Preparation method of hemostatic sponge

Abstract

The invention discloses a preparation method of hemostatic sponge. The preparation method comprises the following steps: pouring a hydrophilic chitosan solution into a mold and pre-freezing; a chitosan solution is added into the mold, pre-freezing is continued, and the frozen hemostatic sponge is obtained; the frozen hemostatic sponge is freeze-dried, and after moisture is completely removed, the hemostatic sponge is obtained; the hemostatic sponge is provided with a hydrophilic layer and a blood coagulation layer, the hydrophilic layer is formed by a hydrophilic chitosan solution, and the blood coagulation layer is formed by a chitosan solution. According to the preparation method of the hemostatic sponge, chitosan is used as a raw material for preparation, on the basis that the self blood coagulation performance of the chitosan is kept, a hydrophilic layer is further introduced, and when the hemostatic sponge is used, the hydrophilic layer is attached to the skin, and a blood coagulation layer is arranged outside, so that blood can rapidly permeate into the material when making contact with the material, and rapid blood coagulation is achieved under the action of the chitosan.

Description

[0001] This application is a divisional application of the invention patent application with application date of December 29, 2022, application number 2022117116508, and invention name “A double-layer structured chitosan hemostatic sponge and its preparation method”. Technical Field

[0002] The invention relates to the technical field of medical materials, and in particular to a method for preparing a double-layer chitosan hemostatic sponge. Background Art

[0003] Hemostasis is an important step in emergency medical treatment. When patients undergo surgery or suffer sudden trauma in daily life, rapid hemostasis is required. It is particularly important to achieve rapid and effective hemostasis in harsh war environments and complex emergencies during emergency treatment of war and trauma.

[0004] The hemostatic products currently sold and used on the market can be roughly divided into three categories based on their mechanism of action: the first category absorbs moisture from the blood near the wound through the physical or chemical action of the material, causing the coagulation components of the blood at the wound to concentrate and aggregate, thereby accelerating coagulation, or the material improves the adhesion and aggregation ability of red blood cells or platelets through the electrostatic effect between the surface charge and cells, such as gelatin hemostatic sponge and quick gauze; the second category uses the adhesion of the material to the tissue to directly seal the wound, thereby achieving hemostasis, such as α-cyanoacrylate tissue glue; the third category directly or indirectly provides hemostatic active substances to react with the effective components of the blood, initiates or enhances the intrinsic and exogenous coagulation pathways, and then accelerates coagulation, such as snake venom hemocoagulase.

[0005] Chitosan is a natural cationic polysaccharide extracted from shrimp and crab shells. It has multiple functions such as coagulation, antibacterial, wound healing, and scar inhibition. It is a multifunctional material with good biocompatibility, non-immunogenicity, and non-irritation. It was confirmed by the US FDA as a GRAS (generally recognized as safe) substance in 2001. Chitosan can adsorb and aggregate red blood cells through electrostatic action, while activating and adhering platelets, promoting thrombosis.

[0006] At present, most of the chitosan-based medical device products registered in China are in the form of non-woven dressings, gels, powders, etc., and there are relatively few sponge products. Among the existing sponge products, the chitosan functional layer is mostly a single-layer structure. Due to the poor wettability of chitosan to water, when it is actually applied to wounds, wound bleeding is not easy to penetrate into the material, and even side leakage may occur when the amount of bleeding is large, affecting the coagulation effect. Summary of the invention

[0007] In view of this, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for preparing a double-layer chitosan hemostatic sponge.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for preparing a chitosan hemostatic sponge comprises the following steps:

[0010] mixing chitosan with acetic acid solution and dissolving the chitosan to obtain a chitosan solution;

[0011] Adding a hydrophilic substance to part of the chitosan solution and mixing, dissolving and obtaining a hydrophilic chitosan solution;

[0012] pouring the hydrophilic chitosan solution into a mold and pre-freezing it;

[0013] adding a portion of the chitosan solution into the mold, and continuing pre-freezing to obtain a frozen hemostatic sponge;

[0014] The frozen hemostatic sponge is freeze-dried to completely remove water, thereby obtaining the double-layer chitosan hemostatic sponge.

[0015] According to some preferred implementation aspects of the present invention, the mass ratio of the hydrophilic substance to chitosan is 1:5 to 1:20.

[0016] According to some preferred embodiments of the present invention, the hydrophilic substance is one or more selected from polyethylene glycol, benzalkonium bromide, polyvinyl pyrrolidone, polyvinyl alcohol, Tween 80, poloxamer, and sodium lauryl sulfate.

