A platelet-responsive self-gelling hemostatic material and methods of making and using the same

Abstract

The application provides a platelet-responsive self-gelation hemostatic material and a preparation method and application thereof, and belongs to the medical field. Based on a multiple cross-linking strategy of dynamic covalent bonds and non-covalent interactions, a hemostatic material capable of realizing self-gelation quickly is designed by using modified sodium alginate and hydroxylated porcine small intestinal submucosa as raw materials. The hemostatic material has good hydrophilicity and liquid absorption capacity, can realize powder-gelation transformation in a short time, form a stable physical barrier, has platelet response characteristics, can promote platelet adhesion and activation, accelerate the blood coagulation process of the body, and improve the mechanical properties and tissue adhesion of the material, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a platelet-responsive self-gelation hemostatic material and a preparation method and use thereof. BACKGROUND

[0002] Acute trauma often occurs in military training and daily life, and uncontrollable bleeding and wound infection after tissue damage are the main causes of casualties. Therefore, efficient hemostasis in first aid is crucial to improve the survival rate of patients. Powder materials have a large specific surface area and excellent liquid absorption capacity, can concentrate blood coagulation-related substances, and promote the formation of blood clots, making them an ideal choice for hemostatic materials. However, traditional hemostatic powders are easily washed away by blood flow, and cannot form a stable physical barrier on the wound surface, affecting their hemostatic ability for severe bleeding wounds.

[0003] In physiological hemostasis, the role of platelets is extremely important. They quickly "recognize" the damaged site and adhere to the exposed subendothelial collagen, activating the rapid formation of platelet hemostatic plugs, and the activation of platelets is a necessary condition for the activation of coagulation factors. Developing a platelet-responsive hemostatic powder is expected to accelerate the hemostatic process and achieve good hemostatic effect.

[0004] Patent (CN115814148B) reports a hemostatic material (AM7-CS3) prepared from sodium alginate and carboxymethyl chitosan. This material has both hydrogel and powder application forms. However, the adhesion strength of this material is not ideal, and the enhancement of blood on its gelation process and other properties has not been reported. SUMMARY

[0005] To solve the problems existing in the prior art, the purpose of the present application is to provide a platelet-responsive self-gelation hemostatic material and a preparation method and use thereof.

[0006] The present application provides a self-gelation hemostatic material, which is prepared from the following raw materials in parts by weight: boronate ester bond grafted modified oxidized alginate 1-5 parts, and hydroxylated small intestinal submucosa 0.01-1 parts.

[0007] Further, it is prepared from the following raw materials in parts by weight: boronate ester bond grafted modified oxidized alginate 1 part, and hydroxylated small intestinal submucosa 0.3 parts.

[0008] Further, the self-gelation hemostatic material is a self-gelation hemostatic hydrogel material or a self-gelation hemostatic powder material.

[0009] Further, the oxidized alginate grafted by borate ester linkage is an oxidized alginate grafted by 3-aminobenzene boronic acid, and the oxidized alginate is preferably sodium alginate; the hydroxylated small intestinal submucosa is a small intestinal submucosa modified by 3-amino-1,2-propanediol, and the small intestinal submucosa is preferably pig small intestinal submucosa.

[0010] Further, the synthesis method of the oxidized alginate grafted by borate ester linkage comprises the following steps:

[0011] (1) dissolving the alginate and an oxidizing agent in a solvent to obtain the oxidized alginate after reaction;

[0012] (2) dissolving the oxidized alginate and an activating agent in a solvent, adding a 3-aminobenzene boronic acid solution, and obtaining the oxidized alginate grafted by borate ester linkage after reaction.

[0013] Further, in step (1), the oxidizing agent is sodium periodate; the mass ratio of the alginate to the oxidizing agent is 1:0.1-0.5; the solvent of the reaction is an organic solvent; the temperature of the reaction is 10-40℃, and the time is 4-8 hours.

[0014] Preferably, the mass ratio of the alginate to the oxidizing agent is 1:0.25; the solvent of the reaction is anhydrous ethanol; the temperature of the reaction is 20-30℃, and the time is 6 hours.

[0015] Further, after the reaction in step (1) is completed, dialysis treatment is needed, the dialysis bag has a molecular weight cut-off of 2-5KDa; the dialysis is performed in deionized water for 2-4 days, and the water is changed 2-4 times per day.

[0016] Preferably, the dialysis bag has a molecular weight cut-off of 3.5KDa; the dialysis is performed in deionized water for 3 days, and the water is changed 3 times per day.

