The specific steps include: S1. Add chitosan to an aqueous acetic acid solution to prepare a chitosan solution. Mix the amidated modified carboxymethyl cellulose and the chitosan solution by ultrasonication and adjust the pH to 4.5-5.5 to obtain component A. S2. Chitosan is added to an aqueous acetic acid solution to prepare a chitosan solution. Polylactic acid copolymer crosslinked polyaspartic acid is added to dichloromethane. Chitosan solution is added, ultrasonic emulsified, and dichloromethane is removed by rotary evaporation under reduced pressure to obtain component B. S3. Inject component A into the mold and pre-freeze it. Slowly add component B to the top layer of the pre-frozen component A, continue freezing, and finally freeze-dry to obtain the target chitosan hemostatic material.

2. The preparation process of the chitosan hemostatic material for rapid arterial hemostasis as described in claim 1, characterized in that, In steps S1 and S2, the chitosan has a molecular weight of 20-30 kDa and a degree of deacetylation ≥85%; the chitosan solution has a mass concentration of 30%-50%.

3. The preparation process of the chitosan hemostatic material for rapid arterial hemostasis as described in claim 1, characterized in that, In step S1, the preparation method of the amidated modified carboxymethyl cellulose includes: Carboxymethyl cellulose was added to anhydrous ethanol, and hexamethylenediamine was added to react the mixture. The mixture was filtered and washed until neutral, and then freeze-dried to obtain amidated modified carboxymethyl cellulose.

4. The preparation process of a chitosan hemostatic material for rapid arterial hemostasis as described in claim 3, characterized in that, The solid-liquid ratio of carboxymethyl cellulose to anhydrous ethanol is 1:10-20 g / mL; the amount of hexamethylenediamine added is 1-1.5 times the mass of carboxymethyl cellulose; the reaction temperature is 50-70℃ and the reaction time is 2-4 h.

5. The preparation process of a chitosan hemostatic material for rapid arterial hemostasis as described in claim 1, characterized in that, In step S1, the solid-liquid ratio of the amidated modified carboxymethyl cellulose and chitosan solution is 1:20-30 g / mL; the ultrasonic mixing is performed at 15-20 KHz and 30-40℃ for 1-2 hours.

6. The preparation process of a chitosan hemostatic material for rapid arterial hemostasis as described in claim 1, characterized in that, In step S2, the solid-liquid ratio of the polylactic acid copolymer crosslinked polyaspartic acid to dichloromethane is 1:5-7 g / mL; the solid-liquid ratio of the polylactic acid copolymer crosslinked polyaspartic acid to chitosan solution is 1:20-30 g / mL; the ultrasonic emulsification specifically involves adding surfactant Tween-80 and intermittently ultrasonicating at 25-30℃ and 50-80 rpm for 30-60 seconds each time, for a total of 5-10 minutes. The amount of surfactant added is 0.8-1.2 times the mass of the polylactic acid copolymer crosslinked polyaspartic acid.

7. The preparation process of a chitosan hemostatic material for rapid arterial hemostasis as described in claim 1, characterized in that, In step S3, the mass ratio of component A to component B is 1:1-3.

8. The preparation process of a chitosan hemostatic material for rapid arterial hemostasis as described in claim 1, characterized in that, In step S3, the pre-freezing temperature is -15℃ to -10℃, and the time is 2-3 hours; the continued freezing time is 4-5 hours.

9. A chitosan hemostatic material for rapid arterial hemostasis, characterized in that, It is prepared by the preparation process described in any one of claims 1-7.

10. The application of the chitosan hemostatic material for rapid arterial hemostasis as described in claim 9 in the preparation of rapid arterial hemostatic materials.

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