A water-sensitive in-situ ultrafast self-gelling hemostatic powder and a preparation method and use thereof

Abstract

The present application provides a kind of water-sensitive in-situ ultrafast self-gelling hemostatic powder, which is prepared by mixing and low-temperature grinding the following raw materials in the following weight ratio: grafted mercapto gelatin 1-2 parts, grafted catechol group hyaluronic acid 1-2 parts.The present application also provides a preparation method and use of the water-sensitive in-situ ultrafast self-gelling hemostatic powder.The hemostatic powder combines the characteristics of rapid water absorption, strong adhesion and biocompatibility, making it an ideal candidate for a wide range of medical applications, including hemostasis, wound sealing and tissue regeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to a water-sensitive in-situ ultrafast self-gelling hemostatic powder, belonging to the field of biomaterials. BACKGROUND

[0002] Uncontrolled bleeding after severe tissue trauma is one of the main lethal causes of military and civilian casualties. Timely hemostasis and suture of the wound is the key to reducing the amount of bleeding and effectively improving the survival rate. Conventional methods such as suturing and anastomat are clinically used as the gold standard for wound closure and hemostasis, but due to preoperative anesthesia, long operation time, secondary tissue damage, etc., they are not suitable for on-site first aid treatment of trauma. In addition, bleeding and tissue damage caused by infection, such as surgical bleeding of burns, have anti-infection and regeneration requirements. Therefore, in order to meet the increasingly urgent clinical needs, it is necessary to develop a multifunctional hemostatic material that can quickly stop bleeding, adhere well, has good mechanical properties as a cover for the hemostatic site to maintain blood pressure, meets the requirements of biocompatibility, has good anti-infection ability and promotes healing.

[0003] The main hemostatic materials at present are gauze, sponge, hydrogel and powder. Traditional gauze and sponge mainly stop bleeding by absorbing the liquid in the blood to concentrate the clotting factors. Various hemostatic hydrogels based on natural or synthetic materials are developed for use in incompressible, irregular, deep, narrow or perforated wounds due to their excellent fluidity and shape plasticity. Notably, hemostatic powder composed of tiny particles not only can completely cover and penetrate into irregular, narrow, deep, incompressible bleeding wounds to achieve rapid hemostasis in a short time, but also can quickly hydrate and form a gel in situ to achieve wound sealing and anti-pollutant purposes. At present, many self-gelling powders have recently been developed to quickly form in-situ hydrogels after hydration, effectively sealing bleeding wounds. For example, Bian et al. reported a super-fast self-gel wet-adhesive polyethyleneimine / polyacrylic acid / quaternized chitosan (PEI / PAA / QCS) powder as a hemostatic material and wound dressing. The PEI / PAA / QCS powder deposited on the bleeding wound can quickly absorb a large amount of blood and concentrate the clotting factors. At the same time, the powder can form an adherent hydrogel in situ within 4s after hydration, forming a pressure-resistant physical barrier.

[0004] Frequent replacement of gauze and compression of bleeding sites limits its application in incompressible internal bleeding (such as heart, liver, brain bleeding). Although adhesive hydrogels have developed rapidly, the problem of wet adhesion performance to achieve rapid adhesion and hemostasis of tissues under flowing blood still needs to be solved.

[0005] Application No. 202210558775.5, Invention Name: A hemostatic antibacterial healing-promoting hydrogel with real-time monitoring function and preparation method, a hemostatic antibacterial healing-promoting hydrogel with real-time monitoring function and preparation method, using natural polysaccharide as the main raw material, the polysaccharide is sequentially quaternized, double-bonded and polyphenol grafted modified, and inorganic nanoparticles and chromogenic substances are compounded to obtain a composite hydrogel. The obtained hydrogel can be injected into the bleeding or wound site by a syringe, and can rapidly gel under light, which can realize real-time monitoring of the inflammatory response of the wound site, and can promote hemostasis and wound healing at the bleeding site. The patent document uses polyphenol grafted double bond quaternary ammonium polysaccharide to load inorganic nano materials, and the hydrogel dressing is formed by light-induced crosslinking. It needs to prepare a pre-polymer solution, and then light crosslinking, the application conditions are complex, and it cannot achieve hemostasis in cases of massive bleeding, etc. Because it contains a large amount of water itself, the amount of blood absorption is limited, and due to the existence of interfacial water, the tissue adhesion is usually poor, and effective sealing cannot be achieved.

