Preparation method of a hydrogel hemostatic patch with underwater adhesiveness

Abstract

The present invention provides a method for preparing a hydrogel hemostatic patch with underwater adhesiveness. The preparation method of the patch includes: mixing hyaluronic acid modified with o-aminophenylboronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water to prepare an HPAA hydrogel precursor solution; adding the hydrogel precursor solution into a mold and irradiating it with ultraviolet light to obtain the product. The weight part ratio of the hyaluronic acid modified with o-aminophenylboronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water is (13-17):(9-11):(140-160):(0.5-1.5):(300-350). The purpose of the present invention is to provide a method for preparing a hydrogel hemostatic patch with underwater adhesiveness for rapid and strong underwater tissue adhesion.

Description

Technical Field

[0001] The present invention relates to the technical field of hemostatic patches, and particularly relates to a preparation method of a hydrogel hemostatic patch with underwater adhesiveness. Background Art

[0002] The main cause of death during surgical operations and trauma processes is uncontrollable bleeding, accounting for more than 30% of trauma-related deaths. When the body's natural blood coagulation process cannot control rapid bleeding, timely use of hemostatic materials is crucial for rapid hemostasis and saving lives. Traditional hemostatic materials include dry sponges, gauzes, bandages or tourniquets, and then the wound is sutured with sutures or staples. However, combining these dry hemostatic materials with sutures or staples has some disadvantages, including time-consuming, complex operation and causing additional tissue damage. These disadvantages reduce the survival rate of patients in emergency situations and significantly limit the efficiency of bleeding treatment.

[0003] Hydrogel patches can provide strong wet tissue adhesiveness, biomimetic mechanical strength and elasticity. In the prior art, a polyacrylic acid-related dry cross-linked tape is disclosed. This hydrogel tape removes the interfacial water of tissues through the mechanical action of pressure and self-swelling, so that the carboxyl groups on the surface of PAA therein can form strong ionic cross-links with the amine groups in tissues within 5 seconds, and then covalent cross-links gradually form between the NHS ester groups therein and the primary amine groups of tissues within a few minutes. In recent years, researchers have also combined various interactions into hydrogel patches according to the adhesion mechanism of PAA. By adding charge interaction, π-π interaction, cation-π interaction, hydrogen bond and hydrophobic effect, etc., to enhance the cross-linking of hydrogel patches, significant progress has been made in improving toughness, controlling swelling, reducing potential inflammation and improving biocompatibility. These improvements have promoted the better clinical application of PAA-related hydrogel patches.

[0004] However, in a multi-aqueous environment, most current hydrogel patches cannot quickly remove the interfacial water in contact with the patch, resulting in slow adhesion activation and unstable adhesion performance. This remains a challenge that limits the use of sticky patches in real scenarios. In real surgical operations or underwater injuries, the injured site is often immersed in water or body fluids, generating not only a wet interface, but a highly tissue-hydrated interface. Most PAA patches in the prior art take more than 5 seconds to drain the wet interfacial water layer through pressure, and the drainage effect in an underwater environment is not good. In addition, it is difficult to apply sufficient external pressure to these previously reported patches when the operating space and manpower are limited. That is to say, if the patch cannot quickly remove the interfacial water between tissues, it will lose adhesion due to surface hydration and cannot adhere completely, which greatly limits the success rate of most existing hemostatic patches.

[0005] Currently, most existing wet tissue hydrogel patches mainly rely on physical compression and swelling to exclude and absorb interfacial water during the adhesion process. In a highly hydrated environment, this method is usually slow and inefficient. In view of this, the present invention proposes a method for preparing a hydrogel hemostatic patch with underwater adhesion performance. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing a hydrogel hemostatic patch with underwater adhesion for rapid and strong underwater tissue adhesion.

[0007] The present invention provides a method for preparing a hydrogel hemostatic patch with underwater adhesion. The preparation method of the patch includes: mixing hyaluronic acid modified with o-aminophenylboronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water to prepare an HPAA hydrogel precursor solution; adding the hydrogel precursor solution into a mold and irradiating it with ultraviolet light to obtain the product.

[0008] Further, the weight part ratio of the hyaluronic acid modified with o-aminophenylboronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water is (13 - 17):(9 - 11):(140 - 160):(0.5 - 1.5):(300 - 350).

[0009] Further, the weight part ratio of the hyaluronic acid modified with o-aminophenylboronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water is 15:10:150:1:324.

[0010] Further, the wavelength of the ultraviolet light is 360 - 370 nm, and the irradiation time is 9 - 11 min.

