Injectable adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel and application thereof

Abstract

The invention relates to the field of biomedical materials, and particularly provides injectable adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel and application thereof.The hemostatic hydrogel is formed by carboxylated chitosan sol and dialdehyde starch size through Schiff base reaction, hydrogen bond construction and in-situ rapid crosslinking by means of a bidirectional pumping and injecting delivery strategy, and the hemostatic hydrogel is prepared through a two-way pumping and injecting delivery strategy, and the injectable adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel is prepared from the carboxylated chitosan sol, the dialdehyde starch size and the injectable adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel. The hydrogel has the advantages of good biocompatibility, injectability, adhesion, personalized adaptation and the like, integrates multiple hemostasis mechanisms, a physical barrier effect of rapid gelation, an expansion compression effect of water absorption swelling, an electrical neutralization effect of carboxylated chitosan amino and a tissue adhesion effect of dialdehyde starch aldehyde group, realizes multi-scene rapid and efficient hemostasis, and has a wide application prospect. The preparation method of the injectable adhesive hemostatic hydrogel is low in cost, innovative in technology, mild in condition, convenient to store and beneficial to large-scale batch production, and has a wide application prospect in the field of efficient hemostatic hydrogel.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to an injectable, adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel and its applications. Background Technology

[0002] Traumatic bleeding is a common and highly threatening emergency in clinical practice. Whether caused by external trauma, surgery, or endogenous bleeding, the rupture of blood vessels leads to blood leakage, disrupting the body's vascular barrier function and further causing imbalances in the body's physiological homeostasis. This imbalance not only affects normal blood circulation but also makes it easier for various pathogens and harmful substances to invade the body, increasing the risk of complications such as infection and thus worsening the patient's condition. Especially in cases of massive bleeding, hemorrhagic shock and the resulting coagulation disorders will further deteriorate the clinical situation, and in severe cases, may even lead to death. Therefore, timely and effective hemostasis of traumatic bleeding is not only a crucial link in trauma management and emergency care but also an important guarantee for preventing secondary damage and improving survival rates.

[0003] Currently, there are many types of common hemostatic materials, mainly including cotton gauze, sponges, films, powders, dry gels, and hydrogels. Among them, hydrogels, as a highly promising hemostatic material, are gradually becoming an important candidate material in the field of hemostasis due to their unique physicochemical properties and biomedical performance, such as good hydration, biocompatibility, plasticity, modifiability, and multifunctionality. However, with the development of modern complex economic life and the continuous changes in the needs of trauma treatment, the pathological environment of traumatic bleeding is becoming increasingly severe, and the sites of traumatic bleeding are becoming increasingly complex. Existing hydrogel hemostatic materials, which are usually in block form, are gradually unable to meet the increasingly high clinical requirements. Injectable hydrogels, as a new type of hydrogel hemostatic material, have attracted much attention, with advantages such as being non-invasive, easy to operate, and flexible in form. Moreover, they can be customized according to the shape and depth of the wound, precisely covering irregular or deep bleeding points. In addition, the gelation time of injectable hydrogels can be adjusted, which means that their gelation rate in vivo can be controlled according to clinical needs to achieve a more ideal hemostatic effect.

[0004] Currently, injectable hemostatic hydrogels are mainly made of materials such as chitosan, gelatin, and cellulose. They typically gel rapidly at the wound site through physicochemical mechanisms, forming a protective film or structure. Utilizing physical barrier effects and expansion compression, they reduce blood loss and achieve hemostasis. However, these traditional injectable hemostatic hydrogels suffer from drawbacks such as poor adhesion, complex gelation reaction conditions, unstable gelation time, limited hemostatic effect, single hemostatic mechanism, superficial application site, and numerous side effects, failing to meet the ever-increasing demands for hemostatic materials. This suggests that the biocompatibility, adhesion, and gelation properties of novel injectable hemostatic hydrogels need further improvement, and they need to be endowed with deeper hemostatic mechanisms to achieve rapid and efficient hemostasis in multiple scenarios. Strategic material selection, innovative delivery strategies, and integrated hemostatic models are crucial.