[0017] According to some preferred implementation aspects of the present invention, the mass concentration of the acetic acid solution is 0.1% to 10%.

[0018] According to some preferred implementation aspects of the present invention, the mass concentration of chitosan in the chitosan solution is 0.1% to 8%.

[0019] According to some preferred implementation aspects of the present invention, the mass concentration of the hydrophilic substance in the hydrophilic chitosan solution is 0.01% to 5%.

[0020] According to some preferred embodiments of the present invention, when preparing the frozen hemostatic sponge, the mass ratio of the hydrophilic chitosan solution to the chitosan solution is 1:4 to 1:10 to control the thickness of the hydrophilic layer and the coagulation layer.

[0021] According to some preferred implementation aspects of the present invention, the pre-freezing temperature is -15°C to -70°C, and the pre-freezing time is 2-12 hours.

[0022] According to some preferred embodiments of the present invention, the freeze-drying parameters are: vacuum degree below 40 Pa, temperature 8-12° C. maintained for 36-60 h, preferably temperature 10° C. maintained for 48 h.

[0023] The present invention also provides a chitosan hemostatic sponge prepared by the preparation method as described above, which has a double-layer structure of a hydrophilic layer and a coagulation layer, wherein the hydrophilic layer is formed by a hydrophilic chitosan solution, and the coagulation layer is formed by a chitosan solution. While retaining the coagulation performance of chitosan, wound bleeding can be quickly introduced into the chitosan sponge to achieve rapid coagulation and reduce the amount of bleeding. The thickness ratio of the hydrophilic layer to the coagulation layer is 1:4 to 1:10.

[0024] Due to the implementation of the above technical solutions, the present invention has the following advantages over the prior art: the preparation method of the double-layer chitosan hemostatic sponge in the present invention is prepared with chitosan as the raw material, and on the basis of maintaining the coagulation performance of chitosan itself, a hydrophilic layer is further introduced. When in use, the hydrophilic layer fits the skin and the coagulation layer is outside, so that blood can quickly penetrate into the material when in contact with the material, and rapid coagulation is achieved under the action of chitosan. At the same time, the material can quickly turn into a viscous gel after contacting blood, and cooperate to block the wound site. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] The preparation method of the double-layer chitosan hemostatic sponge in this embodiment comprises the following steps:

[0027] Step 1: Mix chitosan and acetic acid solution and dissolve them to obtain a chitosan solution.

[0028] The mass concentration of the acetic acid solution used is 0.1% to 10%. The mass concentration of chitosan in the chitosan solution is 0.1% to 8%.

[0029] Step 2: adding a hydrophilic substance to a part of the chitosan solution and mixing, and obtaining a hydrophilic chitosan solution after dissolution.

[0030] The hydrophilic substance is one or more selected from polyethylene glycol, benzalkonium bromide, polyvinyl pyrrolidone, polyvinyl alcohol, Tween 80, poloxamer, sodium lauryl sulfate. The mass concentration of the hydrophilic substance in the hydrophilic chitosan solution is 0.01% to 5%; the mass ratio of the hydrophilic substance to chitosan is 1:5 to 1:20.

[0031] Step 3: Pour the hydrophilic chitosan solution into a mold and pre-freeze it until it is completely frozen.

[0032] The pre-freezing temperature is -15°C to -70°C, and the pre-freezing time is 2 - 12 h.

[0033] Step 4: Add part of the chitosan solution into the mold, and continue pre-freezing until it is completely frozen to obtain a frozen hemostatic sponge.

[0034] The mass ratio of the hydrophilic chitosan solution to the chitosan solution is 1:4 to 1:10. The pre-freezing temperature is -15°C to -70°C, and the pre-freezing time is 2 - 12 h.

[0035] Step 5: Perform freeze-drying on the frozen hemostatic sponge. After completely removing the moisture, a double-layer chitosan hemostatic sponge is obtained.

[0036] The parameters of freeze-drying are a vacuum degree below 40 Pa and a temperature of 10°C maintained for 48 h.

