Purified polysaccharide hemostatic sponge and its preparation method and application

Abstract

This invention provides a pullulan polysaccharide hemostatic sponge and its preparation method. The sponge comprises a double-network structure consisting of a skeletal layer and an interpenetrating layer. The skeletal layer is formed by aldol condensation of pullulan polysaccharide, constituting the outer network; the interpenetrating layer is formed by ionic crosslinking of sodium alginate and gallium ions, constituting the inner network; the skeletal layer and the interpenetrating layer are connected by borate ester bonds, thus forming the pullulan polysaccharide hemostatic sponge. The sponge of this invention not only has high porosity and water absorption rate, enabling it to quickly absorb excess blood from the wound surface for rapid and effective hemostasis and promoting wound coagulation, but also possesses antibacterial and hemostatic properties and shape memory function, making it applicable to various environments.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a pullulan polysaccharide hemostatic sponge, its preparation method, and its application. Background Technology

[0002] In the treatment of trauma from sudden accidents and in hemostasis during clinical surgery, especially in the on-site care of wounded personnel during wartime, rapid and efficient hemostasis can effectively reduce casualties and alleviate subsequent complications. Traditional hemostatic materials are not ideal for hemostasis of irregularly shaped, deep, narrow, or ruptured arteries. Therefore, there is a need for a fast, safe, and efficient hemostatic material suitable for on-site and clinical emergency use to replace traditional hemostatic materials.

[0003] Currently reported rapid hemostatic materials mainly include powders (zeolite, collagen powder, and potato starch), oxidized regenerated cellulose, fibrin glue, and porous sponges (gelatin sponges and collagen sponges). Each of them has certain shortcomings. For example, porous zeolite and potato starch release a lot of heat after absorbing water from the blood, which can easily lead to wound inflammation; fibrin glue originates from blood and may cause viral infection; gelatin and collagen have poor tissue adhesion, and their hemostatic function depends on sufficient platelets and clotting factors. Furthermore, hemostatic sponges currently suffer from problems such as simple structure and function, poor hemostatic performance, and unclear coagulation and antibacterial mechanisms.

[0004] Therefore, it is necessary to research new hemostatic sponges with high performance and multiple properties. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a pullulan polysaccharide hemostatic sponge and its preparation method. This sponge not only has high porosity and water absorption rate, enabling it to quickly absorb excess blood from the wound surface and achieve rapid and effective hemostasis and promote wound coagulation, but also possesses antibacterial and hemostatic properties and shape memory function, making it applicable to various environments.

[0006] According to a first aspect of the present invention, a pullulan hemostatic sponge is provided, comprising a double network structure consisting of a skeletal layer and an interpenetrating layer, wherein the skeletal layer is formed by pullulan polysaccharide through aldol condensation to form an outer network; the interpenetrating layer is formed by sodium alginate and gallium ions through ionic crosslinking to form an inner network; the skeletal layer and the interpenetrating layer are connected by borate ester bonds to form the pullulan hemostatic sponge.

[0007] As an optional implementation, the strength and absorbency of the pullulan polysaccharide hemostatic sponge can be altered by adjusting the number of borate ester bonds.

[0008] According to a second aspect of the present invention, a method for preparing pullulan polysaccharide hemostatic sponge is provided, comprising the following steps:

[0009] S1. Dissolve pullulan in distilled water. After it is completely dissolved, add sodium periodate and react in the dark to obtain the first solution. Then refrigerate the first solution in a refrigerator.

[0010] S2. Thaw the first solution after refrigeration, then add borax, gallium ions and sodium alginate, mix well and adjust the pH to alkaline, stir thoroughly to achieve cross-linking reaction, and freeze dry to obtain pullulan polysaccharide hemostatic sponge.

[0011] As an optional implementation, the molar ratio of pullulan to sodium periodate is (1:0.5) to (1:3).

[0012] As an optional implementation, in step S1, the reaction time in the dark is 20 to 120 minutes, the temperature of the refrigerated container after the reaction is -4 to -160°C, and the refrigeration time is 6 to 48 hours.

[0013] As an optional implementation, the molar ratio of borax, gallium ions, and sodium alginate is (1:1:1) to (1:3:3).

[0014] As an optional implementation, in step S2, the pH is adjusted to 8-10.

[0015] As an optional implementation, pullulan has a molecular weight of 100-2000 kDa and sodium alginate has a viscosity of 200 ± 20 mPa·s.