[0005] To address the aforementioned technical challenges, the inventors developed an injectable, adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel. This hydrogel is formed by in-situ rapid cross-linking of carboxylated chitosan sol and dialdehyde starch slurry via a bidirectional injection delivery strategy, through Schiff base reaction and hydrogen bonding. It possesses advantages such as good biocompatibility, injectability, adhesiveness, and personalized adaptability. It integrates multiple hemostatic mechanisms, including the physical barrier effect of rapid gelation, the swelling and compression effect of water absorption and swelling, the charge neutralization effect of carboxylated chitosan amino groups, and the tissue adhesion effect of dialdehyde starch aldehyde groups, achieving rapid and efficient hemostasis in various scenarios. This injectable, adhesive hemostatic hydrogel preparation method is low-cost, technologically innovative, operates under mild conditions, and is easy to store, facilitating large-scale mass production and showing broad application prospects in the field of high-efficiency hemostatic hydrogels. Summary of the Invention

[0006] The present invention achieves the above objectives through the following technical solutions. The primary objective of the present invention is to provide an injectable, adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel, which is prepared by the following method, including the following steps:

[0007] (1) At room temperature, carboxylated chitosan is dissolved in a solvent to prepare carboxylated chitosan sol;

[0008] (2) Dissolve the dialdehyde starch in a solvent to form a dialdehyde starch slurry;

[0009] (3) The carboxylated chitosan sol prepared in step (1) and the dialdehyde starch slurry prepared in step (2) are respectively injected into a bidirectional syringe to form a hydrogel precursor;

[0010] (4) Sterilize the hydrogel precursor prepared in step (3) to obtain the sterilized hydrogel precursor;

[0011] (5) The sterilized hydrogel precursor prepared in step (4) is delivered to the bleeding point, adhered, and cross-linked in situ to obtain carboxylated chitosan / dialdehyde starch hemostatic hydrogel.

[0012] Preferably, the mass-to-volume ratio of carboxylated chitosan to solvent in step (1) is 6-12:100.

[0013] Preferably, the degree of carboxylation of the carboxylated chitosan in step (1) is ≥60%.

[0014] Preferably, the dialdehyde starch in step (2) is prepared by the following method: starch is uniformly dispersed in water, sodium periodate solution is added, the mixture is stirred and reacted, excess ethylene glycol is added to remove the remaining sodium periodate, the mixture is filtered, washed until no chloride ions are detected by silver nitrate standard solution, repeatedly washed and dehydrated with anhydrous ethanol, and then freeze-dried under vacuum for 20-30 hours to obtain dialdehyde starch; the degree of aldehyde substitution of the dialdehyde starch is 30-95%.

[0015] Preferably, the mass-to-volume ratio of the dialdehyde starch to the solvent in step (2) is 12:100, and the temperature is set to 60-75℃.

[0016] Preferably, the solvent in steps (1) and (2) is one or more of water, physiological saline, PBS buffer, etc.

[0017] Preferably, the volume ratio of carboxylated chitosan sol to dialdehyde starch slurry in the bidirectional syringe described in step (3) is 1:1.

[0018] A second objective of this invention is to provide the application of the hemostatic hydrogel in the preparation of hemostatic materials.

[0019] A third objective of this invention is to provide the application of the hemostatic hydrogel in the preparation of medical hemostatic materials.

[0020] A fourth objective of this invention is to provide the application of the hemostatic hydrogel in the preparation of hemostatic materials for in vivo bleeding.

[0021] The beneficial effects of this invention are:

[0022] (1) The present invention provides an injectable, adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel. The hemostatic hydrogel is composed of carboxylated chitosan sol and dialdehyde starch slurry. It is formed by in-situ rapid cross-linking through Schiff base reaction and hydrogen bonding using a bidirectional injection delivery strategy. It can adapt to the shape and depth of the wound, accurately cover irregular or deep bleeding points, and achieve a more ideal and adaptive hemostatic effect by adjusting the gelation state and rate according to clinical needs.

[0023] (2) The hemostatic hydrogel described in this invention has good biocompatibility, can maintain normal coagulation function over time, has good hemostatic potential, and has better hemostatic performance. It highlights that the injectable hemostatic hydrogel studied has a greater competitive advantage than hemostatic membranes, and also has certain advantages over conventional commercial hemostatic materials. It also has the potential for efficient and rapid hemostasis in multiple scenarios.

[0024] (3) The hemostatic hydrogel described in this invention has the advantages of being injectable, adhesive, and adaptable to individual needs, effectively solving the drawbacks of traditional injectable hemostatic hydrogels. It integrates multiple hemostatic mechanisms, such as the physical barrier effect of rapid gelation, the expansion and compression effect of water absorption and swelling, the electrical neutralization effect of carboxylated chitosan amino groups, and the tissue adhesion effect of dialdehyde starch aldehyde groups, so as to achieve rapid and efficient hemostasis in multiple scenarios.