[0011] Further, the preparation method of the hyaluronic acid modified with o-aminophenylboronic acid includes: dissolving hyaluronic acid in deionized water, sequentially adding N-hydroxysuccinimide and 1-(3-dimethylpropyl)-3-ethylcarbodiimide for mixing reaction, then adding 2-aminophenylboronic acid hydrochloride to obtain a mixed solution, adjusting the pH of the mixed solution to 4 - 5 for reaction, then adjusting it to pH 7.5 - 8.5 to stop the reaction, then performing dialysis, freezing and then putting it into a freeze dryer for freeze-drying to obtain the product.

[0012] Further, the weight part ratio of the hyaluronic acid, deionized water, N-hydroxysuccinimide, 1-(3-dimethylpropyl)-3-ethylcarbodiimide and 2-aminophenylboronic acid hydrochloride is (8 - 12):(180 - 220):(6 - 8):(10 - 14):(4 - 6).

[0013] Further, the pH is adjusted by adding an aqueous sodium hydroxide solution with a concentration of 0.8 - 1.2 M. After the pH reaches 4 - 5, the reaction needs to be stirred at room temperature for 11 - 13 h.

[0014] Further, the dialysis step includes: using a dialysis bag with a molecular weight cut-off MWCO of 3000 - 4000 Da, dialyzing with an aqueous sodium chloride solution with a mass concentration of 0.8 - 1% for 46 - 50 h, and then further dialyzing with deionized water for 22 - 26 h.

[0015] Further, the preparation method of the acetylated β-cyclodextrin includes: dissolving β-cyclodextrin and triethylamine in anhydrous N,N-dimethylformamide, stirring and reacting at 0 - 1 °C under nitrogen protection at 250 - 350 r / min for 0.8 - 1.2 h, then adding acryloyl chloride and continuing to stir and react at room temperature at 250 - 350 r / min for 10 - 14 h. After filtration, the precipitate is washed, redissolved in deionized water, frozen, and then placed in a freeze dryer for freeze-drying into a sponge-like shape to obtain the product.

[0016] Further, the weight ratio of β-cyclodextrin, triethylamine, anhydrous N,N-dimethylformamide, and acryloyl chloride is (8 - 12):(6 - 8):(55 - 66):(4 - 8).

[0017] Further, the washing method of the precipitate is to precipitate with ethyl acetate. The amount of ethyl acetate added is more than 50 times the volume of the precipitate, and it is filtered and washed more than three times.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention proposes a novel HPAA hydrogel patch. After the patch contacts the tissue surface, the phenylboronic acid ester bonds on the surface will quickly break and absorb water, thereby significantly enhancing water absorption and improving the adhesion speed and performance. By introducing phenylboronic acid ester bonds into the polyacrylic acid system, the patch can quickly adhere to the wet tissue surface within 1 - 2 seconds and exhibit a strong adhesion strength of up to 163.2 kPa. In addition, the phenylboronic acid ester bonds endow the patch with detachable performance, and the patch can be easily detached from the adhered tissue using a glucose solution. At the same time, the mechanical properties of the patch can be optimized by adjusting the ratio of covalent and non-covalent interactions, thereby achieving high strain (2296%) and high toughness (82 kPa). The in vivo and in vitro experimental results both show that the patch performs excellently in rapid organ hemostasis and sutureless stitching. Therefore, the HPAA patch shows broad biomedical application prospects in the preparation of hemostatic and wound treatment materials due to its excellent underwater adhesion, mild on-demand peeling performance, adjustable mechanical strength, and good biocompatibility.

[0020] In addition, the dynamic bonding characteristics of the phenylborate ester bond enable the glucose solution to gently and painlessly separate the patch on demand, thus effectively reducing the potential inflammatory response associated with the slow degradation of PAA. In addition, the HPAA1 patch adopts a multi-network crosslinking system, combining dynamic bonds (such as phenylborate ester bonds and hydrogen bonds) with a small amount of non-dynamic bonds (such as covalent C-C bonds), providing easily adjustable toughness and strength to adapt to the softness and hardness of biological tissues. Preliminary in vitro and in vivo animal experiments have verified the potential of the HPAA1 patch in a variety of medical applications, including rapid hemostasis and wound closure without sutures.

[0021] In this invention, HA-PBA, Ac-βCD and AA are also used as dynamic physical crosslinking agents, and α-ketoglutaric acid is used as an initiator to initiate polymerization under 365nm ultraviolet light, successfully preparing the HPAA1 hydrogel patch. The formulation of this patch is stable, and the preparation method is simple, with significant economic value. The HPAA1 hydrogel shows extensive biomedical application potential in the preparation of hemostatic and wound treatment patches due to its excellent underwater adhesion, gentle on-demand peeling performance, adjustable mechanical strength and good biocompatibility. At the same time, introducing phenylborate ester bonds into the PAA adhesion system can quickly remove interfacial water, thus improving the underwater adhesion speed and stability. Brief Description of the Drawings

[0022] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the embodiments or technical descriptions. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 Schematic diagrams for the formation, adhesion and application of the HPAA hydrogel; among them, 1A is the hydrogel network diagram, 1B is the adhesion principle diagram, 1C is the glucose solution release principle diagram, and 1D is the application prospect diagram of the HPAA patch.

