Bimorphological material for non-compressible hemostasis and preparation method thereof

Abstract

The invention relates to the technical field of biological materials and medical products, in particular to a bimorphological material for hemostasis without pressing and a preparation method. The chemically crosslinked hemostatic material based on gelatin and carboxymethyl cellulose developed by the invention can realize an excellent hemostatic effect in two forms, and has good rheological property, adhesion, injectability and biocompatibility. Compared with a traditional hemostatic material, the hemostatic material can utilize the good biocompatibility of gelatin and the good water absorption of carboxymethyl cellulose, and has great application advantages in irregular wounds and deeper wounds.

Description

Technical Field

[0001] The present invention relates to the field of biomaterial technology and medical product technology, and in particular to a dual-morphology material for non-pressible hemostasis and a preparation method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] During surgical operations, bleeding is a common condition that may seriously affect the success of the operation and the safety of the patient's life. Although the human body can stop bleeding through the mechanism of physiological hemostasis, in the case of massive bleeding, the physiological hemostasis is slow and has limited effect. If the amount of bleeding cannot be effectively controlled in time, the operation may not be carried out normally, thus affecting the surgical effect and the patient's health. Therefore, rapid and effective hemostasis is very important to ensure the quality of surgery and save lives.

[0004] Although traditional hemostatic materials can help control bleeding to a certain extent, they also have some shortcomings. For example, hemostatic materials such as gauze and bandages can only simply cover the surface of the wound, and are often limited in effect for large-area bleeding or deep bleeding, and there is a risk of forming blood clots in the blood vessels. Secondly, some traditional hemostatic materials such as cotton wool and gauze are easily adhered to the secretions at the wound during use, which can easily cause secondary damage to the wound when removing and changing the dressing, prolonging the hemostasis time. At the same time, hemostatic materials such as polysaccharides or minerals may cause allergic reactions, organ damage or increase the risk of infection in patients, affecting postoperative recovery. Therefore, seeking more advanced and more effective hemostatic materials has become an important research direction in the current field of hemostasis to solve the limitations and defects of traditional hemostatic materials in minimally invasive surgery and incompressible hemostasis.

[0005] Carboxymethyl cellulose (CMC) is a natural macromolecular polymer derived from nature with strong water absorption capacity. Materials composed of CMC will not adhere to the wound and can be removed painlessly to avoid secondary damage, so they can be placed in situ for a longer time. Gelatin, as a natural protein-derived material, has good biocompatibility, non-immunogenicity and the ability to promote cell adhesion. Its amino acid components combine with fibrinogen (coagulation factor I) in the blood to form fibrin, which can play a coagulation function. Both have been widely used in the preparation of medical hydrogels. Based on this, we cross-linked gelatin and carboxymethyl cellulose in a solution to prepare a hemostatic composite hydrogel. Summary of the invention

[0007] The purpose of the present invention is to provide a dual-morphological material for rapid hemostasis and a preparation method thereof, wherein the material can achieve excellent hemostatic effects in both forms and has good rheology, adhesion, injectability and biocompatibility. Compared with traditional hemostatic materials, it has great application advantages in irregular wounds and deeper wounds. The chemically cross-linked hemostatic material based on gelatin and carboxymethyl cellulose developed by the present invention can utilize the good biocompatibility of gelatin and the good water absorption of carboxymethyl cellulose. At the same time, the structural characteristics of the two, which are rich in amino groups and rich in carboxyl groups, are used to achieve chemical cross-linking, thereby obtaining a dual-morphological hemostatic material with excellent hemostatic effects.

[0008] In a first aspect, the present invention provides a gelatin / carboxymethyl cellulose rapid hemostatic material, wherein the hemostatic material uses gelatin and carboxymethyl cellulose as raw materials, and is cross-linked through an amidation reaction catalyzed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to obtain a gelatin / carboxymethyl cellulose composite.