[0025] (4) The hemostatic hydrogel preparation method of the present invention is low in cost, innovative in technology, mild in conditions, convenient in storage, and conducive to large-scale mass production. It has broad application prospects in the field of high-efficiency hemostatic hydrogel. Attached Figure Description

[0026] Figure 1 Fourier transform infrared absorption spectra of DS, CC, and CCDS;

[0027] Figure 2 The image shows a 95 / 5 Film product.

[0028] Note: A. Gelation of acidic chitosan / dialdehyde starch (weight ratio 95:5) within 16 hours; B. Actual image of the acidic chitosan / dialdehyde starch (weight ratio 95:5) hemostatic membrane;

[0029] Figure 3 The flowability of CC and CS at different concentrations;

[0030] Note: A. Initial state and flow state after 30 seconds for CC-12, CC-10, CC-8, and CC-6; B. Initial state and flow state after 30 seconds for CS-10, CS-8, CS-6, and CS-4;

[0031] Figure 4 The gelation time and pH value for different concentrations of CCDS, CSDS, and TCSDS;

[0032] Note: A. Gelation time of CCDS-6, CCDS-8, CCDS-10, CCDS-12, CSDS-6, and CSDS-4; B. pH values ​​of CC-10, CS-6, TCS-6 and CCDS-10, CSDS-6, and TCSDS-6;

[0033] Figure 5Images of the gelled products of CCDS-10, CSDS-6, and TCSDS-6;

[0034] Figure 6 Cell compatibility and hemolysis assays for 95 / 5Flim, CSDS-6, and CCDS-10;

[0035] Note: AB. MTT assay of 95 / 5Flim, CSDS-6, and CCDS-10; C. Live and dead cell staining of 95 / 5Flim, CSDS-6, and CCDS-10; D. Hemolysis rate of 95 / 5Flim, CSDS-6, and CCDS-10; E. Actual images of hemolysis experiments using 95 / 5Flim, CSDS-6, and CCDS-10.

[0036] Figure 7 Coagulation test for 95 / 5Flim and CCDS-10;

[0037] Note: A. OD values ​​of 95 / 5Flim and CCDS-10 at different time periods; B. BCI index of 95 / 5Flim and CCDS-10 at different time periods;

[0038] Figure 8 Hemostasis experiment for a liver defect model in SD rats;

[0039] Note: A. Actual image of the hemostasis experiment in the SD rat liver defect model; B. Hemostasis time after different treatments; C. Blood loss after different treatments;

[0040] Figure 9 A physical image of whole blood coagulation from a CCDS-10 device;

[0041] Note: A. Whole blood clotting in CCDS-10 after 25 seconds; B. Whole blood clotting in the untreated Control group;

[0042] Figure 10 The adhesion properties of CCDS-10;

[0043] Note: A. Adhesion of CCDS-10 to various internal organs of SD rats; B. Shear adhesion strength of CCDS-10 to pigskin.

[0044] Figure 11 Swelling and degradation tests for CCDS-10;

[0045] Note: A. Swelling test of CCDS-10; B. Degradation test of CCDS-10. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] It should be noted that the methods described in the following embodiments are all conventional methods, and the reagents described can be purchased or prepared commercially.

[0048] The chitosan / dialdehyde starch hemostatic membrane described in Example 2 below is the optimal solution disclosed by the inventor using the closest prior art CN1434072A, and has the best hemostatic effect.

[0049] Example 1: Preparation of dialdehyde starch

[0050] (1) Weigh 4g of sodium periodate and dissolve it in 150mL of water. Adjust the pH value to 4 with 1mol / L hydrochloric acid buffer to form a sodium periodate solution.

[0051] (2) Weigh 8g of potato starch and disperse it evenly in 50mL of water to form a starch slurry;

[0052] (3) Add the sodium periodate solution prepared in step (1) to the starch slurry prepared in step (2) within 0.5 hours. Set the temperature to 40℃ and stir for 4 hours. Add excess ethylene glycol to remove the remaining sodium periodate. Filter and wash repeatedly with water until no chloride ions are detected by silver nitrate standard solution. Then wash repeatedly with anhydrous ethanol to dehydrate. Finally, freeze dry under vacuum for 20-30 hours to obtain dialdehyde starch, named DS.

[0053] like Figure 1 As shown, the chemical composition and intermolecular interactions were characterized by Fourier transform infrared absorption spectroscopy. DS retained the basic characteristic peaks of natural starch, including the OH stretching peak (3371 cm⁻¹). -1 (left and right) and curved (1637cm) -1 Left and right), CH stretch (2901cm) -1 (left and right) and curved (1453cm) -1 CO stretching (1015cm) -1 (Left and right) etc., and at 1730cm -1 The presence of aldehyde characteristic peaks on the left and right indicates the successful synthesis of dialdehyde starch.

