Tannic acid modified gelatin-based styptic powder as well as preparation method and application thereof

Abstract

The invention provides styptic powder based on tannic acid modified gelatin as well as a preparation method and application of the styptic powder. The modified gelatin is a compound formed by chemically crosslinking and blending tannic acid and pre-crosslinked gelatin, the tannic acid can be uniformly distributed in a gelatin network, and the tannic acid serving as an exogenous hemostatic active substance can be subjected to rapid non-covalent interaction such as H bonds and pi-pi stacking with proteins in blood to form microclots; gelatin particles are promoted to rapidly infiltrate blood and settle, so that aggregation of platelets and red blood cells is accelerated, and gelatin can accelerate activation of an endogenous hemostasis pathway, so that a synergistic effect is formed, and rapid hemostasis is realized. In addition, tannic acid also has good antibacterial activity, wound infection after hemostasis can be effectively avoided, and the tannic acid is especially suitable for emergency hemostasis in outdoor first aid.

Description

Technical Field

[0001] The invention relates to the field of absorbable medical hemostatic materials, and in particular to a hemostatic powder based on tannic acid modified gelatin, and a preparation method and application thereof. Background Art

[0002] Uncontrollable bleeding has always been one of the biggest challenges in surgery. Effective hemostasis can not only significantly reduce the patient's pain, but also greatly improve the success rate of surgery and the patient's survival rate. However, there are still many problems with existing hemostatic materials. For example, hemostatic sponges and hemostatic gauze must be pressed with assistance, and blood clots are easily destroyed when removed, causing secondary damage; long-term use of tourniquets can easily cause excessive pressure on surrounding tissues, leading to nerve damage, local skin damage, and even limb necrosis; the preparation process of hemostatic gel is cumbersome, and the precursor solution is also easily washed away or diluted by the blood flow during application, resulting in hemostasis failure. Compared with the above materials, hemostatic powder has been widely used in emergency hemostasis in surgical operations, outdoor first aid and other scenarios because of its advantages such as portability, ease of use, large-area spraying, and adaptability to complex wound shapes. However, the current hemostatic powder products have a single hemostatic mechanism, mainly relying on endogenous hemostatic pathways, and the hemostasis speed is slow.

[0003] Tannic acid is a plant-based natural polyphenol with a wide range of sources and low prices. Because of its rich catechol groups in its structure, it can interact with blood cells, plasma proteins, etc., thereby promoting vasoconstriction and blood coagulation. It has been widely used in the preparation of hemostatic materials. At the same time, tannic acid also has excellent antibacterial properties and can effectively prevent wound infection after application.

[0004] Patent CN 118217442 discloses "a multifunctional ready-to-use collagen hemostatic powder and its preparation method and application", which is prepared by using tannic acid to cross-link type II collagen under moist conditions to form a hydrogel, freeze-drying and grinding into powder.

[0005] Patent CN 114887111 discloses "a bioabsorbable composite hemostatic material and its preparation method and application". The composite material includes polyvinyl alcohol, gelatin and tannic acid. The polyvinyl alcohol and gelatin solution are mixed and then tannic acid is added for self-assembly, followed by vacuum drying and grinding into powder.

[0006] Patent CN 114887111 discloses a "method for preparing hemostatic powder with strong adhesion, antibacterial properties and biodegradability". The main materials include quaternized chitosan, gelatin and tannic acid. The quaternized chitosan and gelatin solution are mixed and then tannic acid is added and stirred thoroughly to obtain a hydrogel, which is then freeze-dried and ground into powder.

[0007] Thanks to the extremely high reactivity of tannic acid, it can self-assemble to form a composite material after simply mixing with biological macromolecules such as proteins and polysaccharides. However, due to its extremely fast reaction speed, it is easy for the reactants to precipitate or react with some macromolecules to aggregate, resulting in the inability to obtain a homogeneous tannic acid composite material. It is also easy for the local concentration of tannic acid in the material to be too high, which is prone to sudden release during application, which may damage tissues and cause strong inflammatory reactions.