[0009] Furthermore, a method for preparing a rapid hemostatic material based on gelatin / carboxymethyl cellulose is provided, which specifically comprises the following steps: S1. Weigh a certain amount of gelatin particles and place them in a beaker, add an appropriate amount of ultrapure water and heat to prepare a gelatin solution; S2. Weigh a certain amount of EDC, NHS and 2-morpholineethanesulfonic acid (2-MES) and fully dissolve them in an appropriate amount of ultrapure water; S3, adding the mixed solution in S2 to the gelatin solution in S1, and then adding different amounts of carboxymethyl cellulose, stirring and reacting at room temperature for 20-24 hours, and the obtained products are named CG30, CG60, CG90, and CG120 respectively; S4, quenching the reaction, adding a certain amount of disodium hydrogen phosphate dodecahydrate and sodium chloride to the product of the reaction in S3, adjusting the pH with sodium hydroxide solution, and stirring sufficiently to obtain a suspension; S5, centrifuging the suspension obtained in S4 in a high-speed centrifuge, removing the supernatant and adding ultrapure water for washing, repeating the above operation four times to remove excess water, placing the sample in a -80°C medical freezer for pre-freezing for 3 h, placing the pre-frozen gel in a freeze dryer for at least 48 h, taking out the freeze-dried sample, and grinding it to obtain a hemostatic powder; S6. Mix the sample powder in S5 with 4-6 times the mass of physiological saline to obtain a fluid hemostatic gel.

[0010] The gelatin solution in S1 is usually 1 g of gelatin plus 50 mL of ultrapure water, and the reaction condition is to fully stir and dissolve at 60°C.

[0011] Furthermore, the molar ratio of NHS to EDC in S2 is 0.2:1. The function of adding 2-MES is to provide an acidic environment. The volume of ultrapure water added is 50 mL.

[0012] Furthermore, the specific preparation methods of the products in S3 are as follows: CG30: the mass ratio of carboxymethyl cellulose to gelatin is 0.3; CG60: the mass ratio of carboxymethyl cellulose to gelatin is 0.6; CG90: the mass ratio of carboxymethyl cellulose to gelatin is 0.9; CG120: the mass ratio of carboxymethyl cellulose to gelatin is 1.2.

[0013] Furthermore, the substances required for quenching the reaction in S4 are generally disodium hydrogen phosphate dodecahydrate and sodium chloride, with a molar ratio of 1:20, and the pH of the solution is adjusted to about 8.5.

[0014] Furthermore, the rotation speed of the high-speed centrifuge in S5 is 5500 rpm, and the reaction time is 5 min.

[0015] Furthermore, the mass ratio of the physiological saline to the lyophilized powder added in S6 is 4:1-6:1.

[0016] In another aspect of the present invention, the prepared gelatin / carboxymethyl cellulose rapid hemostatic material has two different hemostatic forms, which are gel and powder.

[0017] Another aspect of the present invention provides a gelatin / carboxymethyl cellulose rapid hemostatic material for use in non-pressable hemostasis.

[0018] Another aspect of the present invention provides a gelatin / carboxymethyl cellulose rapid hemostatic material that can achieve hemostatic function under different environments.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The advantages of the present invention are that the preparation process is simple, it is easy to prepare on a large scale and market it, and different forms of hemostatic materials, such as powder and gel, can be easily obtained in the preparation process, and dual selection can be provided according to demand; it is easy to operate when used, which optimizes the cumbersome operation steps of traditional hemostatic materials and shortens the operation time. At the same time, the present invention has excellent hemostatic ability, certain adhesion and good fluidity, and is easy to store for a long time. It is an ideal clinical hemostatic material with broad market prospects.

[0020] (2) The chemically cross-linked hemostatic material of gelatin / carboxymethyl cellulose of the present invention has a good hemostatic effect, low biological toxicity, good biocompatibility, safe use, and meets the requirements for application in incompressible hemostatic materials. Among them, the hemostatic powder can be applied to a variety of irregular shapes such as diffuse wound hemostasis due to its shape adaptability, large specific surface area and good water absorption performance, and the injectable fluid properties of the hemostatic gel enable it to penetrate into the tiny cavities in the body, be accurately injected and closely fit to the bleeding wound, and achieve high contact area, high efficiency, and precise hemostasis. It can be prepared into a hemostatic drug with clinical value, which is of great significance to the development of incompressible hemostatic materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 : Graph showing the hemostatic effect during liver hemostasis in mice in the hemostatic gel group.

[0023] Figure 2 : Graph of bleeding volume in the mouse liver experiment in the hemostatic gel group.

[0024] Figure 3 : Image of hemostatic effect during hemostasis of pig spinal microvenules in the hemostatic gel group.

[0025] Figure 4 : Graph showing the amount of bleeding during hemostasis of the spinal microvenules in the hemostatic gel group.