[0016] As an optional implementation, the gallium ion is one or more of gallium nitrate, gallium sulfate, and gallium acetate.

[0017] In a third aspect of the present invention, a hemostatic dressing is provided, which is obtained by encapsulating hemostatic active molecules in the aforementioned pullulan polysaccharide hemostatic sponge.

[0018] Compared with the prior art, the significant advantages of the present invention are as follows:

[0019] The pullulan polysaccharide hemostatic sponge of the present invention comes into direct contact with the wound. Due to its high porosity and water absorption rate, it can quickly absorb excess water from the blood on the wound surface to form a gel, which seals the wound and achieves initial hemostasis. The concentrated blood penetrates the surface of the gel and enters the inner layer, where it further coagulates rapidly under the action of gallium ions, thereby achieving rapid and effective hemostasis and promoting wound coagulation. In addition, the hemostatic sponge also has a certain antibacterial function due to gallium ions.

[0020] The pullulan polysaccharide hemostatic sponge of the present invention has excellent mechanical properties and shape memory function, strong water absorption and swelling function and water retention and moisturizing performance, and can be applied to various environments.

[0021] The pullulan polysaccharide hemostatic sponge prepared by this invention has a certain amount of cavity after gallium ions are chelated with sodium alginate, which can encapsulate and release drugs.

[0022] The preparation process of this invention is simple and easy to scale up for mass production. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the pullulan polysaccharide hemostatic sponge of the present invention.

[0024] Figure 2 This is a picture of a sample from Embodiment 1 of the present invention.

[0025] Figure 3 This is a scanning electron microscope image of the skeleton layer of the sample in Example 1 of the present invention.

[0026] Figure 4 This is a scanning electron microscope image of the intercalation layer of the sample in Embodiment 1 of the present invention.

[0027] Figure 5 This is a sample drug release diagram from Example 1 of the present invention. Detailed Implementation

[0028] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0029] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0030] Combination Figure 1 As shown, the pullulan polysaccharide hemostatic sponge of the present invention includes a double network structure composed of a skeleton layer 1 and an interpenetrating layer 2. The skeleton layer is formed by pullulan polysaccharide through aldol condensation 11, forming an outer network; the interpenetrating layer is formed by sodium alginate and gallium ions through ionic crosslinking 21, forming an inner network; the skeleton layer and the interpenetrating layer are connected by borate ester bonds 3, forming the pullulan polysaccharide hemostatic sponge.

[0031] As an optional implementation, the strength and absorbency of the pullulan polysaccharide hemostatic sponge can be changed by adjusting the number of borate ester bonds. Within a certain range, as the number of borate ester bonds increases, chemical cross-linking and pores both tend to increase, thereby increasing the compressive strength and absorbency of the skeleton layer. However, when the number of borate ester bonds exceeds a certain range, it will lead to a decrease in solubility, making it difficult for the interpenetrating layer to enter the sponge network, ultimately affecting the hemostatic and absorbency properties of the sponge.

[0032] In another exemplary embodiment of the present invention, a method for preparing pullulan polysaccharide hemostatic sponge is provided, comprising the following steps:

[0033] S1. Dissolve pullulan in distilled water. After complete dissolution, add sodium periodate and react in the dark for 20-120 min, preferably 30 min, to obtain a first solution. Then, refrigerate the first solution in a refrigerator at -4 to -160°C, preferably -20°C, for 6-48 h, preferably 12 h.

[0034] Sodium periodate oxidizes the hydroxyl groups on pullulan into aldehyde groups. Then, the unoxidized hydroxyl groups on pullulan undergo aldol condensation with the oxidized aldehyde groups, thus forming the overall skeletal layer of the sponge.

[0035] S2. The first solution after refrigeration is placed at 4-25°C, preferably 25°C, for 1-8 hours, preferably 2 hours. Then, borax, gallium ions and sodium alginate are added to it, mixed evenly, and the pH is adjusted to alkaline. The cross-linking reaction is carried out for 1 hour by stirring thoroughly. The solution is then freeze-dried to obtain pullulan polysaccharide hemostatic sponge.

[0036] The sodium alginate-gallium ion system is combined with the sponge's skeletal layer by borate ester bonds formed by borax under alkaline conditions. The strength and absorbency of the hemostatic sponge can be adjusted by controlling the amount of borax to meet different needs.

[0037] As an optional implementation, the molar ratio of pullulan to sodium periodate is (1:0.5) to (1:3).