[0037] The double-layer chitosan hemostatic sponge prepared by the above preparation method has a hydrophilic layer and a coagulation layer. The hydrophilic layer is a chitosan freeze-dried sponge loaded with hydrophilic substances, and the coagulation layer is a pure chitosan freeze-dried sponge. While retaining the coagulation performance of chitosan, the double-layer chitosan hemostatic sponge can quickly introduce the wound bleeding into the interior of the chitosan sponge to achieve rapid coagulation. In addition, the chitosan hemostatic sponge in this example can also slowly gelate after contacting with blood, reducing the blood fluidity of the wound and further reducing the bleeding volume. The thickness ratio of the hydrophilic layer to the coagulation layer is 1:4 to 1:10.

[0038] Example 1

[0039] The double-layer chitosan hemostatic sponge in this example is prepared by the following method:

[0040] Weigh 0.5 g of chitosan raw material and add it to 100 ml of 0.5% acetic acid solution. After stirring and completely dissolving, a chitosan solution is obtained. Take out 20 ml of the chitosan solution, add 0.04 g of Tween 80 to it, and after complete dissolution, a hydrophilic chitosan solution is obtained. Pour the hydrophilic chitosan solution into a freeze-drying container, pre-freeze it at -18°C until it is completely frozen, then pour the remaining chitosan solution into the container and continue pre-freezing until it is completely frozen. Take out the above container and place it in a freeze-drying device. After completely removing the moisture, take it out to obtain the double-layer chitosan hemostatic sponge.

[0041] Example 2

[0042] The double-layer chitosan hemostatic sponge in this example is prepared by the following method:

[0043] Weigh 2g of chitosan raw material and add it to 100ml of 1% acetic acid solution, stir and completely dissolve to obtain a chitosan solution. Take out 10ml of the chitosan solution, add 0.01g of polyethylene glycol thereto, and after it is completely dissolved, obtain a hydrophilic chitosan solution. Pour the hydrophilic chitosan solution into a freeze drying container, prefreeze it at -30°C until it is completely frozen, pour the remaining chitosan solution into the container, and continue to prefreeze it until it is completely frozen. Take out the above container and place it in a freeze drying device, take it out after the water is completely removed, and obtain the double-layer chitosan hemostatic sponge.

[0044] Example 3

[0045] The double-layer chitosan hemostatic sponge in this embodiment is prepared by the following method:

[0046] Weigh 4g chitosan raw material and add it to 100ml 1.5% acetic acid solution, stir and completely dissolve to obtain chitosan solution. Take out 25ml chitosan solution, add 0.05g polyvinyl pyrrolidone thereto, and after it is completely dissolved, obtain hydrophilic chitosan solution. Pour the hydrophilic chitosan solution into a freeze drying container, pre-freeze it at -18°C until it is completely frozen, pour the remaining chitosan solution into the container, and continue to pre-freeze it until it is completely frozen. Take out the above container and place it in a freeze drying device, take it out after the water is completely removed, and obtain the double-layer chitosan hemostatic sponge.

[0047] Example 4

[0048] The double-layer chitosan hemostatic sponge in this embodiment is prepared by the following method:

[0049] Weigh 1g of chitosan raw material and add it to 100ml of 0.5% acetic acid solution, stir and completely dissolve to obtain a chitosan solution. Take out 15ml of the chitosan solution, add 0.03g of sodium 120,000 sulfate thereto, and after it is completely dissolved, obtain a hydrophilic chitosan solution. Pour the hydrophilic chitosan solution into a freeze drying container, pre-freeze it at -70°C until it is completely frozen, pour the remaining chitosan solution into the container, and continue to pre-freeze it until it is completely frozen. Take out the above container and place it in a freeze drying device, take it out after the water is completely removed, and obtain the double-layer chitosan hemostatic sponge.

[0050] Example 5

[0051] The double-layer chitosan hemostatic sponge in this embodiment is prepared by the following method:

[0052] Weigh 1.5g chitosan raw material and add it to 100ml 1% acetic acid solution, stir and completely dissolve to obtain chitosan solution. Take out 12ml chitosan solution, add 0.06g benzalkonium bromide thereto, and after it is completely dissolved, obtain hydrophilic chitosan solution. Pour the hydrophilic chitosan solution into a freeze drying container, pre-freeze it at -40°C until it is completely frozen, pour the remaining chitosan solution into the container, and continue to pre-freeze it until it is completely frozen. Take out the above container and place it in a freeze drying device, take it out after the water is completely removed, and obtain the double-layer chitosan hemostatic sponge.