[0006] Application No. CN202310516735.9, Invention Name: Preparation method and application of body temperature-induced adhesive gelatin-based hydrogel, discloses a preparation method and application of body temperature-induced adhesive gelatin-based hydrogel, belonging to the technical field of biomedical high polymer materials. The patent is to crosslink natural polysaccharide grafted with mercapto and gelatin grafted with mercapto with silver ions and load natural active small molecules to finally obtain a multifunctional hydrogel. The hydrogel is mainly used for wound healing. The gel is constructed by a double network, one is a coordination network crosslinked by silver ions, and the other is a physical crosslinking network of gelatin; among them, the gelatin network has strong tissue adhesion at body temperature (37℃), but has no tissue adhesion at low temperature (5℃), so the hydrogel dressing can be conveniently removed as needed. The patent discloses a silver ion crosslinked polysaccharide and gelatin loaded with active small molecules through a thiol mechanism, and a hydrogel dressing is formed by light-induced crosslinking. The hydrogel dressing realizes wound regeneration, and only serves as a wound dressing, and cannot achieve the function of stopping bleeding in wounds. In addition, the prepared hydrogel cannot achieve hemostasis in cases of massive bleeding, etc. Because it contains a large amount of water itself, the amount of blood absorption is limited, and due to the existence of interfacial water, the tissue adhesion is usually poor, and effective sealing cannot be achieved.

[0007] The current light hemostatic powder still cannot firmly adhere to the wound with severe bleeding to achieve complete sealing effect, and may even dissolve or diffuse into the blood, increasing the risk of vascular embolism. In addition, the main component of the hemostatic powder is a synthetic polymer, which has the characteristics of long degradation time and lack of biological activity. SUMMARY

[0008] The technical scheme of the present application provides a water-sensitive in-situ ultrafast self-gelling hemostatic powder, which is a multifunctional biomimetic hemostatic powder, especially suitable for incompressible internal hemostasis.

[0009] The present application provides a water-sensitive in-situ super-fast self-gelling hemostatic powder, which is prepared by mixing and low-temperature grinding the following raw materials in the following proportions:

[0010] grafted mercapto gelatin 1-2 parts, grafted catechol group hyaluronic acid 1-2 parts.

[0011] Preferably, it is prepared by mixing and low-temperature grinding the following raw materials in the following proportions:

[0012] grafted mercapto gelatin 1 part, grafted catechol group hyaluronic acid 1 part.

[0013] The grafted mercapto gelatin is prepared from gelatin and cysteine hydrochloride, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts.

[0014] The grafted catechol group hyaluronic acid is prepared from hyaluronic acid and dopamine, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts.

[0015] Preferably, in the preparation method of the grafted mercapto gelatin, the mass ratio of gelatin, cysteine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is:

[0016] gelatin 1 part, cysteine hydrochloride 1.86 parts, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) 4.81 parts, N-hydroxysuccinimide (NHS) 2.93 parts;

[0017] In the preparation method of the grafted catechol group hyaluronic acid, the mass ratio of hyaluronic acid, dopamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is:

[0018] hyaluronic acid 1 part, dopamine 1.2 parts, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) 1.8 parts, N-hydroxysuccinimide (NHS) 0.73 parts.

[0019] The preparation method of the grafted mercapto gelatin is as follows:

[0020] The gelatin is completely dissolved in deionized water at 60℃, and then cooled to room temperature, and then EDC, NHS and cysteine hydrochloride are added in sequence, the pH value of the solution is adjusted to 5 using 1M sodium hydroxide and hydrochloric acid, and the reaction is carried out overnight; finally, the purified dialysate is centrifuged to remove insoluble impurities, and the supernatant is freeze-dried, to obtain the grafted thiol gelatin;

[0021] The preparation method of the hyaluronic acid grafted with catechol groups is as follows:

[0022] The HA is dissolved in deionized water, and then EDC and NHS are added, and the pH value is kept at 4.75-5.0; under the protection of nitrogen, dopamine DOPA-HCl dissolved in deionized water is added to the reaction mixture, and the pH value is kept at 4.75-5.0 for 12 hours; then, the reaction solution is dialyzed with deionized water for three days to remove unreacted reagents and by-products; finally, the solution is freeze-dried to obtain the hyaluronic acid grafted with catechol groups.

[0023] The application provides a preparation method of the water-sensitive in-situ ultrafast self-gelling hemostatic powder.