[0026] Figure 5 : Graph showing the hemostatic effect during liver hemostasis in mice in the hemostatic powder group.

[0027] Figure 6 : Graph of bleeding volume in the mouse liver experiment in the hemostatic gel group.

[0028] Figure 7 : Fourier transform infrared spectra of gelatin, carboxymethyl cellulose and CG30, CG60, CG90 and CG120 prepared by the present invention.

[0029] Figure 8 : C element diagram of the prepared CG60 obtained in the XPS test.

[0030] Fig. 9 : N element diagram of the prepared CG60 obtained in the XPS test.

[0031] Fig.10 : O element map of the prepared CG60 obtained in the XPS test.

[0032] Fig.11: Blood compatibility diagram of prepared CG60. DETAILED DESCRIPTION

[0033] The present invention proposes a chemically cross-linked hemostatic material based on gelatin / carboxymethyl cellulose and a preparation method thereof. In order to make the advantages and technical solutions of the present invention clearer and more specific, the present invention is described in detail below in conjunction with specific embodiments.

[0034] First, the main raw materials and experimental equipment required for the present invention are described in detail.

[0035] Experimental instruments: electronic balance, water bath magnetic stirrer, vacuum oven, magnetic stirrer, pH meter, high-speed centrifuge, -80 ℃ medical freezer, freeze dryer, grinding mortar, vacuum drying oven, water purification system.

[0036] Experimental drugs: Gelatin, carboxymethyl cellulose (CMC), N-hydroxysuccinimide (NHS), and 2-morpholineethanesulfonic acid (2-MES) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium chloride (NaCl), sodium hydroxide (NaOH), and sodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0038] Example 1. Preparation of chemically cross-linked hemostatic material based on gelatin / carboxymethyl cellulose The target product was obtained by crosslinking gelatin and carboxymethyl cellulose using EDC / NHS as a crosslinking agent. Weigh 1 g of gelatin and place it in a beaker, add 50 mL of ultrapure water and a magnet, and adjust the water bath magnetic stirrer to 60 °C to fully stir and dissolve the gelatin. Weigh 0.767 g of EDC and 0.092 g of NHS and dissolve them in 50 mL of ultrapure water, and add 0.488 g of 2-MES to provide an acidic environment. The gelatin solution was introduced into a beaker equipped with a crosslinking agent, and 0.3 g, 0.6 g, 0.9 g, and 1.2 g of carboxymethyl cellulose were added, respectively, and stirred on a magnetic stirrer at room temperature for 20-24 h. The products were named CG30, CG60, CG90, and CG120, respectively.

[0039] Add 1.8 g Na2HPO4·12H2O and 5.85 g NaCl to the product after the reaction, adjust the pH to 8.5 with NaOH solution, and stir thoroughly to obtain a suspension. Centrifuge the suspension at 5500 rpm for 5 min, remove the supernatant and add ultrapure water for washing, repeat the above operation four times, remove excess water, place the sample in a -80 ℃ refrigerator for 3 h, place the pre-frozen gel in a freeze dryer for at least 48 h, take out the freeze-dried sample, and grind it. The obtained sample is a powdered hemostatic material. Add physiological saline 4-6 times the weight of the powder, and mix thoroughly to obtain a gel-shaped hemostatic material.

[0040] Example 2: Application of chemical cross-linked gel based on gelatin / carboxymethyl cellulose in hemostasis of mouse liver Hemostatic effect test. Healthy 5-6 week old female mice were selected, with 4 mice in each group, including the experimental groups CG30, CG60, and CG90, the positive control group Surgiflo, and the negative control group without wound treatment. After the mice were anesthetized by intraperitoneal injection of 1% w / v pentobarbital solution, the chest hair was removed, and the liver was carefully and fully exposed and placed on a pre-weighed filter paper. Then, a surgical blade was used to make an incision of 3 mm in length and 2 mm in depth on the surface of the liver to create a liver bleeding model. Subsequently, the gel was immediately covered on the wound with a syringe, and the hemostasis time was recorded and the amount of bleeding was calculated (bleeding amount = postoperative weight of the filter paper - preoperative weight). The hemostatic effect is as follows. Figure 1 , Figure 2 As shown. Figure 1 as well as Figure 2 It can be seen that in the above test, CG30, CG60 and CG90 prepared by the present invention all have good hemostatic effects, and are better than the positive control group.