[0008] Therefore, how to stably prepare a composite hemostatic material with controllable tannic acid content and uniform distribution is of great research significance. Summary of the invention

[0009] The purpose of the present invention is to provide a hemostatic powder based on tannic acid modified gelatin, which is a composite of tannic acid and pre-crosslinked gelatin after chemical crosslinking and blending. The present invention also provides a preparation method and application method of the tannic acid modified gelatin.

[0010] To achieve the above object, the technical solution adopted by the present invention is: A hemostatic powder based on tannic acid-modified gelatin, wherein the modified gelatin is pre-cross-linked gelatin modified by tannic acid, wherein the mass percentage of gelatin is 70-95%, and the mass percentage of tannic acid is 5-30%.

[0011] Furthermore, the cross-linking method of the pre-cross-linked gelatin is thermal cross-linking.

[0012] Furthermore, the modified gelatin is a complex obtained by chemically cross-linking tannic acid and pre-cross-linked gelatin, followed by freeze-drying, mechanical crushing and screening. The chemically cross-linked blending is performed by soaking the pre-cross-linked gelatin freeze-dried material in an aqueous solution containing tannic acid or a dimethyl sulfoxide solution containing tannic acid.

[0013] The method for preparing the hemostatic powder based on tannic acid modified gelatin comprises the following steps: (1) Prepare gelatin solution, stir and dissolve at 45-55°C, transfer to room temperature, wait for it to cool and solidify, and then pre-freeze and vacuum freeze-dry; (2) pre-crosslinking the product of step (1); (3) soaking the product in step (2) in an aqueous solution of tannic acid or a dimethyl sulfoxide solution of tannic acid; (4) washing the product in step (3) with pure water, and soaking it in an aqueous solution containing an antioxidant, and then pre-freezing and vacuum freeze-drying; (5) mechanically crushing and sieving the product in step (4) to obtain tannic acid-modified gelatin particles within a target particle size range (50-300 mesh); (6) The tannic acid-modified gelatin particles obtained in step (5) are packaged and sterilized by γ-irradiation before use, with an irradiation dose of 25 to 40 kGy.

[0014] Furthermore, the concentration of the gelatin solution is 1-20 wt / v%.

[0015] Furthermore, the cross-linking method of the pre-cross-linked gelatin is thermal cross-linking, the cross-linking temperature is 120-180° C., and the cross-linking time is 2-48 h.

[0016] Furthermore, the concentration of the tannic acid solution is 0.25-10%, the volume is 50-200 times the mass of the soaked material, and the soaking time is 12-72 hours. After soaking in the tannic acid aqueous solution or dimethyl sulfoxide solution, it is necessary to rinse with pure water several times to remove free tannic acid or replace dimethyl sulfoxide.

[0017] Furthermore, the antioxidant includes one or more of ascorbic acid, citric acid, sodium bisulfite, and sodium pyrosulfite, preferably ascorbic acid or citric acid.

[0018] Furthermore, the concentration of the antioxidant solution is 0.005-0.1 M, the volume is 10-20 times the mass of the soaked material, and the soaking time is 8-24 h.

[0019] The invention discloses an application of gelatin hemostatic powder modified by tannic acid, which can be used for emergency hemostasis of acute wounds or hemostasis of surgical wounds.

[0020] The beneficial effects of the present invention are: (1) The present invention uses pre-cross-linked gelatin and tannic acid for chemical blending and modification, which can avoid the precipitation of gelatin or the aggregation of tannic acid in a certain area of ​​the gelatin network due to the rapid cross-linking reaction between the two, thereby ensuring the uniform distribution of tannic acid and the controllable regulation of its content, and also avoiding the problem of sudden release caused by excessive local concentration of tannic acid during application.