[0024] a. weigh the grafted thiol gelatin and the hyaluronic acid grafted with catechol groups according to the weight ratio;

[0025] b. mix, add into a low-temperature grinder, and grind, to obtain the product.

[0026] The grinding condition is that the frequency is 30 Hz and the grinding time is 15 minutes.

[0027] The application provides the use of the water-sensitive in-situ ultrafast self-gelling hemostatic powder in the preparation of an external medicine with hemostatic effect.

[0028] The water-sensitive in-situ super-fast self-gelling hemostatic powder of the present application can form a chemically cross-linked hydrogel in-situ by absorbing interfacial water within 5 seconds, and has excellent blood absorption. The thiol and catechol groups can be cross-linked with the amino groups on the tissue surface, further improving the adhesion strength of the powder to the tissue. The need for additional bandages or sutures is reduced. In addition, this hemostatic powder designed based on the components of the biomimetic extracellular matrix has good biocompatibility and has a certain effect on clearing inflammation. In a full-thickness skin defect rat model, the powder not only can quickly stop bleeding and seal the broken incision, but also can reduce the inflammatory response and accelerate wound healing. The material of the hemostatic powder of the present application is the reaction between thiol-thiol, thiol-dopamine and dopamine quinone-amino to realize cross-linking. By spraying the powder on the surface of the wound, absorbing blood, forming gel in-situ, and forming tissue adhesion, hemostasis and repair are realized. The powder in dry state can effectively absorb interfacial water, enhance adhesion to tissue, and be more easily made into commercial products. The prepared powder can be stored for a long time and used as needed. In summary, the hemostatic powder of the present application combines the characteristics of rapid water absorption, strong adhesion and biocompatibility, making it an ideal candidate for a wide range of medical applications, including hemostasis, wound sealing and tissue regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Preparation and characterization of GSHD powder. (A) Fourier transform infrared spectra and (B) 1H NMR spectra of Gel-SH and HA-DA. Scanning electron microscope images (C) and particle size distribution (D) of Gel-SH. Scale bar = 10 μm. SEM images (E) and particle size distribution (F) of HA-DA. Scale bar = 10 μm. (G) Schematic diagram of powder preparation using a convenient liquid nitrogen-assisted mechanical grinding method. (H) Evaluation of PBS absorption of GSHD hydrogel and GSHD powder. (I) Liquid absorption rate (%) of GSHD hydrogel and GSHD powder in PBS and blood (n = 3);

[0030] Figure 2Rapid gelation conversion, tissue wet-adhesion and sealing of GSHD powder. (A) Storage modulus (G') and loss modulus (G") of GS1HD2, GH1HD1 and GS2HD1 as a function of frequency. (B) Stress-strain curves in compression test and (C) compressive strength (KPa) of three groups (n = 3). (D) Rheological curves of three groups in an alternating step strain sweep mode. (F) Shape adaptability of GS1HD1 was evaluated by "S", "C", "U" shaped models. (G) Self-gelation effect in blood was demonstrated by the inverted tube method. (H) GS1HD1 fixed on glass adhered to wet pig skin in a short time. (I) Pig skin adhered on GS1HD1 was washed by water. (J) SEM images of the adhesive interface, scale bar = 30 μm. (L) Adhesion strength (KPa) and (M) burst pressure (mmHg) of GS1HD2, GS1HD1 and GS1HD2 (n = 3);

[0031] Figure 3 Biocompatibility and antioxidant properties of GSHD powder. (A) Live / dead staining images of NIH-3T3 and (B) L929 cells after incubation with GSHD powder for 1, 3 and 5 days. Scale bar, 200 μm. Cell viability of (C) NIH-3T3 and (D) L929 cells after incubation with GSHD powder for 1, 3 and 5 days was evaluated by MTT method (n = 3). Immunofluorescence staining of (E) IL-6 and (F) IL-10. Scale bar, 200 μm (ns: no significant difference);

[0032] Figure 4 Blood compatibility and hemostatic performance of GSHD powder. (A) Hemolysis test and (B) hemolysis ratio of HA-DA, Gel-SH and GSHD powder. (C) Schematic diagram of inducing liver hemorrhage of rats and hemostasis using GSHD powder. (D) Photographs of hemorrhage and hemostasis after using GSHD powder. (E) Quantification of liver hemostasis time and blood loss amount (n = 3). (F) Photographs of acute hemorrhage and hemostasis using GSHD powder in rat tail vein, (G) rat heart and (H) rat femoral artery hemorrhage model;