[0041] Example 3: Application of chemical cross-linked gel based on gelatin / carboxymethyl cellulose in hemostasis of porcine spinal microvenules Healthy 1-year-old Tibetan pigs weighing about 30 kg were selected. The experimental materials were CG60 in the experimental group, Surgiflo in the positive control group, and no treatment was given to the negative control group. Propofol and Shutai were used for general anesthesia by endotracheal intubation and intramuscular injection. The skin was cut longitudinally with 2-3 spinous processes upward and downward, with the 3rd and 4th lumbar vertebrae as the center. The skin retractor was used to pull the incision open. The electrosurgical knife was used to cut the attachment of the supraspinal ligament and the atrial muscle in the midline of the spinous process, and the subcutaneous fascia and muscle were separated. The lateral sacrospinal muscle was separated under the periosteum, and the S retractor was pulled open to expose the lumbar vertebral plate. The tissue on the vertebral plate was removed with a bone rongeur, and the spinal canal was further exposed to find the venous plexus on both sides of the spinal canal. In the negative control group, the blood vessel was punctured by a needle and the gauze weighed in advance was quickly placed on the wound. After one minute, the gauze was removed and weighed again to record the amount of bleeding. After the second puncture, 300 μL Surgiflo and CG60 were immediately added through a syringe to cover the wound as the positive control group and the experimental group, and the above operation was repeated. If there is less bleeding during the operation, use a suction device to absorb it in time, and be careful not to damage the blood vessels or nerves; if there is more bleeding, Surgiflo can be injected into the corresponding area. After sufficient hemostasis, add normal saline to flush and use the suction device to make the field of vision clear. Figure 3 , Figure 4 As shown, it can be observed that CG60 significantly reduced the amount of bleeding compared with the negative control and had a good hemostatic effect similar to that of Surgiflo.

[0042] Example 4: Application of chemically cross-linked powder based on gelatin / carboxymethyl cellulose in hemostasis of mouse liver Healthy 5-6 week old female mice were selected, 3 mice in each group. The experimental group was treated with CG60 hemostatic powder, the positive control group was treated with Surgicel hemostatic powder, and the negative control group did not treat the wound. After intraperitoneal injection of 1% w / v pentobarbital solution, the mice were anesthetized, the chest hair was removed, and the liver was carefully and fully exposed and placed on a pre-weighed filter paper. Then, a surgical blade was used to make an incision of 3 mm in length and 2 mm in depth on the surface of the liver to create a liver bleeding model. Subsequently, the hemostatic powder was immediately used to evenly cover the wound, and the hemostasis time was recorded and the amount of bleeding was calculated (bleeding amount = postoperative weight of the filter paper - preoperative weight). The hemostatic effect is as follows: Figure 5 , Figure 6 As shown. Figure 5 as well as Figure 6 It can be seen that in the above test, the CG60 hemostatic powder prepared by the present invention has a good hemostatic effect that is no less than that of Surgicel, and is significantly better than the positive control group.

[0043] Example 5: Fourier transform infrared spectroscopy structure analysis of gelatin / carboxymethyl cellulose rapid hemostatic material Weigh 5 mg of freeze-dried CG30, CG60, CG90, CG120 and gelatin samples respectively using a balance and grind them into powder in a mortar. Press the freeze-dried samples and carboxymethyl cellulose powder into transparent discs using a tabletting method. −1 The spectral range is scanned, and potassium bromide is used as a reference substance. The FTIR pattern of the sample is obtained by Fourier transform and the chemical bonds and group composition of the sample are analyzed to determine the cross-linking of carboxymethyl cellulose and gelatin. The measurement results are as follows: Figure 7 As shown, at 1640 cm -1 and 1540 cm -1 The peaks at 1585 cm -1 The peak at the center is the characteristic peak of carboxymethyl, which indicates that CG30, CG60, CG90 and CG120 have successfully cross-linked carboxymethyl cellulose.

[0044] Example 6. XPS analysis of CG60 samples To ensure that the sample is fully dried, the freeze-dried sample is ground and placed in a vacuum oven at 60 °C for 12 h. A layer of double-sided tape of about 0.5 cm × 0.5 cm is attached to a 2 cm × 3 cm aluminum foil, and then the sample is slowly sprinkled on the double-sided tape. Fold the aluminum foil in half and place it on a tablet press for about 10 s. Tear open the aluminum foil, cut off the excess foil with scissors, and finally blow it gently with a nitrogen gun. Analyze the C, N, and O elements in the sample, and the experimental results are as follows: Figure 8 , 9 As shown in 10, it can be observed Figure 8 and Fig.10 The carboxyl groups in the Fig. 9 The presence of amino groups from gelatin, Fig. 9 The amide bond formed by the two can be clearly observed, proving that the cross-linking is successful.