[0021] (2) The present invention introduces tannic acid as an exogenous hemostatic active substance, which can form microclots by rapid non-covalent interactions such as H bonds and π-π stacking with proteins in the blood, prompting gelatin particles to quickly infiltrate the blood and settle, thereby accelerating the aggregation of platelets and red blood cells. Gelatin itself can also accelerate the activation of endogenous hemostatic pathways, thereby forming a synergistic effect and achieving rapid hemostasis. In addition, tannic acid itself also has good antibacterial activity, which can effectively prevent wound infection after hemostasis, and is particularly suitable for emergency hemostasis in outdoor first aid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Attached Figure 1 It is the tannic acid modified gelatin complex prepared in different groups in Example 2.

[0023] Attached Figure 2 It is the tannic acid modified gelatin complex prepared in Example 3 and Comparative Example 1.

[0024] Attached Figure 3 This is a photo of an in vitro coagulation test.

[0025] Attached Figure 4 This is a photograph of the blood absorption rate of gelatin particles.

[0026] Attached Figure 5 This is a photo of a blood clot formed when gelatin particles are mixed with blood. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention. Example 1

[0028] (1) Prepare a gelatin solution with a concentration of 2.5 wt / v%, stir and dissolve it at 50°C, then transfer it to room temperature, wait for it to cool and solidify, and then pre-freeze and vacuum freeze-dry it; (2) cross-linking the product in step (1) at 150° C. in vacuum for 4 h; (3) soaking the product in step (2) in a dimethyl sulfoxide solution of tannic acid at a mass volume ratio of 1:100 and a concentration of 1 wt / v% for 24 h; (4) The product in step (3) was washed with pure water for multiple times to replace DMSO, and then immersed in a 0.01 M ascorbic acid aqueous solution at a mass volume ratio of 1:20 for 24 hours before pre-freezing and vacuum freeze-drying; (5) The product of step (4) is mechanically crushed and sieved to obtain tannic acid-modified gelatin particles within the target particle size range (50-300 mesh), which are packaged and sterilized by γ-irradiation for use at a irradiation dose of 25 kGy. Example 2

[0029] Tannic acid-modified gelatin particles were prepared according to steps (1) to (5) in Example 1, and the concentration of the tannic acid soaking solution was adjusted to investigate its effect on the composite content of gelatin. The concentrations of all groups were as follows: Group 1 Group 2 Group 3 Group 4 Tannic acid concentration (wt / v%) 0.5% 1% 2% 4% Group 2 is the preparation parameters in Example 1.

[0030] Weigh the initial mass of the gelatin lyophilized product as m 1 The mass of the final product is m 2 , tannic acid content (%) = (m 2 -m1 )*100 / m 1 The freeze-dried tannic acid modified gelatin in each group Figure 1 There were 4 parallel samples in each group. The results of tannic acid content are shown in Table 1.

[0031] Table 1. Tannic acid content in modified gelatin Serial number Tannic acid soaking solution concentration Tannic acid content in modified gelatin Group 1 0.5% 6.4% Group 2 1.0% 8.3% Group 3 2.0% 11.4% Group 4 4.0% 13.4% From the above results, it can be seen that the tannic acid content in the modified gelatin can be adjusted by changing the concentration of the tannic acid soaking solution. The higher the concentration of the soaking solution, the higher the tannic acid content in the modified gelatin, and the darker the color of the corresponding sample. Example 3

[0032] (1) Prepare a gelatin solution with a concentration of 2.5 wt / v%, stir and dissolve it at 50°C, then transfer it to room temperature, wait for it to cool and solidify, and then pre-freeze and vacuum freeze-dry it; (2) cross-linking the product in step (1) at 150° C. in vacuum for 4 h; (3) soaking the product in step (2) in an aqueous solution of tannic acid at a mass volume ratio of 1:100 and a concentration of 1 wt / v% for 24 h; (4) The product of step (3) is rinsed with pure water for multiple times to remove free tannic acid, and then immersed in a 0.01 M ascorbic acid aqueous solution at a mass volume ratio of 1:20 for 24 hours before being pre-frozen and vacuum freeze-dried; (5) The product of step (4) is mechanically crushed and sieved to obtain tannic acid-modified gelatin particles within the target particle size range (50-300 mesh), which are packaged and sterilized by γ-irradiation for use at a irradiation dose of 25 kGy.