[0054] Example 2: Preparation of Chitosan / Dialdehyde Starch Hemostatic Membrane

[0055] (1) At room temperature, prepare 100 mL of acetic acid aqueous solution with a volume ratio of 2%, weigh 3 g of chitosan and dissolve it in the solution to prepare acidic chitosan sol;

[0056] (2) Weigh 5g of the dialdehyde starch prepared in Example 1, dissolve it in 100mL of water, and prepare a dialdehyde starch slurry by water bath at 70℃.

[0057] (3) The acidic chitosan sol prepared in step (1) and step (2) is thoroughly mixed with the dialdehyde starch slurry at a weight ratio of 95:5, and centrifuged to remove bubbles, to obtain the acidic chitosan / dialdehyde starch hemostatic membrane hydrogel precursor.

[0058] (4) Using a straight glass tube with copper wires at both ends, scrape the acidic chitosan / dialdehyde starch hemostatic membrane hydrogel precursor prepared in step (3) onto a clean glass plate, dry it, and obtain the acidic chitosan / dialdehyde starch hemostatic membrane.

[0059] (5) The acidic chitosan / dialdehyde starch hemostatic membrane prepared in step (4) was neutralized with 2wt% sodium hydroxide solution, and repeatedly rinsed and dialyzed with water. Finally, it was dried to obtain the chitosan / dialdehyde starch hemostatic membrane, named 95 / 5Film.

[0060] like Figure 2 As shown, the prepared acidic chitosan sol did not directly gel after being uniformly mixed with dialdehyde starch slurry. After being prepared into a 95 / 5 film, its surface was flat, transparent, smooth, and flexible, and it can be used as a candidate material for hemostatic barrier.

[0061] Example 3: Preparation of carboxylated chitosan / dialdehyde starch hemostatic hydrogel

[0062] (1) At room temperature, 6, 8, 10 and 12 g of carboxylated chitosan were weighed and dissolved in 100 mL of water to prepare carboxylated chitosan sols of different concentrations, named CC. Among them, the carboxylated chitosan sol with a concentration of 6 g / 100 mL was named CC-6, the carboxylated chitosan sol with a concentration of 8 g / 100 mL was named CC-8, the carboxylated chitosan sol with a concentration of 10 g / 100 mL was named CC-10, and the carboxylated chitosan sol with a concentration of 12 g / 100 mL was named CC-12.

[0063] (2) Dissolve 12g of the dialdehyde starch prepared in Example 1 in 100mL of water, set the temperature to 70℃, and gelatinize the dialdehyde starch to form a dialdehyde starch slurry.

[0064] (3) The carboxylated chitosan sol and dialdehyde starch slurry of different concentrations prepared in steps (1) and (2) are injected into a bidirectional syringe to form hydrogel precursors of different concentrations.

[0065] (4) The hydrogel precursors of different concentrations prepared in step (3) are used... 60 Co radiation sterilization (20-25 kGy) yielded hydrogel precursors of different concentrations after sterilization;

[0066] (5) Using a bidirectional injection and delivery strategy, the sterilized hydrogel precursors of different concentrations prepared in step (4) are delivered to the bleeding point, adhere, and crosslink in situ to form carboxylated chitosan / dialdehyde starch hemostatic hydrogels of different concentrations, named CCDS, to achieve hemostasis. Among them, the carboxylated chitosan / dialdehyde starch hemostatic hydrogel prepared with a concentration of 6g / 100mL of carboxylated chitosan sol is named CCDS-6, the carboxylated chitosan / dialdehyde starch hemostatic hydrogel prepared with a concentration of 8g / 100mL of carboxylated chitosan sol is named CCDS-8, the carboxylated chitosan / dialdehyde starch hemostatic hydrogel prepared with a concentration of 10g / 100mL of carboxylated chitosan sol is named CCDS-10, and the carboxylated chitosan / dialdehyde starch hemostatic hydrogel prepared with a concentration of 12g / 100mL of carboxylated chitosan sol is named CCDS-12.

[0067] like Figure 1 As shown, the chemical composition and intermolecular interactions were characterized by Fourier transform infrared absorption spectroscopy. The CCDS hydrogel retained the characteristic peaks of DS and CC, and peaked at 1147 cm⁻¹. -1 New characteristic peaks appear on the left and right sides, and at 1735 cm⁻¹ -1 No characteristic peaks of aldehyde groups were observed at the left and right positions, indicating that the Schiff base reaction occurred successfully. (3271 cm⁻¹) -1 The redshift of the characteristic peaks at the left and right and the 1586 cm⁻¹ -1 The intensity changes of the characteristic peaks on the left and right indicate the construction and strengthening of intermolecular hydrogen bonds, proving at the molecular level that DS slurry and CC sol can crosslink in situ to form CCDS hydrogel.