[0033] Figure 5In vivo skin incision adhesion and sealing performance of GSHD powder. (A) Schematic diagram of GSHD powder treatment of rat full-thickness skin incision. (B) Experimental images and (C) wound healing rate of skin incision in different treatment groups (n = 3). Scale bar is 1 centimeter. (D) H&E staining images show the tissue recovery of incisions in different treatment groups at day 14. Scale bar: 200 pm. (E) Masson's trichrome staining images show the collagen recovery of incisions in different treatment groups at day 14. Scale bar, 200 pm. (F) IL-6 and (G) TNF-a immunofluorescence staining images of different treatment groups at day 14 to evaluate inflammatory infiltration. Scale bar, 100 pm. DETAILED DESCRIPTION

[0034] Example 1 Preparation of the water-sensitive in-situ ultrafast self-gelling hemostatic powder of the present application

[0035] Preparation of Gel-SH: 1 gram of gelatin was completely dissolved in 100 milliliters of deionized water at 60 °C, then cooled to room temperature, and EDC (4.81 grams), NHS (2.93 grams) and cysteamine hydrochloride (1.86 grams) were added in turn. The pH value of the solution was adjusted to 5 using 1M sodium hydroxide and hydrochloric acid, and reacted overnight. Finally, the purified dialysate was centrifuged to remove insoluble impurities, and the supernatant was freeze-dried to obtain Gel-SH.

[0036] Preparation of HA-DA: 1 gram of HA was dissolved in 100 milliliters of deionized water. EDC (1.8 grams) and NHS (0.73 grams) were then added, and the pH value was maintained at 4.75-5.0. Under nitrogen protection, 1.2 grams of dopamine DOPA-HCl dissolved in 50 milliliters of deionized water was added to the reaction mixture, and the pH value was maintained at 4.75-5.0 for 12 hours. After that, the reaction solution was dialyzed with deionized water for three days to remove unreacted reagents and byproducts. Finally, the solution was freeze-dried to obtain HA-DA.

[0037] Preparation of GSHD powder and its hydrogel: Freeze-dried Gel-SH and HA-DA were added to a cryogenic grinder and ground at a frequency of 30 hertz for 15 minutes to obtain fine and uniform Gel-SH and HA-DA powders, respectively. GS1HD2, GS1HD1 and GS2HD1 powders were prepared by mixing Gel-SH and HA-DA powders in a mass ratio of 1:2, 1:1 and 2:1, respectively. The prepared powders were stored in a dry environment.

[0038] GSHD hydrogel control group was prepared by adding PBS solution to GSHD powder for self-gelation.

[0039] Example 2 Preparation and characterization of GSHD powder

[0040] In order to obtain a component with a biomimetic extracellular matrix composition, Gel-SH (gelatin grafted with thiol groups) and HA-DA (hyaluronic acid grafted with catechol groups) were first synthesized. Specifically, an amidation reaction was carried out under EDC / NHS catalysis to introduce thiol groups and catechol groups into gelatin and hyaluronic acid, respectively. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance ( 1 The chemical structures of Gel-SH and HA-DA were evaluated by Fourier transform infrared spectroscopy (H NMR). Figure 1 A), 1553cm -1 and 1613cm -1 The peak at 1731 cm indicates that the amidation is successful. -1 The peak at represents the phenolic hydroxyl group. Similarly, the amide bond peaks (1630 and 1540 cm -1 ) and thiol at 970 cm -1 The bending vibration peak at Figure 1 As shown in Figure B, the proton peaks (δ = 6.8, 6.9 ppm) in HA-DA and the peak (δ = 3.13 ppm) in the Gel-SH spectrum further confirm the successful grafting of functional groups. Gel-SH and HA-DA powders were prepared using a convenient liquid nitrogen-assisted mechanical grinding method, which is conducive to large-scale production and product conversion ( Figure 1 G). Scanning electron microscope image ( Figure 1 C&E) showed that the shape of the powder was mainly irregular polyhedron. Further statistical analysis revealed that the size distribution of the ground powder was very wide. The median particle size (D50, the corresponding particle size when the cumulative particle size distribution percentage of the sample reached 50%) of Gel-SH and HA-DA powders was 31 and 40 μm, respectively, indicating that the smaller the particle size ( Figure 1 D&F). As we all know, powdered hemostatic materials have the advantages of adapting to the irregular topography of wounds and high water absorption, so they are widely used. Therefore, we mainly evaluated the water absorption capacity between different forms (hydrogel and powder) of the same material. Figure 1 As shown in Figure H, GSHD hydrogel and powder of equal weight absorbed 300 μL of dyed PBS. Unlike the residual PBS in the hydrogel group, the powder-based material absorbed PBS within 1 minute. The liquid absorption behavior of the GSHD hydrogel and powder in PBS and blood was also evaluated. Figure 1 The results in Figure 1 showed that the absorption rates of GSHD powder for PBS and blood were 518.3±50.7% and 569.2±46.3%, respectively, while those for the GSHD hydrogel control group were 26±5% and 31.4±3.6%, respectively.