[0033] Weigh the initial mass of the gelatin lyophilized product as m 1 The mass of the final product is m 2 , tannic acid content (%) = (m 2 -m 1 )*100 / m 1 , after calculation, the tannic acid content is 14.8%, which is close to the tannic acid content of the modified gelatin in Example 1, indicating that there is no significant difference between the two soaking methods.

[0034] Comparative Example 1 (1) Prepare a gelatin solution with a concentration of 2.5 wt / v%, stir and dissolve it at 50°C, then transfer it to room temperature, wait for it to cool and solidify, and then pre-freeze and vacuum freeze-dry it; (2) soaking the product in step (1) in an aqueous solution of tannic acid at a mass volume ratio of 1:100 and a concentration of 1 wt / v% for 24 h; (3) The product of step (2) is rinsed with pure water for multiple times to remove free tannic acid, and then immersed in a 0.01 M ascorbic acid aqueous solution at a mass volume ratio of 1:20. After immersion for 24 hours, it is pre-frozen and vacuum freeze-dried to obtain a gelatin / tannic acid mixture, which is packaged and sterilized by γ irradiation before use, with an irradiation dose of 25 kGy.

[0035] See the picture of the gelatin / tannic acid mixture after freeze drying. Figure 2 , 4 parallel samples in each group. The gelatin in Comparative Example 1 is yellow / white, and the tannic acid is concentrated in a certain area or a certain surface, and the distribution is extremely uneven, while the gelatin component in Example 3 is light yellow as a whole, indicating that the tannic acid is distributed more evenly. Therefore, cross-linking the gelatin to a certain extent in advance is conducive to its uniform distribution when soaking in tannic acid.

[0036] Comparative Example 2 (1) Prepare a gelatin solution with a concentration of 10 wt / v%, stir and dissolve it at 50°C, then transfer it to room temperature, wait for it to cool and solidify, and then pre-freeze and vacuum freeze-dry it; (2) cross-linking the product in step (1) under vacuum at 150° C. for 24 h; (3) Mechanically crushing and sieving the product of step (2) to obtain gelatin particles within the target particle size range (50-300 mesh), which are packaged and sterilized by gamma irradiation with an irradiation dose of 25 kGy.

[0037] Comparative Example 3 (1) Prepare a 2.5 wt / v% gelatin solution and dissolve it by stirring at 50°C; (2) Prepare a 2.5 wt / v% tannic acid solution and stir to dissolve; (3) mixing the gelatin solution in step (1) and the tannic acid solution in step (2) at a volume ratio of 10:1 to obtain a gelatin / tannic acid mixture having a tannic acid content of 10%; (4) pre-freezing and freeze-drying the product of step (3); (5) The product of step (4) is mechanically crushed and sieved to obtain gelatin / tannic acid mixture particles within the target particle size range (50-300 mesh), which are packaged and sterilized by gamma irradiation with an irradiation dose of 25 kGy.

[0038] Saturated water absorption test of gelatin particles Take the samples of all groups in Example 2, Example 3, Comparative Example 2 and Comparative Example 3, and weigh the initial mass of gelatin particles as m 1 , mixed with normal saline at a mass-volume ratio of 1:10, and the mass of normal saline is m 2After shaking, let it stand for 24 hours and then pour it onto a 400-mesh stainless steel sieve to collect the filtrate, the mass of which is m 3 , then the saturated water absorption rate (%) of gelatin particles is (m 2 -m 3 )*100 / m 1 .