[0017] In step (2), the activating agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; the solvent of the reaction is an inorganic solvent; the mass ratio of the oxidized alginate, the activating agent and 3-aminobenzene boronic acid is 1:1-5:1-3; the solvent in the 3-aminobenzene boronic acid solution is an organic solvent; the temperature of the reaction is 10-40℃, and the time is 20-30 hours.

[0018] Preferably, in step (2), the solvent of the reaction is water; the mass ratio of the oxidized alginate, the activating agent and 3-aminobenzene boronic acid is 1:3:2; the solvent in the 3-aminobenzene boronic acid solution is anhydrous ethanol; the temperature of the reaction is 20-30℃, and the time is 24 hours.

[0019] Further, the synthesis method of the hydroxylated small intestinal submucosa comprises the following steps:

[0020] (i) reacting small intestinal submucosa, acid and enzyme to obtain small intestinal submucosa digestion solution;

[0021] (ii) dissolving small intestinal submucosa digestion solution, 3-amino-1,2-propanediol and activating agent in solvent, and after reaction, the hydroxylated small intestinal submucosa is obtained.

[0022] Further, in step (i), the acid is hydrochloric acid; the enzyme is pepsin; the mass ratio of small intestinal submucosa to enzyme is 1:0.05-0.3; the reaction temperature is 20-30℃, and the reaction time is 20-100 hours.

[0023] Preferably, in step (i), the mass ratio of small intestinal submucosa to enzyme is 1:0.01-0.25, preferably 1:0.2; the reaction temperature is 25℃, and the reaction time is 24-96 hours, preferably 72 hours.

[0024] Further, before the reaction in step (i) starts, the small intestinal submucosa needs to be powdered and sieved, and the sieve size is 100-300 mesh; in step (i), the pH needs to be adjusted, and the pH range is 1-3.

[0025] Preferably, in step (i), the sieve size is 200 mesh; and the pH range is 2.

[0026] In step (ii), the activating agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; the mass ratio of small intestinal submucosa to 3-amino-1,2-propanediol is 1:0.5-6; the molar ratio of 3-amino-1,2-propanediol to activating agent is 1:0.5-5; the solvent is inorganic solvent; the reaction temperature is 10-40℃, and the reaction time is 20-30 hours.

[0027] Further, in step (ii), the mass ratio of small intestinal submucosa to 3-amino-1,2-propanediol is 1:1-5, preferably 1:3; the molar ratio of 3-amino-1,2-propanediol to activating agent is 1:1-3, preferably 1:1.5; the solvent is water; the pH is adjusted to 5-6 by using inorganic sodium salt solution or inorganic acid solution; the reaction temperature is 20-30℃, and the reaction time is 24 hours.

[0028] Further, in step (ii), the pH needs to be adjusted, and the pH is adjusted to 4-7 by using alkali solution or acid solution.

[0029] The application further provides a method for preparing the self-gelling hemostatic material, which comprises the following steps: dissolving the boronate ester bond grafted modified oxidized alginic acid and the hydroxylated small intestinal submucosa in a solvent respectively, mixing the boronate ester bond grafted modified oxidized alginic acid solution and the hydroxylated small intestinal submucosa solution, adjusting the pH value, and forming a self-gelling hemostatic hydrogel material; or the method further comprises the following steps: freeze-drying and grinding the hydrogel to obtain a self-gelling hemostatic powder material.

[0030] Further, the solvent is an inorganic solvent or an organic solvent; the volume ratio of the boronate ester bond grafted modified oxidized alginic acid solution and the hydroxylated small intestinal submucosa solution is 1:0.05-2.5; and the pH value is adjusted to a range of 6-10.

[0031] Further, the solvent is a phosphate buffer solution, water or normal saline, preferably, the solvent is a phosphate buffer solution; the volume ratio of the boronate ester bond grafted modified oxidized alginic acid solution and the hydroxylated small intestinal submucosa solution is 1:0.1-2, preferably 1:1; and the pH value is adjusted to a range of 7-9, preferably 8.

[0032] The application further provides the use of the self-gelling hemostatic material in the preparation of a hemostatic material.

[0033] Compared with the patent (CN115814148B), the application has the following beneficial effects:

[0034] (1) The adhesion strength of the AP / SIS-OH hydrogel of the application is 80.07 KPa, while the adhesion strength of the AM7-CS3 hydrogel is about 9.50 KPa, so the AP / SIS-OH hydrogel of the application has better adhesion strength;

[0035] (2) When the AP / SIS-OH of the application is used as a medium for triggering gelation, the adhesion strength is increased to 109.7 KPa, while there is no related report on the AM7-CS3 hemostatic powder, so the AP / SIS-OH of the application has the performance of platelet activation-mediated adhesion enhancement;

[0036] (3) The burst pressure of the AP / SIS-OH hemostatic powder of the application is 154.90 mmHg, while the burst pressure of the AM7-CS3 hemostatic powder is 129.00 mmHg, which basically meets the application in the case of first-degree hypertension (systolic pressure 140-159 mmHg, diastolic pressure 90-99 mmHg), so the AP / SIS-OH hemostatic powder of the application has better burst pressure.