[0041] The powder is composed of natural biopolymers, contains rich hydroxyl, amino, carboxyl and other hydrophilic groups, has a larger specific surface area compared with hydrogel, and therefore has excellent liquid absorption capacity, suitable for treating massive hemorrhage.

[0042] Rapid gelation conversion, tissue wet adhesion and sealing of GSHD powder

[0043] After absorbing blood, the thiol and catechol groups in the powder will undergo a click reaction and rapidly convert into a stable gel-like structure. It not only forms a physical barrier to prevent blood from flowing out, but also prevents the powder from entering the blood, thereby reducing the risk of other complications during material use.

[0044] To better understand the influence of powder composition content on structure and performance, we set up three different powder compositions. According to the different contents of Gel-SH and HA-DA in GSHD powder, we designed three groups of powder, GS1HD2 (Gel-SH 20mg, HA-DA 40mg), GS1HD1 (Gel-SH 30mg, HA-DA 30mg) and GS2HD1 (Gel-SH 40mg, HA-DA 20mg), with mass ratios of 1:2, 1:1 and 2:1 respectively, and the final mass of each group of powder was 60mg. First, we evaluated the mechanical properties of the powder after self-gelation using a rheometer. In the frequency sweep mode (A), the hydrogel formed after self-gelation showed typical viscoelastic behavior, characterized by a storage modulus (G') greater than a loss modulus (G"). The G' of GS1HD1 was 2448Pa, about 2.89 times and 1.48 times that of GS1HD2 and GS2HD1, indicating that a denser hydrogel network was formed in GS1HD1 powder. Figure 2 Figure 2 B and 2C, the compressive strengths of GS1HD2, GS1HD1 and GS2HD1 were 23.72±5.88, 40.55±3.31 and 25.61±4.67KPa respectively, indicating that the powder could form a firm three-dimensional network structure after absorbing water, which could conduct external stress. Scanning electron microscopy further observed the morphology of the hydrogel after self-gelation, as shown in Figure 2 D, GS1HD1 formed a more uniform and clear pore structure, which was conducive to better hemostatic performance. To apply the hydrogel to massive bleeding situations and address damage caused by blood pressure, tearing and external forces, we further explored the self-healing behavior of GS1HD2, GS1HD1 and GS2HD1. As shown in Figure 2 ​E shows that under low strain (1%) conditions, the hydrogel network is maintained (G' > G"), while under excessive strain (200%) conditions, the hydrogel network is destroyed (G' < G"), and during the alternating size strain process, the integrity of the hydrogel can be repeatedly restored. This self-healing behavior can be attributed to the dynamic thiol-catechol crosslinking reaction. In addition, in order to test the shape adaptability of the hydrogel, we also used "S" shape, "C" shape and "U" shape models to simulate irregular and deeper wound surfaces, which are more similar to the actual situation. As shown in Figure 2 F shows that after removing the models of different shapes, all samples maintain the original shape intact and are not damaged, which is mainly due to the rapid self-gelation and self-healing ability of the powder. In order to verify the self-gelation effect of the powder on blood, we took GS1HD1 as an example to carry out the inverted tube experiment. As shown in Figure 2 G shows that after placing GS1HD1 into the test tube containing blood, it immediately absorbs a large amount of liquid. After inverting the test tube for 5 seconds, GS1HD1 has been converted into a hydrogel, forming a complete liquid flow barrier.