[0039] The results are shown in Table 2: Table 2. Water absorption of gelatin particles Group Group 1 Group 2 Group 3 Group 4 Example 3 Comparative Example 2 Comparative Example 3 Water absorption 961% 873% 771% 614% 624% 599% 2360% It can be seen from the results of Group 1 to Group 4 that the increase in the tannic acid concentration in the composite will lead to a decrease in its water absorption rate. It can be seen from Group 4 and Example 3 that the tannic acid-modified gelatin prepared by the two methods has a similar water absorption rate when the tannic acid content is close. In vitro coagulation test of gelatin particles

[0040] Take fresh rabbit blood into a sodium citrate blood collection tube and add 1 / 4 of the rabbit blood volume of CaCl 2 Solution (0.2 M) was added to neutralize the anticoagulant in the blood, and then the gelatin particles and rabbit blood were mixed at a mass / volume ratio of 1:10. The centrifuge tube was inverted every 10 seconds for observation. If the blood did not slide off, it was determined that the coagulation was successful. After testing, the coagulation time of the blank group was 5 minutes, so it was set that the coagulation failed if it did not coagulate for more than 2 minutes. There were 5 groups in total, namely, the samples in Group 1 and Group 4 in Example 2, Example 3, Comparative Example 2 and Comparative Example 3. The results are shown in Table 3 and Figure 3 .

[0041] Table 3. In vitro coagulation test of gelatin particles Group A: Group 1 B: Group 4 C: Example 3 D: Comparative Example 2 E: Comparative Example 3 In vitro clotting time 20 s 30 s 20 s fail fail The water absorption rates of the gelatin particles in Group 4, Example 3 and Comparative Example 2 are similar, but the coagulation rates of the first two groups are significantly faster than that of the third group, indicating that the introduction of tannic acid into the gelatin particles can significantly improve the hemostasis rate; the modified gelatin particles in Group 4 and Example 3 are prepared in different ways, but there are no obvious differences in water absorption, tannic acid content and in vitro coagulation rate, indicating that the products obtained by the two preparation methods are similar; the sample in Comparative Example 3 is obtained by simply mixing and freeze-drying an uncross-linked gelatin solution and a tannic acid solution, and its tannic acid content is between that of Group 1 and Group 4, but it has no obvious hemostatic activity, indicating the necessity of using pre-cross-linked gelatin for tannic acid chemical modification. Blood absorption rate test of gelatin particles

[0042] Take fresh rabbit blood into a sodium citrate blood collection tube and add 1 / 4 of the rabbit blood volume of CaCl 2Solution (0.2 M) was added to neutralize the anticoagulant in the blood. The gelatin particles were naturally sprinkled on the fresh rabbit blood at a mass volume ratio of 1:10 and the timing was started. After 1 minute, the particles were turned over to observe whether the blood coagulated. Then the time required for all the particles to be soaked by the blood was recorded. There were 2 groups in total, namely Example 3 and Comparative Example 2. The results are shown in Table 4 and Figure 4 .

[0043] Table 4. Blood absorption rate test of gelatin particles Group The time it takes for gelatin particles to be completely soaked by blood Example 3 5 min Comparative Example 2 9 min Example 3 is tannic acid modified gelatin particles, and Comparative Example 2 is pure gelatin particles. The water absorption of the two groups is similar, but the blood absorption rate of the tannic acid modified gelatin particles is significantly faster. The blood has coagulated at 1 minute, and most of the gelatin powder has been infiltrated by the blood, while the blood in Comparative Example 2 has not coagulated and still has good fluidity, and most of the gelatin particles are still floating above the blood without being infiltrated. Therefore, the introduction of tannic acid into gelatin particles can increase its blood absorption rate. It is speculated that the interaction between tannic acid and proteins in the blood increases its ability to capture blood.