[0068] Example 4: Preparation of acidic chitosan / dialdehyde starch hemostatic hydrogel

[0069] (1) At room temperature, prepare 100 mL of acetic acid aqueous solution with a volume ratio of 2%, and weigh 4, 6, 8 and 10 g of chitosan respectively and dissolve them in it to prepare acidic chitosan sols of different concentrations, named CS. Among them, the acidic chitosan sol with a concentration of 4 g / 100 mL is named CS-4, the acidic chitosan sol with a concentration of 6 g / 100 mL is named CS-6, the acidic chitosan sol with a concentration of 8 g / 100 mL is named CS-8, and the acidic chitosan sol with a concentration of 10 g / 100 mL is named CS-10.

[0070] (2) Dissolve 12g of the dialdehyde starch prepared in Example 1 in 100mL of water, set the temperature to 70℃, and gelatinize the dialdehyde starch to form a dialdehyde starch slurry.

[0071] (3) The acidic chitosan sol and dialdehyde starch slurry of different concentrations prepared in steps (1) and (2) are injected into a bidirectional syringe at a volume ratio of 1:1 to form hydrogel precursors of different concentrations.

[0072] (4) The hydrogel precursors of different concentrations prepared in step (3) are used... 60 Co radiation sterilization (20-25 kGy) yielded hydrogel precursors of different concentrations after sterilization;

[0073] (5) Using a bidirectional injection and delivery strategy, the sterilized hydrogel precursors of different concentrations prepared in step (4) are delivered to the bleeding point, adhere, and crosslink in situ to form acidic chitosan / dialdehyde starch hemostatic hydrogels of different concentrations, named CSDS, to achieve hemostasis. Among them, the acidic chitosan / dialdehyde starch hemostatic hydrogel prepared with chitosan sol at a concentration of 4g / 100mL is named CSDS-4, the acidic chitosan / dialdehyde starch hemostatic hydrogel prepared with chitosan sol at a concentration of 6g / 100mL is named CSDS-6, the acidic chitosan / dialdehyde starch hemostatic hydrogel prepared with chitosan sol at a concentration of 8g / 100mL is named CSDS-8, and the acidic chitosan / dialdehyde starch hemostatic hydrogel prepared with chitosan sol at a concentration of 10g / 100mL is named CSDS-10.

[0074] Example 5: Preparation of Chitosan / Dialdehyde Starch Hemostatic Hydrogel

[0075] (1) At room temperature, 6g of chitosan was weighed into 100mL of acetic acid aqueous solution with a volume ratio of 2% and dissolved in it. The solution was dialyzed to neutral using a dialysis bag to prepare chitosan sol, which was named TCS-6.

[0076] (2) Dissolve 12g of the dialdehyde starch prepared in Example 1 in 100mL of water, set the temperature to 70℃, and gelatinize the dialdehyde starch to form a dialdehyde starch slurry.

[0077] (3) The chitosan sol and dialdehyde starch slurry prepared in steps (1) and (2) are injected into a bidirectional syringe at a volume ratio of 1:1 to form a hydrogel precursor.

[0078] (4) The hydrogel precursor prepared in step (3) is used 60 Co radiation sterilization (20-25 kGy) yields sterilized hydrogel precursor;

[0079] (5) Using a bidirectional injection and delivery strategy, the sterilized hydrogel precursor prepared in step (4) is delivered to the bleeding point, adheres, and crosslinks in situ to form a chitosan / dialdehyde starch hemostatic hydrogel, named TCSDS-6.

[0080] Example 6

[0081] This embodiment uses CC, CCDS, CS, CSDS, TCS-6, and TCSDS-6 from embodiments three, four, and five as the implementation objects for the following experiments:

[0082] (1) The flowability of CC and CS was characterized by the tube tilting method;

[0083] (2) The gelation time of CCDS and CSDS was recorded using the tube tilting method;

[0084] (3) Test the pH values ​​of CC-10, CCDS-10, CS-6, CSDS-6, TCS-6, and TCSDS-6.

[0085] like Figure 3 As shown in AB, the flowability of CC and CS sols gradually decreased with the increase of carboxylated chitosan and chitosan concentration. CC-6, CC-8, CC-10, CC-12, CS-4, and CS-6 sols with good flowability were selected for the gelation experiment to maintain suitable injectability.