[0038] As an optional implementation, the molar ratio of pullulan to sodium alginate is 1:1.

[0039] As an optional implementation, the molar ratio of borax, gallium ions, and sodium alginate is (1:1:1) to (1:3:3).

[0040] As an optional implementation, in step S2, the pH is adjusted to 8-10.

[0041] As an optional implementation, pullulan has a molecular weight of 100-2000 kDa and sodium alginate has a viscosity of 200 ± 20 mPa·s.

[0042] As an optional implementation, the gallium ion is one or more of gallium nitrate, gallium sulfate, and gallium acetate.

[0043] As an optional implementation, the freeze-thaw process of the first solution can be performed 1 to 3 times.

[0044] In another exemplary embodiment of the present invention, a hemostatic dressing is also provided, which is obtained by encapsulating hemostatic active molecules in the aforementioned pullulan polysaccharide hemostatic sponge; wherein the hemostatic active molecules include one or more of vitamin K1, vitamin K3, vitamin K4, acetylglycine ethylenediamine, carbazoline, sodium carboxysulfonate, Yunnan Baiyao, thrombin, and Chinese and Western medicines and their extracts with hemostatic effects.

[0045] The pullulan polysaccharide hemostatic sponge of the present invention has excellent mechanical properties and shape memory function, strong water absorption and swelling function and water retention and moisturizing performance; it has certain antibacterial and hemostatic functions and can promote wound healing; it has a certain amount of cavity, which can encapsulate and release drugs; thus realizing the multifunctional integration of hemostatic sponge.

[0046] Among them, the shape memory function and strong water absorption and moisturizing properties come from the outer network formed by pullulan polysaccharide through aldol condensation;

[0047] Its antibacterial and hemostatic functions, and its ability to encapsulate drugs, come from the cross-linking of sodium alginate with gallium ions;

[0048] The excellent mechanical properties are due to the double network system formed by the introduction of borate ester bonds after the incorporation of borax.

[0049] The coagulation and antibacterial mechanism of the pullulan polysaccharide hemostatic sponge of the present invention is as follows:

[0050] Coagulation mechanism

[0051] The hemostatic sponge of this invention comes into direct contact with the wound, absorbs moisture from the blood to form a gel, seals the wound, and achieves initial hemostasis; the concentrated blood penetrates the surface of the gel and enters the inner layer, where it further and rapidly coagulates under the action of gallium ions.

[0052] Antibacterial mechanism

[0053] The hemostatic sponge of the present invention relies on gallium ions to have certain antibacterial functions. Gallium ions weaken bacterial iron metabolism through a "Trojan horse" strategy, which is a novel strategy to combat bacterial infection and is expected to become a candidate to replace antibiotics in the fight against drug-resistant bacteria.

[0054] The pullulan polysaccharide hemostatic sponge of the present invention can be used as a hemostatic material for treating combat wounds, trauma emergency hemostasis, or wound healing hemostasis.

[0055] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0056] Unless otherwise specified, all reagents used in the following examples are commercially available reagents.

[0057] Example 1

[0058] Dissolve 10.00g pullulan in 100.00mL of distilled water. After it is fully dissolved, add 4.00g of sodium periodate and react in the dark for 2 hours. Then refrigerate at -20℃ for 12 hours.

[0059] The refrigerated solution was thawed at room temperature for 2 hours. Then, 7.16g of borax, 2.53g of gallium nitrate, and 7.96g of sodium alginate were added and mixed thoroughly. The pH of the solution was adjusted to approximately 9, and the cross-linking reaction was carried out with thorough stirring for 1 hour. The solution was then freeze-dried to obtain the hemostatic dressing. Figure 2 As shown.

[0060] Example 2

[0061] Dissolve 10.00g pullulan in 100.00mL of distilled water. After it is fully dissolved, add sodium periodate and react in the dark, then refrigerate.

[0062] The refrigerated solution was thawed at room temperature for 2 hours, and then 7.16g of borax, 2.53g of gallium nitrate and 7.96g of sodium alginate were added and mixed evenly. The pH of the solution was adjusted to 9, and the cross-linking reaction was carried out for 1 hour. The solution was then freeze-dried to obtain the hemostatic dressing.