[0037] In summary, the present application provides a platelet-responsive self-gelation hemostatic material, a preparation method and use thereof. Based on the multi-crosslinking strategy of dynamic covalent bond and non-covalent interaction, the present application designs a hemostatic material capable of realizing self-gelation by using the sodium alginate grafted with phenylboronic acid and the pig small intestinal submucosa after hydroxylation as raw materials. The hemostatic material has good hydrophilicity and liquid absorption capacity, can realize powder-gelation transformation in a short time, form a stable physical barrier, has platelet-responsive characteristics, can promote platelet adhesion and activation, accelerate the blood coagulation process of the body, and improve the mechanical properties and tissue adhesion of the material, and has good application prospect.

[0038] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.

[0039] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as the scope of the above subject matter of the present application being limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 (a) synthesis schematic diagram and (b-c) nuclear magnetic hydrogen spectrum of ADA-PBA and SIS-OH.

[0041] Figure 2 Infrared spectrum of AP, AP / SIS and AP / SIS-OH.

[0042] Figure 3 Microscopic surface morphology of AP / SIS-OH powder.

[0043] Figure 4 AP / SIS-OH hemostatic powder and Celox TM Absorption of hemostatic powder on (a) PBS and (b) anticoagulant whole blood; (c) liquid absorption rate of different hemostatic powders on PBS and blood; (d) photo of AP / SIS-OH powder realizing powder-gelation transformation quickly (****p<0.0001).

[0044] Figure 5 (a) stress-strain curve and (b) compression strength of AP, AP / SIS and AP / SIS-OH hemostatic powder after realizing gelation transformation; (c) rheological evaluation and (d) photo of self-healing characteristics of AP / SIS-OH (**p<0.01, ns: no significant difference).

[0045] Figure 6(a) CCK-8 results and (b) live / dead staining results of BRL cells treated with AP, AP / SIS and AP / SIS-OH on days 1, 3 and 5 (scale bar = 200 μm) (ns: no significant difference).

[0046] Figure 7 SSC-A and CD62p of platelets after different treatments + (a) Flow cytometry results and (b, c) corresponding statistical results; ELISA results of platelet cell membrane (d) P-selectin and (e) GP IIb / IIIa expression levels after 1 and 5 min of different material treatments (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference).

[0047] Figure 8 The results included (a) the sealing experiment of AP / SIS-OH on damaged pig lungs and stomach; (b) the puncture strength of different groups of hemostatic powder; (c) the rheological properties of materials after PRP-triggered gelation of different hemostatic powders; and (d) scanning electron microscopy images of the AP / SIS-OH group (low magnification: scale bar = 30 μm; high magnification: scale bar = 10 μm); and (e) the tissue adhesion strength of AP / SIS-OH after contact with PBS, PPP, PRP and anticoagulated whole blood (*p<0.05, **p<0.01, ***p<0.001, ns: no significant difference).

[0048] Figure 9 (a) Images of the hemostasis process of different materials applied to the rabbit liver hemorrhage model and (b) images of filter paper after hemostasis; (c, d) images of bleeding amount and hemostasis time after hemostasis with different materials (*p<0.05, **p<0.01, ***p<0.001).

[0049] Figure 10 (a) Images of the hemostasis process of AP / SIS-OH hemostatic powder applied to a rabbit cardiac perforation hemorrhage model; (b) HE staining results of its adhesion to the heart surface; (c) the sealing and hemostatic effect of AP / SIS-OH hemostatic powder on femoral artery perforation hemorrhage in rabbits; (d) bleeding status and (e) bleeding volume (**p<0.01). Detailed Implementation

[0050] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0051] The "room temperature" condition referred to in this invention is 25±5℃.

[0052] Example 1: Preparation of platelet-responsive self-gelling hemostatic material

[0053] 1. Synthesis of modified sodium alginate

[0054] (1) Sodium alginate was uniformly dispersed in anhydrous ethanol at a ratio of 2% (unit g / mL) with continuous stirring to obtain a dispersion system of sodium alginate / anhydrous ethanol. NaIO4 was dissolved in water with the same volume as anhydrous ethanol to obtain a NaIO4 solution. The mass of NaIO4 was 0.25 times that of sodium alginate.