[0086] like Figure 4 As shown in Figure A, with the increase of carboxylated chitosan and chitosan concentration, the gelation time of CCDS and CSDS hydrogels is shortened. Among them, the gelation time of CCDS-10, CCDS-12 and CSDS-6 hydrogels is not significantly different and is the shortest, which is more conducive to achieving efficient hemostasis through rapid gelation. Therefore, CCDS-10 and CSDS-6 hydrogels were selected as candidates for injectable hemostatic hydrogels.

[0087] like Figure 4 As shown in Figure B, the CSDS-6 hydrogel exhibits acidity due to the acidity of the CS-6 sol, while the CCDS-10 hydrogel tends to be neutral because the pH value of the CC sol is close to neutral. Furthermore, the pH value of the TCSDS-6 hydrogel prepared using the proportions of the components of the CSDS-6 hydrogel also tends to be neutral because the pH value of the TCS sol is close to neutral. Therefore, the CCDS-10 and TCSDS-6 hydrogels are more in line with the application requirements in the field of biomedical materials.

[0088] like Figure 5As shown, comparing the gelation states of CCDS-10, CSDS-6 and TCSDS-6 hydrogels, CCDS-10 and CSDS-6 exhibit significantly more uniform gelation and better mechanical structure, providing a suitable mechanical structural basis for their hemostatic properties.

[0089] Therefore, considering all factors, CCDS-10 is a more suitable candidate for injectable hemostatic hydrogels. If the acidity of CCDS-6 does not affect the biocompatibility requirements for applications in the biomedical materials field, it can also be considered a candidate for injectable hemostatic hydrogels. However, the poor mechanical structure and gelation state of TCSDS-6 make it unsuitable as a candidate for injectable hemostatic hydrogels.

[0090] Example 7

[0091] This embodiment uses the 95 / 5Film, CCDS-10, and CSDS-6 from Embodiments 2, 3, and 4 as the implementation objects for the following experiments:

[0092] (1) L929 cells were seeded in 96-well plates and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. After strict sterilization, each sample was co-cultured with the cells. After 24 hours, an appropriate amount of MTT reagent was added and incubated at 37°C for 4 hours. Then the supernatant was removed, and dimethyl sulfoxide was added to dissolve the purple crystals formed. The mixture was shaken at room temperature in the dark for 10 minutes to dissolve the crystals evenly. The absorbance value was read at 570 nm using an ELISA reader (Infinite M Nano, TECAN, China). Each group was repeated three times.

[0093] (2) Each group of strictly sterilized samples and L929 cells were co-cultured in 24-well plates. After 24 hours of culture, the cells were stained with a live and dead cell staining kit for 0.5 hours. Then the staining solution was removed to terminate the incubation. An appropriate amount of PBS was added to cover the cells. Live cells (green fluorescence) and dead cells (red fluorescence) were photographed using an inverted fluorescence microscope (VERT1, Zeiss, USA).

[0094] (3) Take an appropriate amount of whole blood from SD rats, add 3.8wt% sodium citrate solution for anticoagulation, dilute the blood with physiological saline at a ratio of 4:5 (volume ratio), incubate the sample in 10mL of physiological saline at 37℃ for 30 minutes, then add 0.2mL of diluted blood and incubate for 1 hour; select physiological saline and deionized water as negative control and positive control, respectively; centrifuge the mixed solution after incubation at 800g speed for 5 minutes, and use an ELISA reader (Infinite M Nano, TECAN, China) to measure the absorbance of the supernatant at 545 nm and calculate the hemolysis rate.

[0095] like Figure 6As shown in AB, except for the CSDS-6 hydrogel, whose cell compatibility was less than 70%, the cell compatibility of the other samples remained above the standard line, meeting the application requirements.

[0096] like Figure 6 As shown in C, in the staining results of live and dead cells in each group of samples, except for the CSDS-6 group, the number of red fluorescent cells representing dead cells in the other groups was relatively small, and the cell proliferation activity was not affected, indicating that the other samples except for the CSDS-6 hydrogel have good cell compatibility.

[0097] like Figure 6 As shown in DE, except for CSDS-6 hydrogel, the other samples showed almost no hemolytic effect in the hemolysis experiment, demonstrating good biocompatibility.

[0098] In summary, only the 95 / 5Film hemostatic membrane and CCDS-10 hydrogel meet the biocompatibility requirements for applications in the field of biomedical materials, and can be used as hemostatic membranes and injectable hemostatic hydrogels, respectively, for subsequent experiments.