[0053] Example 6

[0054] The double-layer chitosan hemostatic sponge in this embodiment is prepared by the following method:

[0055] Weigh 1g of chitosan raw material and add it to 100ml of 1.5% acetic acid solution, stir and completely dissolve to obtain a chitosan solution. Take out 16ml of the chitosan solution, add 0.01g of polyvinyl alcohol thereto, and after it is completely dissolved, obtain a hydrophilic chitosan solution. Pour the hydrophilic chitosan solution into a freeze drying container, pre-freeze it at -18°C until it is completely frozen, pour the remaining chitosan solution into the container, and continue to pre-freeze it until it is completely frozen. Take out the above container and place it in a freeze drying device, take it out after the water is completely removed, and obtain the double-layer chitosan hemostatic sponge.

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 1 is that no hydrophilic layer is provided in this comparative example. The remaining steps and parameters are basically the same as those in Example 1.

[0058] Comparative Example 2

[0059] The difference between this comparative example and Example 1 is that no coagulation layer is provided in this comparative example. The remaining steps and parameters are basically the same as those in Example 1.

[0060] Comparative Example 3

[0061] The difference between this comparative example and Example 1 is that the amount of Tween added in this comparative example is 0.01 g. The remaining steps and parameters are basically the same as those in Example 1.

[0062] Comparative Example 4

[0063] The difference between this comparative example and Example 1 is that the chitosan solution used to prepare the hydrophilic layer in this comparative example is 30 ml. The remaining steps and parameters are basically the same as those in Example 1.

[0064] Test Case

[0065] 1) Determination of in vitro coagulation index (BCI)

[0066] The in vitro coagulation index (BCI) of the hemostatic sponges in the examples and comparative examples was measured as follows: a sponge sample with an area of ​​1.5 cm × 1.5 cm was taken and placed in a 50 ml centrifuge tube. After being placed in a water bath at 37°C for 5 min, fresh rabbit blood (0.1 mL) was gently dripped onto the sample, and then 0.02 ml of 0.2 M CaCl was immediately added. 2 Solution, after 5 minutes, gently add 25mL of deionized water to the centrifuge tube, shake at 37℃ and 50rp / min for 5 minutes, then take out the solution and measure its Abs value at a wavelength of 545nm using a UV spectrophotometer. Set up a control: 0.1ml of blood is added to a centrifuge tube, and then add 25ml of deionized water. The Abs value measured at the same wavelength is assumed to be 100 as a reference value, and the coagulation index BCI is:

[0067] BCI = 100 × A 样品 / A 参考

[0068] The in vitro coagulation indexes of the above examples and comparative examples are shown in Table 1 below:

[0069] Table 1 In vitro coagulation index

[0070] sample BCI Example 1 18.0 Example 2 15.2 Example 3 23.8 Example 4 12.1 Example 5 16.5 Example 6 17.4 Comparative Example 1 44.5 Comparative Example 2 48.8 Comparative Example 3 36.3 Comparative Example 4 40.1

[0071] The smaller the value of the coagulation index BCI, the better the coagulation effect of the corresponding material, and vice versa. The results of Table 1 show that a double-layer chitosan hemostatic sponge with good coagulation performance can be prepared according to the formula and process in the embodiment. It can be seen from Comparative Examples 1 and 2 that the coagulation effect of the double-layer hemostatic sponge is better than that of a single coagulation layer or a hydrophilic layer. Too little content of the hydrophilic component in the hydrophilic layer, or too high a ratio of the hydrophilic layer to the coagulation layer, will lead to a decrease in coagulation performance.

[0072] 2) Rabbit ear artery hemostasis experiment

[0073] The hemostatic sponge in the embodiment and the comparative example was subjected to a rabbit ear artery hemostasis experiment. The experimental method is as follows: after using medical alcohol to disinfect the back of the rabbit ear, first press the proximal end with your hand, and transversely cut the ear artery 7 cm from the top of the ear tip. Release the pressure, and after 3 seconds of free bleeding, gently wipe the floating blood on the ear wound with sterilized gauze, and immediately press the hemostatic material on the bleeding wound, and vertically pressurize with a weight of 100 g, start timing at the same time, and gently uncover the hemostatic material after 30 seconds to observe whether bleeding continues. If the wound continues to bleed, continue to press to stop bleeding until the bleeding stops, and record the hemostasis time. After hemostasis is completed, calculate the amount of blood loss.