[0055] (2) The NaIO4 solution was added dropwise to the dispersion system of sodium alginate / anhydrous ethanol under light-proof conditions, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, 15 mL of ethylene glycol was added and stirred for 30 min to terminate the reaction.

[0056] (3) The reaction product was loaded into a dialysis bag (molecular weight cut-off 3.5 KDa) and dialyzed in deionized water for 3 days, with water changed 3 times a day. After dialysis, freeze-drying was performed to obtain a sample (referred to as an ADA sample).

[0057] (4) The ADA sample was completely dissolved in water at a concentration of 0.5% (unit g / mL), and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added and stirred until completely dissolved. The molar ratio of ADA to EDC was 1:3.

[0058] (5) 3-Aminobenzeneboronic acid (PBA) was dissolved in anhydrous ethanol and added dropwise to the ADA solution. The pH was adjusted to 7, and the reaction was carried out at room temperature for 24 h. The molar ratio of ADA to PBA was 1:2.

[0059] (6) After the reaction was completed, the dialysis and freeze-drying steps were carried out synchronously as in step (3) to obtain a modified sodium alginate sample (referred to as an ADA-PBA sample).

[0060] 2. Synthesis of hydroxylated porcine small intestinal submucosa

[0061] (1) Preparation of porcine small intestinal submucosa (SIS) digestion solution: The decellularized SIS was ground into powder using a ball mill, and the powder was passed through a 200-mesh sieve to obtain SIS powder. 0.3 g of SIS powder was added to 30 mL of hydrochloric acid solution (pH = 2), and 50 mg of pepsin was added. The mixture was stirred at 25°C for 72 h to obtain a SIS digestion solution.

[0062] (2) 3-Amino-1,2-propanediol (APD, 0.9 g) was dissolved in 10 mL of water to obtain an APD solution.

[0063] (3) 30 mL of SIS digestion solution (containing SIS 0.3 g) was added to 150 mL of water. After the solution was dissolved and diluted, the APD solution was added dropwise. Then, EDC was added to the reaction system, and the amount of EDC was 1.5 times of that of APD. After the pH of the system was adjusted to 5-6 by NaOH solution or HCl solution, the system was reacted at room temperature for 24 h.

[0064] (4) After the reaction was completed, the reaction product was loaded into a dialysis bag (with a molecular weight cut-off of 3.5 KDa), and dialyzed in deionized water for 3 days, with water changed 3 times a day. After dialysis, the hydroxylated porcine small intestinal submucosa sample (referred to as SIS-OH sample) was obtained by freeze-drying.

[0065] 3. Preparation of platelet-responsive self-gelling hemostatic powder

[0066] (1) The freeze-dried ADA-PBA was dissolved in phosphate buffered saline (PBS) at a mass volume ratio (g / mL) of 10%, and was ready for use.

[0067] (2) The freeze-dried SIS-OH was dissolved in phosphate buffered saline (PBS) at a mass volume ratio (g / mL) of 3%, and was ready for use.

[0068] (3) The solutions in steps (1) and (2) were mixed at a volume ratio of 1:1, and the pH value was adjusted to 8, thereby forming the final hydrogel (referred to as AP / SIS-OH hydrogel).

[0069] (5) After the AP / SIS-OH hydrogel was freeze-dried, a hemostatic powder (referred to as AP / SIS-OH hemostatic powder) was prepared by physical grinding method.

[0070] The following comparative examples were prepared for comparison with the samples.

[0071] Comparative Example 1, preparation of hemostatic powder

[0072] The freeze-dried ADA-PBA of Example 1 was dissolved in phosphate buffered saline (PBS) at a mass volume ratio (g / mL) of 10%, and the pH value was adjusted to 8, thereby obtaining a hydrogel. After freeze-drying, a hemostatic powder was obtained by physical grinding method.

[0073] The hemostatic powder is referred to as AP hemostatic powder.

[0074] Comparative Example 2, preparation of hemostatic powder

[0075] According to the method of Reference Example 1, the only difference is that the SIS-OH is replaced by untreated porcine small intestinal submucosa (SIS) digestion solution, and the amount of SIS is the same as that of SIS-OH in Example 1, and the other conditions are the same, thereby obtaining a hemostatic powder. The hemostatic powder is referred to as AP / SIS hemostatic powder.

[0076] The beneficial effects of the present application are demonstrated below by experimental examples.