[0099] Example 8

[0100] This embodiment uses the 95 / 5Film and CCDS-10 from Embodiments 2 and 3 as the implementation objects for the following experiments:

[0101] Each sample was preheated to 37°C, and then 50 μL of recalcified SD rat whole blood solution (0.2 M CaCl2) was added to the sample surface. After incubation at 37°C for 1 min, 3 min and 5 min respectively, 10 mL of water was added to hemolyze the non-coagulated blood cells. The absorbance of the supernatant was measured at 545 nm using an ELISA reader (Infinite M Nano, TECAN, China). 50 μL of recalcified whole blood with 10 mL of water served as the positive control group, and an equal volume of water served as the blank control group. The in vitro coagulation index (BCI) was calculated.

[0102] like Figure 7 As shown in AB, the BCI index of both 95 / 5Film and CCDS-10 hydrogel gradually decreased over time, indicating that both 95 / 5Film and CCDS-10 hydrogel maintained normal coagulation function and had good hemostatic potential over time, with CCDS-10 hydrogel exhibiting better coagulation performance.

[0103] Example 9

[0104] This embodiment uses the 95 / 5Film and CCDS-10 from Embodiments 2 and 3 as the implementation objects for the following experiments:

[0105] (1) Healthy female SD rats weighing 250-300g were selected. After adequate anesthesia, analgesia, disinfection, and skin preparation, the liver was dissected and exposed. The tissue fluid around the liver was removed with gauze. Then, a disc-shaped full-thickness defect model was constructed using a 6mm diameter circular punch. The blank group served as the negative control group and was named Control. The commercially available injectable hemostatic dressing SurgiFlo was used. @ As a positive control group, it was named SurgiFlo @ The bleeding volume and hemostasis time of 95 / 5Film and CCDS-10 hydrogel were detected to evaluate their hemostatic performance.

[0106] (2) A total of 1 mL of DS and CC were simultaneously delivered into 0.5 mL of fresh whole blood from SD rats. After 25 seconds, the test tube was inverted to observe the hemostatic effect of CCDS-10 hydrogel. The blank group was used as the negative control group and named Control.

[0107] like Figure 8 As shown, the blood loss of CCDS-10 hydrogel was approximately 0.55g, and the hemostasis time was approximately 68s, which was far superior to the other groups. This indicates that CCDS-10 hydrogel has better hemostatic performance, highlighting that the injectable hemostatic hydrogel studied has a greater competitive advantage than hemostatic membranes and also has certain advantages over conventional commercial hemostatic materials.

[0108] like Figure 9 As shown, after 25 seconds, fresh SD rat whole blood and CCDS-10 hydrogel formed a whole. After the CCDS-10 hydrogel gelled, it lost its fluidity, indicating that CCDS-10 hydrogel has good hemostatic properties and has the potential for efficient and rapid hemostasis in multiple scenarios.

[0109] In summary, the experimental groups and performance evaluation statistics involved are shown in the table below:

[0110]

[0111] Example 10

[0112] This embodiment uses the CCDS-10 from Embodiment 3 as the implementation object for the following experiments:

[0113] (1) DS and CC were simultaneously delivered onto a clean, moist glass slide, and then immediately the internal organs of SD rats were adhered to it and the adhesion was observed.

[0114] (2) Cut fresh pig skin into rectangular pieces about 2cm wide and 8cm long. Simultaneously deliver DS and CC to the middle of two pieces of fresh pig skin and apply a pressure of about 98g. After the CCDS-10 hydrogel gels, use a universal testing machine (CMT2102, China, strain rate of 5mm / min at room temperature) to test the adhesion shear strength of the CCDS-10 hydrogel.

[0115] like Figure 10 As shown in Figure A, the CCDS-10 hydrogel can adhere to various internal organs of SD rats.

[0116] like Figure 10 As shown in Figure B, the average adhesive shear strength of CCDS-10 hydrogel is approximately 3.57 kPa, exhibiting good tissue adhesion and making it suitable for efficient and rapid hemostasis in various scenarios.

[0117] Example 11

[0118] This embodiment uses the CCDS-10 from Embodiment 3 as the implementation object for the following experiments:

[0119] Prepare CCDS-10 hydrogel samples of similar size and shape for testing. Set up four parallel groups. Detect the equilibrium swelling ratio of the samples by immersion weighing method and the degradation rate of the samples by degradation weighing method.

[0120] like Figure 11 As shown in Figure A, the swelling rate of CCDS-10 hydrogel gradually increases over time, reaching swelling equilibrium in about 1 hour. The equilibrium swelling rate is about 2.3 times its own weight, demonstrating good potential for hemostasis through expansion and compression.