[0044] Blood clot swelling rate test Take fresh rabbit blood into a sodium citrate blood collection tube and add 1 / 4 of the rabbit blood volume of CaCl 2 Solution (0.2 M) was added to neutralize the anticoagulant in the blood. The gelatin particles were mixed in a 15 mm diameter circular mold at a mass volume ratio of 1:10 and placed at 37 °C for 2 h to form a blood clot, which was weighed as m 1 Then soak in PBS and place at 37℃ for 24 h before taking out and weighing m 2 , swelling ratio = (m 2 -m 1 ) / m 1 , a total of 4 groups, respectively, group 1 in Example 2, Example 3, comparative example 2 and blank group, the results are shown in Table 5 and Figure 5 .

[0045] Table 5. Blood clot swelling rate test A: Group 1 B: Example 3 C: Comparative Example 2 D: Blank group Swelling rate (%) -4 -16 -2 -12 The swelling ratios of all groups were negative, indicating that there was no risk of excessive swelling after application of gelatin particles.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hemostatic powder based on tannic acid modified gelatin, characterized in that: The modified gelatin is pre-cross-linked gelatin modified by tannic acid, wherein the mass percentage of gelatin is 70-95%, and the mass percentage of tannic acid is 5-30%.

2. The hemostatic powder based on tannic acid modified gelatin according to claim 1, characterized in that: The cross-linking method of the pre-cross-linked gelatin is thermal cross-linking.

3. The hemostatic powder based on tannic acid modified gelatin according to claim 1, characterized in that: The modified gelatin is a composite of tannic acid and pre-crosslinked gelatin after chemical crosslinking and blending. The chemical crosslinking and blending method is to soak the pre-crosslinked gelatin freeze-dried material in an aqueous solution of tannic acid, or soak the pre-crosslinked gelatin freeze-dried material in a dimethyl sulfoxide solution of tannic acid.

4. The method for preparing a hemostatic powder based on tannic acid modified gelatin according to claim 1, characterized in that: The steps include: (1) Prepare gelatin solution, stir and dissolve at 45-55°C, transfer to room temperature, wait for it to cool and solidify, and then pre-freeze and vacuum freeze-dry; (2) pre-crosslinking the product of step (1); (3) soaking the product in step (2) in an aqueous solution of tannic acid or a dimethyl sulfoxide solution of tannic acid; (4) washing the product in step (3) with pure water, and soaking it in an aqueous solution containing an antioxidant, and then pre-freezing and vacuum freeze-drying; (5) The product of step (4) is mechanically crushed and sieved to obtain tannic acid-modified gelatin particles within a target particle size range (50-300 meshes), which are packaged and sterilized by γ-irradiation for use at a irradiation dose of 25-40 kGy.

5. The method for preparing hemostatic powder based on tannic acid modified gelatin according to claim 4, characterized in that: The gelatin solution described in step (1) has a concentration of 1-20 wt / v%.

6. The hemostatic powder based on tannic acid modified gelatin according to claim 4, characterized in that: The pre-crosslinking in step (2) is carried out by high-temperature vacuum crosslinking, the crosslinking temperature is 120-180°C, and the crosslinking time is 2-48 h.

7. The method for preparing hemostatic powder based on tannic acid modified gelatin according to claim 4, characterized in that: The tannic acid solution in step (3) has a concentration of 0.25-10%, a volume of 50-200 times the mass of the soaked material, and a soaking time of 12-72 h.

8. The method for preparing hemostatic powder based on tannic acid modified gelatin according to claim 4, characterized in that: The antioxidant described in step (4) includes one or more of ascorbic acid, citric acid, sodium bisulfite, and sodium metabisulfite. The concentration of the antioxidant is 0.005-0.1 M, the volume is 10-20 times the mass of the soaking material, and the soaking time is 8-24 h.

9. Use of the hemostatic powder based on tannic acid modified gelatin as described in claim 1.

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