[0077] Experimental Example 1, Structure characterization of hemostatic powder

[0078] 1. Experimental method

[0079] The successful synthesis of ADA-PBA and SIS-OH was verified by nuclear magnetic resonance spectrum.

[0080] The chemical structure of AP, AP / SIS and AP / SIS-OH was characterized by Fourier transform infrared absorption spectrometer.

[0081] The results ( Figure 1 ) show that there are proton peaks at δ = 7-8 ppm belonging to the hydrogen atoms of benzene ring, proving the successful grafting of PBA ( Figure 1 b); a strong proton peak is formed at δ = 2.81 ppm, which belongs to the methylene contained in APD, proving the successful hydroxylation of SIS ( Figure 1 c).

[0082] It can be seen from Figure 2 that compared with the infrared spectrum of AP, AP / SIS has a new absorption peak at 3372 cm -1 , which is a characteristic absorption peak of hydroxyl group, because SIS contains substances containing hydroxyl groups. After the reaction of AP and SIS-OH, a clear B-O bond absorption peak appears at 1376 cm -1 , because after the modification of SIS with two hydroxyl groups, more borate ester bonds can be formed with ADA-PBA. Further, the absorption peak of the hydroxyl group (3372 cm -1 ) is stronger than that of the hydrogen bond (3281 cm -1 ), indicating that the hydroxyl group reacts more with the dihydroxyphenyl boron group, reducing the hydrogen bonds formed between free hydroxyl groups. The experimental results show that ADA-PBA and SIS-OH have been successfully synthesized, and the hydroxyl group in AP / SIS-OH forms a borate ester bond with the dihydroxyphenyl boron group.

[0083] Experimental Example 2, Performance test of hemostatic powder

[0084] 1. Experimental method

[0085] (1) The microstructure of AP / SIS-OH powder was observed by atomic force microscope, and the test method was as follows: AP / SIS-OH powder was evenly spread on a glass substrate, and was gelled with PBS and PRP respectively, and was stabilized at 37℃ for 5 min.

[0086] (2) Liquid Absorption Capacity Test: One of the outstanding advantages of powdered hemostatic materials is their large specific surface area, which allows for rapid liquid absorption. Therefore, the liquid absorption capacity of AP / SIS-OH was evaluated using a static water contact angle meter, with commercial hemostatic powder Celox as a benchmark. TM Zeolite and Yunnan Baiyao hemostatic powder (YB) were used as reference standards.

[0087] (3) Mechanical property test: The mechanical properties of AP / SIS-OH were determined by uniaxial compression test.

[0088] (4) Self-healing performance test: The self-healing properties of AP / SIS-OH were evaluated using the step-by-step strain scanning mode of a rheometer.

[0089] (5) Procoagulant Activity Assay: Platelet P-selectin (CD62p) and glycoprotein GP IIb / IIIa are considered markers of platelet activation. When platelets are activated, the expression site of P-selectin rapidly changes from the α-granule membrane surface to the cell membrane surface, subsequently mediating the adhesion of granulocytes and monocytes to platelets. Similarly, in the resting state, platelet GP IIb and GP IIIa are independent glycoproteins. After activation, GP IIb and GP IIIa form a heterodimer, GP IIb / IIIa. It is a receptor for fibrinogen and von Willebrand factor (vWF), mediating platelet aggregation. Therefore, procoagulant activity is evaluated through a platelet activation assay. First, AP, AP / SIS, and AP / SIS-OH are incubated with platelets, and platelet granule size and CD62p expression are analyzed by flow cytometry. + The expression of SSC-A was analyzed, and then the fluorescence signal intensity of SSC-A was statistically analyzed.

[0090] (6) Animal Hemostatic Efficacy Test: A rabbit liver defect hemorrhage model was used to evaluate the hemostatic effect of the adhesive hydrogel. A 10 mm diameter and 5 mm deep notch was created on the rabbit liver using a skin biopsy device, and the material was immediately placed over the notch. The bleeding process was recorded until complete hemostasis was achieved. In addition to the AP, AP / SIS, and AP / SIS-OH groups, clinical fibrin glue (FB) and Celox were used. TM The hemostatic powder served as the positive control group, while the group without hemostatic treatment served as the blank control group. The effectiveness of the material in controlling massive hemorrhage was evaluated using rabbit cardiac perforation and femoral artery perforation models.

[0091] 2. Experimental Results

[0092] from Figure 3It can be seen that in the PBS group, the gel exhibits a flat and uniform morphology; while in the PRP group, a fibrous network structure was observed on the gel surface, proving that AP / SIS-OH powder activates platelets and, together with fibrinogen, constitutes a fibrin network.