[0121] like Figure 11 As shown in Figure B, the structure of CCDS-10 hydrogel gradually disintegrates over time, and its structure is almost completely disintegrated after 72 hours. It exhibits good degradation performance, environmental friendliness, long-term structural stability and suitable degradation performance, which is beneficial for efficient and rapid hemostasis in multiple scenarios and degradation and removal after hemostasis.

[0122] In summary, this invention provides an injectable, adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel. The hydrogel is composed of carboxylated chitosan sol and dialdehyde starch slurry, delivered via a bidirectional injection strategy. It is formed through Schiff base reaction and hydrogen bonding, resulting in rapid in-situ cross-linking. This allows it to adapt to the shape and depth of wounds, precisely covering irregular or deep bleeding points. Furthermore, by adjusting the gelation state and rate according to clinical needs, it achieves a more ideal and adaptive hemostatic effect. The hemostatic hydrogel of this invention exhibits good biocompatibility, maintains normal coagulation function over time, demonstrates excellent hemostatic potential, and possesses superior hemostatic performance. This injectable hemostatic hydrogel demonstrates a greater competitive advantage over hemostatic membranes and also has certain advantages over conventional commercial hemostatic materials, possessing the potential for efficient and rapid hemostasis in multiple scenarios. The hemostatic hydrogel described in this invention offers advantages such as injectability, adhesiveness, and personalized adaptability, effectively overcoming the drawbacks of traditional injectable hemostatic hydrogels. It integrates multiple hemostatic mechanisms, including the physical barrier effect of rapid gelation, the swelling and compression effect of water absorption and swelling, the charge neutralization effect of carboxylated chitosan amino groups, and the tissue adhesion effect of dialdehyde starch aldehyde groups, achieving rapid and efficient hemostasis in various scenarios. The hemostatic hydrogel preparation method described in this invention is low-cost, technologically innovative, operates under mild conditions, and is convenient to store, facilitating large-scale mass production and possessing broad application prospects in the field of high-efficiency hemostatic hydrogels.

[0123] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An injectable, adhesive carboxylated chitosan / dialdehyde starch hemostatic hydrogel, characterized in that, The hemostatic hydrogel is prepared by the following method, including the following steps: (1) At room temperature, carboxylated chitosan is dissolved in a solvent to prepare carboxylated chitosan sol; (2) Dissolve the dialdehyde starch in a solvent to form a dialdehyde starch slurry; (3) The carboxylated chitosan sol prepared in step (1) and the dialdehyde starch slurry prepared in step (2) are respectively injected into a bidirectional syringe to form a hydrogel precursor; (4) Sterilize the hydrogel precursor prepared in step (3) to obtain the sterilized hydrogel precursor; (5) The sterilized hydrogel precursor prepared in step (4) is delivered to the bleeding point, adhered, and cross-linked in situ to obtain carboxylated chitosan / dialdehyde starch hemostatic hydrogel.

2. The hemostatic hydrogel as described in claim 1, characterized in that, The mass-volume ratio of carboxylated chitosan to solvent in step (1) is 6-12:

100.

3. The hemostatic hydrogel as described in claim 1, characterized in that, The degree of carboxylation of the carboxylated chitosan in step (1) is ≥60%.

4. The hemostatic hydrogel as described in claim 1, characterized in that, The dialdehyde starch described in step (2) is prepared by the following method: starch is uniformly dispersed in water, sodium periodate solution is added, the mixture is stirred and reacted, excess ethylene glycol is added to remove the remaining sodium periodate, the mixture is filtered, washed, and the reaction continues until no chloride ions are detected by silver nitrate standard solution. The mixture is then repeatedly washed and dehydrated with anhydrous ethanol, and then freeze-dried under vacuum for 20-30 hours to obtain dialdehyde starch. The degree of aldehyde substitution of the dialdehyde starch is 30-95%.

5. The hemostatic hydrogel as described in claim 1, characterized in that, In step (2), the mass-volume ratio of the dialdehyde starch to the solvent is 12:100, and the temperature is set to 60-75℃.

6. The hemostatic hydrogel as described in claim 1, characterized in that, The solvent mentioned in steps (1) and (2) is one or more of water, physiological saline, PBS buffer, etc.

7. The hemostatic hydrogel as described in claim 1, characterized in that, In step (3), the volume ratio of carboxylated chitosan sol to dialdehyde starch slurry in the bidirectional syringe is 1:

1.

8. The use of the hemostatic hydrogel as described in any one of claims 1-7 in the preparation of hemostatic materials.

9. The use of the hemostatic hydrogel according to any one of claims 1-7 in the preparation of medical hemostatic materials.

10. The use of the hemostatic hydrogel according to any one of claims 1-7 in the preparation of hemostatic materials for in vivo bleeding.

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