[0045] Tissue wet adhesion is very important in the treatment of bleeding. Figure 2 H shows that the main purpose is to evaluate the wet bioadhesion effect using pigskin. In order to create a wet environment, PBS and GS1HD1 powder were added to the pigskin, and interestingly, the pigskin was lifted by the glass slide within 6 seconds, which proves good tissue wet adhesion. In addition, considering the use in severe bleeding situations, the hemostatic powder will be impacted by the fast flowing blood. Figure 2 I evaluates the anti-blood flushing ability of the powder. The powder after self-gelation can tightly adhere to the tissue even under the impact of violent water flow. Scanning electron microscope observation of the tight adhesion of the hydrogel to the tissue interface shows tight connection Figure 2 J) under the microscope.

[0046] In order to quantitatively evaluate the tissue adhesion, a lap shear test was carried out. In Figure 2 L, the adhesion strength of GS1HD1 is as high as 28.29 ± 2.98 KPa, which is significantly higher than the other two groups of powder. In addition, a burst test was also used to determine the tissue sealing ability. After 30 minutes of stabilization, the burst pressure was increased using a syringe pump. As shown in Figure 3As shown in M, the burst pressure of GS1HD1 is 216.25 ± 10.5 mmHg, which is nearly 2 times of GS1HD2 and GS2HD1, indicating that it has stronger resistance to blood flushing. In addition, the burst pressure of tGS1HD1 is even higher than the normal systolic pressure of human body (120 mmHg), which may be related to the optimal cohesive force and adhesive force of GS1HD1. In the above experiments, GS1HD1 obtained the optimal adhesive strength and burst pressure, which indicates that the appropriate combination of thiol and catechol groups can generate more adhesion sites and form stronger chemical cross-linking with tissues. In summary, our self-adhesive powder has excellent self-healing, shape remodeling, rapid self-adhesion and stronger wet tissue adhesion performance, which is very suitable for use in wound hemostasis and sealing.

[0047] The beneficial effects of the present application are demonstrated below by efficacy tests.

[0048] Test Example 1 Cell compatibility and antioxidant performance of GSHD powder

[0049] As a biomaterial for wound hemostasis and sealing, it is crucial to evaluate its biocompatibility. To evaluate the cell compatibility of the material, we used MTT detection method and live / dead staining method to evaluate cell proliferation and cell growth activity. As shown in Figure 3 As shown in A and B, after co-culturing with GSHD powder material for 1, 3 and 5 days, the cell growth activity of NIH-3T3 and L929 mouse fibroblasts was very high, and almost no dead cells were found by PI staining. The MTT detection results Figure 3 C&D) showed that the cell proliferation ability co-cultured with GSHD powder was comparable to the control group, with no statistically significant difference. These results indicate that the powder has good in vitro biocompatibility and can maintain the normal physiological state of cells.

[0050] During the wound healing process, macrophages play a key role in regulating immune response and inflammation. M1 macrophages have pro-inflammatory properties, while M2 macrophages secrete anti-inflammatory cytokines and regulate the production of extracellular matrix, with anti-inflammatory properties. During the wound repair process, the phenotype conversion of macrophages from M1 to M2 is often hindered, leading to persistent inflammatory response and delayed wound healing. Therefore, materials used for wound healing should have excellent inflammation regulation ability, promote the polarization of macrophages from M1 to M2 phenotype, and thus accelerate wound healing. Surprisingly, the catechol molecules contained in GSHD powder have the potential to scavenge free radicals and reduce oxidative stress, which may regulate the behavior of macrophages. To evaluate the effect of GSHD powder on macrophage polarization and inflammation regulation, we used immunofluorescence staining method to evaluate the expression of interleukin-6 (IL-6) and interleukin-10 (IL-10). As shown in Figure 4E and F, compared with the control group, the fluorescence intensity of proinflammatory cytokine IL-6 in GSHA powder treatment group was reduced, while the fluorescence intensity of anti-inflammatory cytokine IL-10 was increased. This may be due to the grafted catechol groups in the powder which have good anti-inflammatory effect in vitro.

[0051] Blood compatibility and hemostatic properties of GSHD powder

[0052] An ideal hemostatic powder should have good blood compatibility. When the hemostatic powder contacts the bleeding wound, it should not or rarely induce hemolysis. Therefore, we need to evaluate the blood compatibility of GSHD powder by in vitro hemolysis test. 0.1% Triton-X 100 is the positive control group. After co-incubation with different materials, the supernatant of each group was collected by centrifugation Figure 4 A). The hemolysis rate of HA-DA group, Gel-SH group and GSHD group was less than 5% Figure 4 B). Therefore, the prepared GSHD powder material meets the safety standards of hemostatic materials.