[0093] result( Figure 4 a) and 4b) show that within 1 second of contact with AP / SIS-OH, droplets of PBS / anticoagulated whole blood were rapidly absorbed. However, within the same timeframe, Celox... TM No obvious liquid absorption was observed. Then, the liquid absorption rate of different materials was further measured. The results are as follows: Figure 4 As shown in c, Celox after 300 seconds TM The PBS absorption rates of Zeolite and YB were 321.2%, 287.9%, and 272.8%, respectively, while the PBS absorption rates of AP, AP / SIS, and AP / SIS-OH were 894.1%, 889.7%, and 828.5%, respectively, significantly superior to the compared commercial hemostatic powders. Similarly, when anticoagulated whole blood was used as the absorbent, the absorbent rates of AP, AP / SIS, and AP / SIS-OH remained significantly higher than the other three groups. These results demonstrate the excellent hydrophilicity and absorbent capacity of AP, AP / SIS, and AP / SIS-OH materials. Figure 4 d illustrates the powder-gel transformation process of AP / SIS-OH. After absorbing liquid, the powder rapidly transforms into a gel-like substance within 3 seconds based on multiple covalent / non-covalent crosslinking methods.

[0094] Figure 5 The results showed that AP, AP / SIS, and AP / SIS-OH hydrogels exhibited similar properties, with no obvious stress abrupt change points during compression, proving that the materials were not damaged. The compressive strengths of AP, AP / SIS, and AP / SIS-OH hydrogels were 50.5 kPa, 59.8 kPa, and 92.5 kPa, respectively. Figure 5 b) AP / SIS-OH hydrogel has the best mechanical properties. Figure 5 The results showed that when 300% strain was applied to the AP / SIS-OH hydrogel, the loss modulus (G”) was greater than the storage modulus (G’), indicating that the hydrogel was destroyed. However, when a small strain (1%) was applied, the storage modulus (G’) of the hydrogel was greater than the loss modulus (G”), restoring its viscoelastic properties, which proved that the AP / SIS-OH hydrogel has good self-healing properties. Figure 5 Figure d shows that the two AP / SIS-OH hydrogels did not break even when subjected to tensile force after contact, which intuitively proves their self-healing properties.

[0095] Figure 6The results showed that the extracts of AP, AP / SIS, and AP / SIS-OH, after co-culturing with rat hepatocyte cell lines (BRL cells) for 1, 3, and 5 days, did not exhibit cytotoxicity compared to the control group, indicating that the materials did not affect cell proliferation. This was confirmed by live / dead staining results (…). Figure 6 (b) It can be seen that the field of view is almost entirely composed of live cells with green fluorescence, with very few dead cells showing red fluorescence. Furthermore, after co-culturing with the hydrogel extract, the cells maintained their elongated spindle shape and were in good condition. In conclusion, AP, AP / SIS, and AP / SIS-OH hydrogels did not affect cell proliferation and activity, indicating good cell compatibility.

[0096] Figure 7 In the results of adjuvant therapy (AD), forward scatter (FSC) and side scatter (SSC) parameters can reflect cell size and complexity, as well as platelet aggregation. Statistical analysis of the fluorescence signal intensity of SSC-A showed that AP / SIS and AP / SIS-OH significantly promoted platelet aggregation. In the control group, i.e., at rest, the intensity of platelet SSC-A was 1.33 × 10³, while the AP, AP / SIS, and AP / SIS-OH groups were 1.69, 3.47, and 3.43 times higher, respectively. Furthermore, the introduction of SIS and hydroxylated SIS components greatly promoted platelet aggregation. The platelet activation marker CD62p was further analyzed. + Statistical analysis showed that SIS possesses procoagulant activity that promotes platelet activation. Furthermore, P-selectin and glycoproteins GP IIb / IIIa on the platelet cell membrane surface were extracted and quantified using ELISA, with results as follows: Figure 7 As shown in e and 7f, significant differences in P-selectin and GP IIb / IIIa levels were observed between the different groups 1 min after platelet contact with the material. These differences were similar to those observed after 5 min of incubation, indicating that the reaction between AP, AP / SIS, and AP / SIS-OH and platelets occurs within a very short time. At 1 min of incubation, the P-selectin content in the control group was 9.5 pg / mL, while the contents in the AP, AP / SIS, and AP / SIS-OH groups were 1.01, 1.50, and 1.63 times higher, respectively. Similarly, the GP IIb / IIIa content in the control group was 458.3 U / mL, while the contents in the AP, AP / SIS, and AP / SIS-OH groups were 1.75, 2.52, and 3.10 times higher, respectively. This suggests that AP has a relatively weak ability to activate platelets, while the addition of the SIS component significantly promoted the platelet activation process, demonstrating its procoagulant activity.