[0045] Example 7. Blood compatibility determination of CG60 samples Specific steps of the hemolysis experiment: Take 500 μL of fresh blood, add 5 mL of PBS, centrifuge (10000 g, 5 min), discard the supernatant, and repeat until the supernatant is clear after centrifugation. Add PBS to 10 mL and mix well, centrifuge (10000 g, 5 min). Add 10 mL of PBS to resuspend the red blood cells. Mix 200 μL of red blood cell suspension with 800 μL of gradient concentration CG60 PBS solution; mix 200 μL of red blood cell suspension with 800 μL of 1% Trton X-100 solution as a positive control; mix 200 μL of red blood cell suspension with 800 μL PBS as a negative control and incubate at 37 °C for 1 hour. Then centrifuge (10000 g, 5 min), take 100 μL of supernatant and transfer it to a 96-well plate. Finally, use an enzyme reader to record the absorbance at 450 nm. The hemolysis rate is calculated according to the formula: Hemolysis rate (%) = (Ab sample-Ab negative) / (Ab positive-Ab negative) × 100%. The experimental results are as follows Fig.11 As shown, the CG60 of the present invention has good blood compatibility.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A quick hemostatic material based on gelatin / carboxymethyl cellulose, characterized in that: The hemostatic material comprises gelatin and carboxymethyl cellulose. The hemostatic material is cross-linked by an amidation reaction catalyzed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a gelatin / carboxymethyl cellulose complex.

2. The method for preparing the rapid hemostatic material according to claim 1, characterized in that: The specific steps are as follows: S1. Weigh a certain amount of gelatin particles and place them in a beaker, add an appropriate amount of ultrapure water and heat to prepare a gelatin solution; S2. Weigh a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 2-morpholineethanesulfonic acid and fully dissolve them in an appropriate amount of ultrapure water; S3, adding the mixed solution in S2 to the gelatin solution in S1, and then adding different amounts of carboxymethyl cellulose, stirring and reacting at room temperature for 20-24 hours, and the obtained products are named CG30, CG60, CG90, and CG120 respectively; S4, quenching the reaction, adding a certain amount of disodium hydrogen phosphate dodecahydrate and sodium chloride to the product of the reaction in S3, adjusting the pH with sodium hydroxide solution, and stirring sufficiently to obtain a suspension; S5, centrifuging the suspension obtained in S4, removing the supernatant and washing, removing excess water, adding ultrapure water and centrifuging again, repeating the above steps four times, freeze-drying the obtained sample, and grinding it to obtain hemostatic powder; S6. Mix the sample powder in S5 with 4-6 times the mass of normal saline to obtain a fluid hemostatic gel.

3. The preparation method according to claim 2, characterized in that: The molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in S2 is 0.2:

1.

4. The preparation method according to claim 2, characterized in that: The specific preparation methods of the products in S3 are as follows: CG30: the mass ratio of carboxymethyl cellulose to gelatin is 0.3; CG60: the mass ratio of carboxymethyl cellulose to gelatin is 0.6; CG90: the mass ratio of carboxymethyl cellulose to gelatin is 0.9; CG120: the mass ratio of carboxymethyl cellulose to gelatin is 1.

2.

5. The preparation method according to claim 2, characterized in that: The substances required for quenching the reaction in S4 are disodium hydrogen phosphate dodecahydrate and sodium chloride, with a molar ratio of 1:20 and a solution pH of 8.

5.

6. The preparation method according to claim 2, characterized in that: The speed of the S5 high-speed centrifuge is 5500 rpm, and the reaction time is 5 min.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the physiological saline to the lyophilized powder added in S6 is 4:1-6:

1.

8. The rapid hemostatic material according to claim 1, characterized in that: There are two different hemostatic forms, gel and powder.

9. Use of the rapid hemostatic material as claimed in claim 1 in non-pressible hemostasis.

10. Use of the rapid hemostatic material as claimed in claim 1 to achieve hemostatic function in different environments.

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