[0024] Figure 2 Schematic diagrams for the synthesis of HAPBA and AC-β-CD; among them, 2A is the HAPBA synthesis reaction formula, 2B is the AC-β-CD synthesis reaction formula, 2C is the NMR results diagram of HA and HA-PBA, and 2D is the NMR results diagram of β-CD and AC-β-CD.

[0025] Figure 3Characterization diagrams of HAAA and HPAA hydrogels; among them, 3A is the Fourier infrared diagram, 3B is the swelling schematic diagram, 3C is the swelling statistical chart, 3D is the statistical chart of elastic modulus (G’) and viscous modulus (G”) at a fixed frequency within the strain range of 0.1% to 10000%, 3E is the statistical chart of dynamic frequency sweep measurement at a constant strain of 1% and a frequency between 0.1 and 100 Hz, 3F is the scanning electron microscopy (SEM) diagram, and 3G is the statistical chart of pore diameter.

[0026] Figure 4 Diagram showing that HPAA1 has good self-healing ability; among them, 4A is the statistical chart of the step oscillation test under continuous alternating strain scanning (1% to 10000%), and 4B to 4D are the schematic diagrams showing the self-healing ability.

[0027] Figure 5 Diagram of the tensile properties of HPAA and HAAA hydrogels; among them, 5A is the schematic diagram of the fracture toughness experiment, 5B is the tensile stress-strain curve diagram, 5C is the application display diagram of the underwater bandage, and 5D is the skin surface adhesion display diagram of the bent joint.

[0028] Figure 6 Diagram of the adhesion and separation of HAAA and HPAA hydrogels underwater; among them, 6A to 6F are the adhesion performance diagrams of wet pig skin, 6G is the underwater pig skin adhesion display diagram, 6H is the schematic diagram of the underwater adhesion principle, and 6I is the representative diagram of painless and non-invasive separation on demand in glucose solution.

[0029] Figure 7 Diagram of the adhesion performance of HAAA and HPAA hydrogels to various materials and organs; among them, 7A is the schematic diagram of the adhesion to iron, rubber, plastic, glass, and polytetrafluoroethylene; 7B is the schematic diagram of the adhesion to wet organ tissues (heart, liver, spleen, lung, kidney), and 7C to 7H are the adhesion curves and statistical charts of wet organs.

[0030] Figure 8 Diagram of the biocompatibility and biodegradability of HPAA hydrogel patches; among them, 8A is the co-culture schematic diagram, 8B is the live-dead staining diagram of co-cultured cells, 8C to 8E are the in vitro and in vivo degradation and statistical charts, and 8F is the in vivo biosafety test diagram.

[0031] Figure 9 Diagram of the application of HPAA patches in wound closure of the full-thickness skin incision model in rats; among them, 9A is the schematic diagram of model establishment, and 9B is the schematic diagram of skin wound healing.

[0032] Figure 10 Diagram of the histological evaluation of wound healing after treatment with HPAA patches; among them, 10A is the HE and Masson staining diagrams, 10B and 10C are the statistical charts of related indicators, 10D is the immunofluorescence staining diagram, and 10E and 10F are the statistical charts of related indicators.

[0033] Figure 11 It is a graph of the hemostatic properties of the HPAA patch in vivo; among them, 11A is a schematic diagram of hemostasis for liver injury, 11B is a schematic diagram of the operation process for hemostasis of liver injury, 11C and 11D are schematic diagrams and statistical graphs of liver bleeding volume, 11E is a schematic diagram of HE staining of liver tissue after hemostasis, 11F is a schematic diagram of hemostasis of the tail, 11G and 11H are schematic diagrams and statistical graphs of tail bleeding volume, 11I is a schematic diagram of hemostasis for heart injury, and 11J is a schematic diagram of the operation process for hemostasis of heart injury.

[0034] Figure 12 It is a schematic diagram of the potential application of the HPAA patch; among them, 12A is a schematic diagram of lung injury repair and experimental results, 12B is a schematic diagram of heart injury repair and experimental results, and 12C is a schematic diagram of gastric injury repair and experimental results. Detailed implementation manners

[0035] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] It should be noted that in the present invention, hyaluronic acid (HA) (Mw = 35 kDa) is purchased from Yuanye (Shanghai, China); N-hydroxysuccinimide (NHS), 1-(3-dimethylpropyl)-3-ethylcarbodiimide (EDC), acrylic acid (AA), and acryloyl chloride are all purchased from Aladdin (Shanghai, China); 2-aminophenylboronic acid hydrochloride (PBA) is purchased from Leyan (Shanghai, China); β-cyclodextrin (β-CD) is purchased from Macklin (Shanghai, China); and α-ketoglutaric acid is purchased from Sigma-Aldrich.