[0097] Effective sealing of the wound is very important in the application of hemostatic materials. The hemostatic powder prepared in this study exhibits a rapid powder-gel transformation property, and a gel barrier with good mechanical properties is quickly formed in a very short time. For example Figure 8 a, its wound sealing ability was first verified by sealing experiments on damaged lungs and stomachs, and air and water were respectively injected into them to check the airtightness. No rupture occurred during the subsequent injection of air and water after the damaged organs were sealed with AP / SIS-OH hemostatic powder, which can meet the normal filling and expansion of the organs. Further, the burst pressures of the Celox TM , AP, AP / SIS and AP / SIS-OH groups were measured, which were 47.06 mmHg, 88.69 mmHg, 95.81 mmHg, 154.9 mmHg( Figure 8 b). Generally, the normal systolic pressure of an adult should be less than 120 mmHg, so AP / SIS-OH can meet the ability to resist normal blood pressure and has application value in heart and artery hemostatic sealing.

[0098] As mentioned earlier, the addition of the SIS component makes the material have good procoagulant activity. This means that the material has the ability to quickly activate the coagulation system and induce the rapid formation of blood clots. Therefore, the rheological properties of the AP, AP / SIS and AP / SIS-OH groups were evaluated when platelet-rich plasma (PRP) was used as a medium to trigger gelation. The results are shown in Figure 8 c, the storage modulus of AP, AP / SIS and AP / SIS-OH groups all increased with time. However, the modulus increase rate of AP / SIS and AP / SIS-OH was significantly better than that of AP, indicating that blood clots were quickly formed in them, reflecting the procoagulant activity of the material. The microstructure of AP / SIS-OH after triggering by PRP was evaluated by scanning electron microscopy. As can be seen from Figure 8 d, on the basis of the typical porous structure of hydrogel, more fibrous structures appear, which are the fibrin network formed by PRP. In order to further explore the effect of different gelation conditions on the tissue adhesion properties of the material, PBS, platelet-poor plasma (PPP), PRP and whole blood were selected as media to trigger the gelation of AP / SIS-OH, and the tissue adhesion strength was evaluated by lap shear test, which were 80.07 KPa, 75.80 KPa, 125.4 KPa, 109.7 KPa( Figure 8e) When PRP was used as the medium to trigger gelation, the material showed the best tissue adhesion strength, while the results of the PPP group and the PBS group were similar. The difference between PRP and PPP mainly lies in the number of platelets, which indicates that platelets have a significant impact on the adhesion performance of the material. The SIS component can promote the activation of platelets, which induces the formation of a fibrin network on the basis of the gel network of the material itself, enhancing the cohesion of the gel system. Moreover, such materials and fibrin networks do not exist in isolation, but rather form an in-situ enhanced double network structure with collagen topological structure and platelet adhesion sites in SIS as the connecting points, embodying the platelet activation-mediated adhesion enhancement performance.

[0099] From the experimental results( Figure 9 a-b), it can be seen that the area of blood on the filter paper in the blank control group is the largest, while the remaining groups show obvious hemostatic effect, among which the AP / SIS-OH group is the smallest. From Figure 9 c-d, the quantitative statistical results of the bleeding amount and the hemostatic time also show that the average bleeding amount of the blank control group is 3.19g, while the FB, Celox TM , AP, AP / SIS and AP / SIS-OH groups are 63.3%, 66.0%, 53.9%, 37.5% and 22.6% of the control group, respectively, which are significantly smaller than the control group. The AP / SIS-OH group has the least bleeding amount (0.72g) and the shortest hemostatic time (138.5s), and the hemostatic effect is the best, which is better than the commercial control fibrin glue and Celox TM hemostatic powder.

[0100] The results( Figure 10 a) show that under very high blood pressure, blood splashes out from the perforation, while AP / SIS-OH can successfully seal the gap. From the HE staining of Figure 10 b, it can also be seen that a gel barrier can be quickly formed after the application of the powder, and it adheres firmly to the surface of the heart. Similarly, AP / SIS-OH also showed excellent hemostatic effect in the rabbit femoral artery perforation bleeding model Figure 10 c-e). Compared with the gauze compression hemostasis method in the control group, the bleeding amount is significantly reduced, only 0.26g.

[0101] From the above experimental results, it can be seen that the AP / SIS-OH material of the present application exhibits excellent hydrophilicity and liquid absorption capacity, excellent mechanical properties and good self-healing characteristics, good cell compatibility, can significantly promote the aggregation of platelets, has good burst pressure, meets the ability to resist normal blood pressure, and has excellent hemostatic effect.