[0063] Sample number pullulan Sodium periodate reaction time Refrigeration time Refrigeration temperature 2-1 10.00g 8.00g 2h 12h -20℃ 2-2 10.00g 12.00g 2h 12h -20℃ 2-3 10.00g 8.00g 1h 12h -20℃ 2-4 10.00g 8.00g 3h 12h -20℃ 2-5 10.00g 8.00g 2h 6h -20℃ 2-6 10.00g 8.00g 2h 24h -20℃ 2-7 10.00g 8.00g 2h 48h -20℃ 2-8 10.00g 8.00g 2h 12h -4℃ 2-9 10.00g 8.00g 2h 12h -80℃ 2-10 10.00g 8.00g 2h 12h -160℃

[0064] The amount of sodium periodate affects the oxidation rate of pullulan, which in turn affects the skeleton of the hemostatic sponge. Experiments showed that sample 2-1 yielded the most complete sponge skeleton under mild reaction conditions. Therefore, in the following examples, the amount of sodium periodate used was 8.00 g, and the reaction time was 2 h.

[0065] Example 3

[0066] Dissolve 10.00g pullulan in 100.00mL of distilled water. After it is fully dissolved, add 8.00g of sodium periodate and react in the dark for 120min. Then refrigerate at -20℃ for 12h.

[0067] The refrigerated solution was thawed at room temperature for 2 hours. Then, borax, gallium nitrate, and sodium alginate were added and mixed evenly. The pH of the solution was adjusted to about 9, and the cross-linking reaction was carried out for 1 hour. The solution was then freeze-dried to obtain the hemostatic dressing.

[0068] Sample number pullulan Borax Gallium nitrate alginic acid reaction time 2-1 10.00g 7.16g 2.53g 7.96g 1h 3-1 10.00g 7.16g 5.06g 15.92g 1h 3-2 10.00g 7.16g 7.59g 23.88g 1h 3-3 10.00g 7.16g 2.53g 7.96g 2h 3-4 10.00g 7.16g 2.53g 7.96g 3h

[0069] Based on photographs and water absorption tests of samples 2-1, 3-1, and 3-2, it can be seen that as the proportions of borax, gallium nitrate, and sodium alginate increase, the number of crosslinking points inside the sponge increases, resulting in a more three-dimensional shape, a more stable skeleton, and stronger compressive strength. At the same time, the number of pores inside the sponge increases, making the sponge more absorbent. However, excessive amounts of borax, gallium nitrate, and sodium alginate can make it difficult for the interpenetrating layer to enter the sponge network, reducing the sponge's hemostatic and absorbent properties.

[0070] As can be seen from the photographs and water absorption tests of samples 2-1, 3-3, and 3-4, the reaction time has little effect on the polymer network.

[0071] Comparative Example 1

[0072] Dissolve 10.00g pullulan in 100.00mL of distilled water. After complete dissolution, add 8.00g sodium periodate and react in the dark for 120min. Then refrigerate at -20℃ for 12h and freeze-dry to obtain hemostatic dressing.

[0073] Example 4

[0074] {Characteristics of the internal and external structures of the hemostatic sponge}

[0075] Take 0.1 g of the sample obtained in Example 1, chop it, place it on a stage, and perform scanning electron microscopy to observe the morphology of its surface structure (skeleton layer) and internal structure (intercalation layer). The results are as follows. Figure 3 , 4 As shown.

[0076] from Figure 3 From this, we can see that the surface of the sponge exhibits a dense and regular structure, proving that the sponge's skeleton is complete; from Figure 4 It can be seen that the interpenetrating layer has larger pores, which can encapsulate and release drugs, and also has strong water absorption.

[0077] Example 5

[0078] {Mechanical Properties and Shape Memory}

[0079] Take 0.1g of the sample from Example 1 and test its compression performance. The specific process is as follows: place the obtained hemostatic sponge under a texture analyzer for compression testing, and the compression rate is 1mm / s.

[0080] Experimental results show that the prepared sponge has good compression-recovery function and can recover under a force of 200 kPa, proving that the hemostatic sponge of the present invention has excellent mechanical properties. At the same time, this also proves that the prepared sponge has shape memory function.

[0081] Example 6

[0082] {Water Absorption Performance Test}

[0083] Take 0.1g of the sample obtained in Example 1 and place it in a container containing 10mL of distilled water. After 5 minutes, take out the sponge and characterize its water absorption performance by testing the change in its weight.

[0084] Experimental results show that the mass of the sponge after absorbing water is 1.5g. Calculation shows that it absorbs 1.4mL of water (1.5-0.1) / 1, indicating that the hemostatic sponge of the present invention has good water absorption.