[0074] The rabbit ear artery hemostasis time and blood loss of the above examples and comparative examples are shown in Table 2 below:

[0075] Table 2 Rabbit ear artery hemostasis time and blood loss

[0076] sample Bleeding time Blood loss Example 1 90s 0.83g Example 2 90s 0.56g Example 3 90s 1.07g Example 4 60s 0.31g Example 5 60s 0.53g Example 6 90s 0.66g Comparative Example 1 150s 2.54g Comparative Example 2 180s 3.27g Comparative Example 3 150s 2.35g Comparative Example 4 150s 2.43g

[0077] The results in Table 2 show that the samples in the examples can achieve hemostasis within 90 seconds, and the amount of blood loss is consistent with the trend of the in vitro coagulation index, that is, the samples with good coagulation performance have less actual blood loss. The hemostasis time of each group of comparative examples is significantly prolonged, and the amount of blood loss is also greater.

[0078] The present application is prepared with chitosan as raw material. On the basis of maintaining the coagulation performance of chitosan itself, a hydrophilic layer is further introduced, so that blood can quickly penetrate into the material when in contact with the material, and rapid coagulation is achieved under the action of the upper layer of chitosan. At the same time, the material can quickly turn into a viscous gel after contact with blood, and cooperate to block the wound site. The preparation method adopted by the present invention has simple process and convenient operation, and can be used for large-scale industrial production. When in use, the hydrophilic layer fits the skin and the coagulation layer is outside.

[0079] The hydrophilic modification method of chitosan in the present invention is physical blending, which has a simple process and does not require chemical cross-linking and separation and purification operations. The present invention also has no special requirements for chitosan raw materials, and does not require water-soluble chitosan or chitosan derivatives. In addition, the chitosan sponge obtained by freeze-drying the acetic acid aqueous solution of chitosan can still adsorb a small amount of acetic acid on its chitosan skeleton, and can slowly gel after contacting with blood, which is beneficial to reducing the fluidity of wound blood and then sealing the wound.

[0080] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a hemostatic sponge, It is characterized in that The steps include: Pour the hydrophilic chitosan solution into the mold and pre-freeze it; adding chitosan solution into the mold and continuing pre-freezing to obtain a frozen hemostatic sponge; freeze-drying the frozen hemostatic sponge to completely remove water to obtain a hemostatic sponge; The hemostatic sponge comprises a hydrophilic layer and a coagulation layer, wherein the hydrophilic layer is formed by a hydrophilic chitosan solution, and the coagulation layer is formed by a chitosan solution.

2. The preparation method according to claim 1, It is characterized in that The pre-freezing temperature is -15°C to -70°C, and the pre-freezing time is 2-12 hours.

3. The preparation method according to claim 1, It is characterized in that The freeze-drying parameters are: vacuum degree below 40Pa, temperature 8-12°C maintained for 36-60h.

4. The preparation method according to claim 1, It is characterized in that The chitosan solution used to form the coagulation layer is prepared by the following steps: chitosan is mixed with acetic acid solution, and the chitosan solution is obtained after dissolving.

5. The preparation method according to claim 4, It is characterized in that The mass concentration of the acetic acid solution is 0.1% to 10%.

6. The preparation method according to claim 1, It is characterized in that The mass concentration of chitosan in the chitosan solution is 0.1% to 8%.

7. The preparation method according to claim 1, It is characterized in that The hydrophilic chitosan solution used to form the hydrophilic layer is prepared by the following steps: adding a hydrophilic substance to the chitosan solution and mixing, and dissolving the hydrophilic substance to obtain the hydrophilic chitosan solution.

8. The preparation method according to claim 7, It is characterized in that The mass concentration of the hydrophilic substance in the hydrophilic chitosan solution is 0.01% to 5%.

9. The preparation method according to claim 7, It is characterized in that The mass ratio of the hydrophilic substance to chitosan in the hydrophilic chitosan solution is 1:5 to 1:

20.

10. The preparation method according to claim 7, It is characterized in that The hydrophilic substance is one or more selected from polyethylene glycol, benzalkonium bromide, polyvinyl pyrrolidone, polyvinyl alcohol, Tween 80, poloxamer, and sodium lauryl sulfate.

11. The preparation method according to claim 1, It is characterized in that When preparing the frozen hemostatic sponge, the mass ratio of the hydrophilic chitosan solution to the chitosan solution is 1:4 to 1:10; the thickness ratio of the hydrophilic layer to the coagulation layer is 1:4 to 1:

10.

12. The preparation method according to any one of claims 1 to 11, It is characterized in that Acetic acid is adsorbed on the chitosan skeleton of the hemostatic sponge, and the hemostatic sponge can be gelled after contacting with blood.