[0053] The in vivo hemostatic performance of GSHD powder was evaluated using a rat liver bleeding model Figure 4 C). As shown in Figure 4 D and 4E, the untreated control group rats had severe liver bleeding and formed a large bloodstain on the filter paper. The hemostatic time was about 120 seconds and the blood loss was about 100 milligrams. In contrast, after applying GSHD powder to the bleeding site, hemostasis was achieved in only 28 seconds, and the blood loss was significantly reduced to 30 milligrams. Compared with the untreated control group, the hemostatic time and blood loss were reduced by about 75% and 70%, respectively, indicating that GSHD powder can quickly control bleeding.

[0054] We further evaluated the in vivo hemostatic performance of GSHD powder in irregularly shaped and non-compressible acute bleeding wounds using rat tail vein, heart and femoral artery bleeding models. The rat tail was cut off to induce acute bleeding of the tail vein, and then GSHD powder was placed on the wound surface, which immediately self-gelated in situ and firmly adhered to the bleeding site. It can quickly absorb blood and achieve hemostasis within 30 seconds Figure 4 F). Similarly, the rat heart and femoral artery were exposed and punctured to induce acute bleeding Figure 4 G and 4H). The use of GSHD powder can achieve rapid and effective hemostasis, sealing the bleeding site within 30 seconds. After hemostasis, these bleeding sites do not bleed again even when stretched. The above results indicate that GSHD powder can effectively exert hemostatic function and prevent secondary bleeding Figure 5 H). In summary, GSHD powder can effectively exert hemostatic effect and avoid secondary bleeding.

[0055] In vivo wound healing performance of GSHD powder on rat skin incision model

[0056] Given the excellent tissue adhesion and rapid hemostatic sealing of GSHD, we further investigated the wound sealing effect of GSHD powder using a rat full-thickness skin incision model Figure 5 A). About 2 cm long skin incision was made on the back of rats, and then treated with glue adhesive, surgical suture or GSHD powder. As shown in Figure 5 B, due to the frequent natural activities of rats, the skin incision of the untreated control group became larger on the first day. In contrast, the incision closure was achieved on the first day for the groups treated with glue adhesive, surgical suture and GSHD powder. By the 14th day after surgery, the incisions of the GSHD powder group and the surgical suture group had healed without scar formation, with a wound closure rate of over 60% Figure 5 C). Although the wound closure effect of the surgical suture group and the GSHD powder treatment group was similar, the GSHD powder treatment achieved seamless closure of the damaged tissue, avoiding secondary damage.

[0057] Histological analysis was performed on the 14th day after surgery to evaluate the healing status of the incision. Hematoxylin and eosin (H&E) staining confirmed the wound healing effect of GSHD powder Figure 5 D). The incision treated with GSHD powder showed epidermal regeneration and new skin appendages such as hair follicles and sebaceous glands. In contrast, the untreated control group incision showed a clear unrecovered dermis layer without skin appendages. Cavitation was still observed at the incision site of the glue adhesive treatment group. The healing effect of the surgical suture group was relatively good; but the presence of the suture line hindered the complete regeneration of the dermis layer. Collagen deposition in the healed tissue was evaluated by Masson's trichrome staining Figure 5 E). On the 14th day, the collagen deposition at the incision site of the untreated control group was the least. The collagen fibers of the glue adhesive treatment group and the surgical suture group were dysplastic and arranged loosely. In contrast, the collagen fibers of the GSHD powder treatment group were regularly oriented and the structure was dense, similar to normal skin.

[0058] Immunostaining of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was performed on the 14th day after surgery ​ F and 5G). The IL-6 and TNF-α secretion level of the untreated control group was high, indicating a severe inflammatory response. The IL-6 and TNF-α level at the skin incision of the GSHD powder treatment group was the lowest, indicating that GSHD powder can effectively reduce the inflammatory response. In summary, GSHD powder can effectively promote wound sealing and tissue regeneration of rat full-thickness skin incision.