[0102] In conclusion, the present application provides a platelet-responsive self-gelling hemostatic material, a preparation method and uses thereof. Based on the multi-crosslinking strategy of dynamic covalent bonds and non-covalent interactions, a hemostatic material capable of rapidly realizing self-gelation is designed by using modified sodium alginate and hydroxylated porcine small intestinal submucosa as raw materials. The hemostatic material has good hydrophilicity and liquid absorption capacity, can realize powder-gelation transformation in a short time, form a stable physical barrier, has platelet-responsive characteristics, can promote platelet adhesion and activation, accelerate the coagulation process of the body, and improve the mechanical properties and tissue adhesion of the material, and has good application prospect.

Claims

1. A self-gelling hemostatic material, characterized in that, It is prepared from the following weight parts of raw materials: boronate ester bond grafted modified oxidized alginic acid salt 1~5 parts, hydroxylated small intestinal submucosa 0.01~1 parts; The synthesis method of the boronate ester bond grafted modified oxidized alginic acid salt comprises the following steps: (1) dissolve the alginic acid salt and oxidizing agent in a solvent, and obtain the oxidized alginic acid salt after reaction; (2) dissolve the oxidized alginic acid salt and activating agent in a solvent, add 3-aminobenzoic acid solution, and obtain the boronate ester bond grafted modified oxidized alginic acid salt after reaction; The synthesis method of the hydroxylated small intestinal submucosa comprises the following steps: (i) react the small intestinal submucosa, acid and enzyme to obtain small intestinal submucosa digestion solution; (ii) dissolve the small intestinal submucosa digestion solution, 3-amino-1,2-propanediol and activating agent in a solvent, and obtain the hydroxylated small intestinal submucosa after reaction.

2. The self-gelling hemostatic material according to claim 1, characterized in that, It is prepared from the following weight parts of raw materials: boronate ester bond grafted modified oxidized alginic acid salt 1 part, hydroxylated small intestinal submucosa 0.3 parts.

3. The self-gelling hemostatic material of claim 1, wherein, The self-gelling hemostatic material is a self-gelling hemostatic hydrogel material or a self-gelling hemostatic powder material.

4. The self-gelling hemostatic material according to any one of claims 1 to 3, characterized in that, The boronate ester bond is 3-aminobenzoic acid, the oxidized alginic acid salt is oxidized sodium alginic acid, the hydroxylation is modified by 3-amino-1,2-propanediol, and the small intestinal submucosa is pig small intestinal submucosa.

5. The self-gelling hemostatic material of claim 1, wherein, In step (1), the oxidizing agent is sodium periodate; the mass ratio of the alginic acid salt to the oxidizing agent is 1:0.1-0.5; the reaction solvent is an organic solvent; the reaction temperature is 10~40℃, and the reaction time is 4~8 hours; In step (2), the activating agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; the reaction solvent is an inorganic solvent; the molar ratio of the oxidized alginic acid salt, the activating agent and 3-aminobenzoic acid is 1:1-5:1-3; the solvent in the 3-aminobenzoic acid solution is an organic solvent; the reaction temperature is 10~40℃, and the reaction time is 20~30 hours.

6. The self-gelling hemostatic material of claim 1, wherein, In step (i), the acid is hydrochloric acid; the enzyme is pepsin; the mass ratio of the small intestinal submucosa to the enzyme is 1:0.05-0.3; the reaction temperature is 20~30℃, and the reaction time is 20~100 hours; In step (ii), the activating agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; the mass ratio of the small intestinal submucosa to 3-amino-1,2-propanediol is 1:0.5-6; the molar ratio of 3-amino-1,2-propanediol to the activating agent is 1:0.5-5; the solvent is an inorganic solvent; the reaction temperature is 10~40℃, and the reaction time is 20~30 hours.

7. A method of preparing the self-gelling hemostatic material according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: dissolving the boronate ester bond grafted modified oxidized alginic acid salt and the hydroxylated small intestinal submucosa in solvents respectively, mixing the boronate ester bond grafted modified oxidized alginic acid salt solution and the hydroxylated small intestinal submucosa solution, adjusting the pH, and forming a self-gelling hemostatic hydrogel material; or, the method further comprises the following steps: freeze-drying and grinding the hydrogel to obtain a self-gelling hemostatic powder material.

8. Use of the self-gelling hemostatic material according to any one of claims 1 to 6 for the preparation of a hemostatic material.

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