[0085] Example 7

[0086] {Drug Encapsulation and Release}

[0087] Take 0.1g of the sample obtained in Example 1 and add 0.1mL of 1mg / mL doxorubicin hydrochloride to the sponge to obtain a drug-loaded hemostatic sponge; perform a drug release test on the drug-loaded hemostatic sponge and detect the amount of drug released by ultraviolet light.

[0088] Experimental results are as follows Figure 5 As shown, the drug is rapidly released within 4 hours, proving that the hemostatic sponge of the present invention has the ability to encapsulate and release drugs.

[0089] Example 8

[0090] {Antibacterial Performance Test}

[0091] Take 0.1g of the sample obtained in Example 1, sterilize it under ultraviolet light for 30min, place the sponge on an agar plate coated with bacterial solution, and incubate it in a 37℃ incubator for 24h before observing the size of the inhibition zone.

[0092] Experimental results showed that only a small number of bacteria were present on the agar plate, proving that the hemostatic sponge of the present invention has good antibacterial properties.

[0093] Example 9

[0094] {Hemostatic Performance Test}

[0095] The hemostatic sponges prepared in Example 1 and Comparative Example 1 were used in a hemostasis experiment on femoral artery injury in rats. The femoral artery of the rats was cut with a scalpel, and the prepared hemostatic sponges were applied to the wound and pressed to stop the bleeding. The results showed that the hemostasis time was 20s and the blood loss was 0.6g, while the hemostasis time of Comparative Example 1 was 100s and the blood loss was 1.1g. This shows that the hemostatic sponge of the present invention has a better hemostatic effect.

[0096] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A pullulan polysaccharide hemostatic sponge, characterized in that, It includes a dual-network structure consisting of a skeleton layer and an interpenetrating layer. The skeleton layer is formed by aldol condensation of pullulan, forming an outer network. The interpenetrating layer is formed by ionic crosslinking of sodium alginate and gallium ions, forming an inner network. The skeleton layer and the interpenetrating layer are connected by borate ester bonds to form a pullulan hemostatic sponge. The preparation method of the pullulan polysaccharide hemostatic sponge includes the following steps: S1. Dissolve pullulan in distilled water. After complete dissolution, add sodium periodate and react in the dark to obtain a first solution. Then, refrigerate the first solution in a refrigerator. The molar ratio of pullulan to sodium periodate is (1:0.5) to (1:3). Sodium periodate oxidizes the hydroxyl groups on pullulan into aldehyde groups. Subsequently, the unoxidized hydroxyl groups in the pullulan molecular chain undergo aldol condensation with the oxidized aldehyde groups. The cross-linked structure formed by this aldol condensation reaction constitutes the skeleton layer of the hemostatic sponge. S2. Thaw the first solution after refrigeration, then add borax, gallium ions and sodium alginate, mix evenly, adjust the pH to alkaline, stir thoroughly to achieve cross-linking reaction, and freeze-dry to obtain pullulan polysaccharide hemostatic sponge; the molar ratio of borax, gallium ions and sodium alginate is (1:1:1)~(1:3:3); Specifically, the borate ester bonds formed by borax under alkaline conditions combine the interpenetrating layer of the sodium alginate-gallium ion system with the skeletal layer of the sponge to form a double network structure; at the same time, the number of borate ester bonds can be controlled by controlling the amount of borax, thereby achieving directional regulation of the mechanical strength and water absorption properties of the hemostatic sponge material.

2. The pullulan polysaccharide hemostatic sponge according to claim 1, characterized in that, In step S1, the reaction time in the dark is 20~120 min, the temperature of the refrigerated container after the reaction is -160~-4℃, and the refrigeration time is 6~48 h.

3. The pullulan polysaccharide hemostatic sponge according to claim 1, characterized in that, In step S2, adjust the pH to 8-10.

4. The pullulan polysaccharide hemostatic sponge according to claim 1, characterized in that, Pullulan has a molecular weight of 100-2000 kDa, and sodium alginate has a viscosity of 200±20 mPa·s.

5. The pullulan polysaccharide hemostatic sponge according to claim 1, characterized in that, Gallium ions are one or more of gallium nitrate, gallium sulfate, and gallium acetate.

6. A hemostatic dressing, characterized in that, The hemostatic active molecules were obtained by encapsulating pullulan polysaccharide hemostatic sponges according to any one of claims 1-5.

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