[0059] The present application provides a multifunctional biomimetic hemostatic powder based on extracellular matrix (ECM) derived biopolymers, which consists of gelatin grafted with thiol groups (Gel-SH) and hyaluronic acid grafted with catechol groups (HA-DA). These components can rapidly form a chemically cross-linked hydrogel in situ by absorbing interfacial water within 5 seconds, thus achieving rapid hemostasis and wound sealing. After rapid blood absorption, the thiol and catechol groups on the GSHD powder can form covalent bonds with the amino groups on the tissue surface, further enhancing the adhesion strength. Vertical shear and burst pressure tests show that the adhesion strength of the GSHD powder after self-gelation is 28.3±3Kpa, and can withstand a burst pressure of up to 216.5±10mmHg. In addition, the powder also has good biocompatibility and anti-inflammatory properties. In a rat full-thickness skin defect model, the powder not only rapidly stops bleeding and seals the wound, but also reduces the inflammatory response and accelerates wound healing. Therefore, due to the strong adhesion, good biocompatibility, rapid hemostasis, and suitability for non-compressible sites and wound sealing of the GSHD powder, we believe that it is a hemostatic material and wound dressing with broad application prospects.

Claims

1. A water-sensitive in-situ ultrafast self-gelling hemostatic powder, characterized by: It is made by mixing and low-temperature grinding the following raw materials in the following weight ratios: 1-2 parts of gelatin grafted with mercapto groups and 1-2 parts of hyaluronic acid grafted with catechol groups.

2. The water-sensitive in-situ ultrafast self-gelling hemostatic powder according to claim 1, characterized in that: It is made by mixing and low-temperature grinding the following raw materials in the following weight ratios: 1 part of gelatin grafted with mercapto groups and 1 part of hyaluronic acid grafted with catechol groups.

3. The water-sensitive in-situ ultrafast self-gelling hemostatic powder according to claim 1 or 2, characterized in that: The grafted thiol gelatin is prepared using gelatin and cysteamine hydrochloride as raw materials and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts; The hyaluronic acid grafted with catechol groups is prepared using hyaluronic acid and dopamine as raw materials and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts.

4. The water-sensitive in-situ ultrafast self-gelling hemostatic powder according to claim 3, characterized in that: In the preparation method of grafted thiol gelatin, the mass ratio of gelatin, cysteamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is: 1 part of gelatin, 1.86 parts of cysteamine hydrochloride, 4.81 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 2.93 parts of N-hydroxysuccinimide (NHS); In the preparation method of the hyaluronic acid grafted with catechol groups, the mass ratio of hyaluronic acid, dopamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is: 1 part of hyaluronic acid, 1.2 parts of dopamine, 1.8 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 0.73 parts of N-hydroxysuccinimide (NHS).

5. The water-sensitive in-situ ultrafast self-gelling hemostatic powder according to claim 3, characterized in that: The preparation method of the grafted thiol gelatin is as follows: Gelatin was completely dissolved in deionized water at 60 °C and then cooled to room temperature. EDC, NHS, and cysteamine hydrochloride were added in sequence. The pH value of the solution was adjusted to 5 using 1 M sodium hydroxide and hydrochloric acid, and the reaction was allowed to proceed overnight. Finally, the purified dialysate was centrifuged to remove insoluble impurities, and the supernatant was lyophilized to obtain the grafted thiol gelatin. The preparation method of the hyaluronic acid grafted with catechol groups is as follows: HA was dissolved in deionized water, followed by the addition of EDC and NHS, with the pH maintained between 4.75 and 5.

0. Under nitrogen protection, dopamine (DOPA-HCl) dissolved in deionized water was added to the reaction mixture, and the pH was maintained between 4.75 and 5.0 for 12 hours. Afterwards, the reaction solution was dialyzed with deionized water for three days to remove unreacted reagents and by-products. Finally, the solution was freeze-dried to obtain hyaluronic acid grafted with catechol groups.

6. A method for preparing the water-sensitive in-situ ultrafast self-gelling hemostatic powder according to any one of claims 1 to 5, characterized in that: It includes the following steps: a. Weighing the weight ratios of thiol-grafted gelatin and catechol-grafted hyaluronic acid; b. Mix, add to low temperature grinder, grind to obtain.

7. The method for preparing the water-sensitive in-situ ultrafast self-gelling hemostatic powder according to claim 6, characterized in that: The grinding conditions are: grinding at a frequency of 30 Hz for 15 minutes.

8. Use of the water-sensitive in-situ ultrafast self-gelling hemostatic powder according to any one of claims 1 to 5 in the preparation of an external-use medicine having a hemostatic effect.

Images