[0037] Embodiment

[0038] Refer to Figures 1 to 3 In this embodiment, a preparation method of a hydrogel patch is provided:

[0039] Preparation of hyaluronic acid modified with o-aminophenylboronic acid (HA-PBA): First, dissolve 10 parts by weight of HA in 200 parts by weight of deionized water, then successively add 7 parts by weight of N-hydroxysuccinimide (NHS) and 12 parts by weight of 1-(3-dimethylpropyl)-3-ethylcarbodiimide (EDC), and mix and react for 1 h; subsequently, add 5 parts by weight of PBA to the solution, adjust the pH to 4.5 using a 1 M sodium hydroxide (NaOH) solution, stir and react at 300 r / min at room temperature for 12 h, then adjust the pH to 8 with 1 M NaOH, put the mixture into a dialysis bag with a molecular weight cut-off MWCO: 3500 Da, dialyze with an aqueous solution containing 0.9% sodium chloride for 48 hours, and then further dialyze with deionized water for 24 hours;

[0040] The modification rate of HA-PBA after dialysis and freeze-drying was determined by 1HNMR to be 30%. Please refer to Figure 2 2A and 2C in

[0041] Preparation of acetylated β-cyclodextrin (AC-β-CD): Dissolve 10 parts by weight of β-CD and 7 parts by weight of triethylamine in 60 parts by weight of anhydrous DMF, then stir and react for 1 h at 0 °C under nitrogen protection. Subsequently, add 5 parts by weight or 7 parts by weight of acryloyl chloride to the solution, stir and react at room temperature for 12 h, filter the liquid through a sintered glass funnel, precipitate with more than 50 times the precipitation volume of ethyl acetate and wash more than 3 times; then, redissolve the solid in 10 times the volume of deionized water, and obtain low-modification-degree AC-β-CD (Ac-β-CD-low) and high-modification-degree AC-β-CD (Ac-β-CD-high) after freeze-drying. The modification rates determined by 1HNMR were 150% and 200% respectively. Please refer to Figure 2 2B and 2D in

[0042] HA-PBA and AC-β-CD synthesized by the above method, together with acrylic acid (AA), α-ketoglutaric acid, and water, are used as the precursor solution for preparing the HPAA hydrogel.

[0043] The precursor solution of the HAAA patch used as a control for the HPAA patch contains HA, Ac-β-CD, acrylic acid (AA), α-ketoglutaric acid, and water (without borate ester bonds).

[0044] The detailed compositions of all hydrogels are listed in Table 1, and all raw materials are in parts by weight values.

[0045] Add the above precursor solution to a polytetrafluoroethylene patch mold according to the ratio in the table, and obtain the formed and dried HPAA patch and HAAA patch after irradiating with 365 nm ultraviolet light for 10 min.

[0046] Compared with the other three groups, the formulation of the HPAA1 patch is stable and has appropriate underwater adhesion and mechanical properties. On the one hand, HAAA1 is used as a control to compare the role of phenylborate ester bonds in HPAA1; on the other hand, HPAA2 is also used as a control to prove that HPAA1 has a more suitable degree of Ac-β-CD substitution (i.e., the concentration of C=C double bonds).

[0047] Table 1 Formulation Material Table

[0048]

[0049] After testing, Fourier transform infrared (FTIR) spectroscopy verified the successful synthesis of the HPAA hydrogel and the presence of phenylborate ester bonds and hydrogen bonds therein, as shown in 3A of Figure 3 .

[0050] Test Example 1

[0051] Characterization of the hydrogel:

[0052] The equilibrium swelling ratio (ESR) was determined by the weighing method. After ultraviolet irradiation, the hydrogel was immersed in pure water and then placed in an incubator at 37 °C. The hydrogel samples were weighed every 1 hour until the swelling equilibrium state was reached.

[0053] The calculation formula of ESR is as follows: ESR = (W t −W d ) / W d × 100%.

[0054] Among them, W t is the weight of the hydrogel at the swelling equilibrium state, and W d is the initial weight of the hydrogel.

[0055] After soaking for 5 h, the swelling degrees of HAAA1 and HPAA1 were about 25 times and 21 times respectively, while the swelling degrees of HAAA2 and HPAA2 were about 7 times and 5 times respectively. During the entire swelling process, the swelling degree of the HAAA1 and HPAA1 patches was faster than that of HAAA2 and HPAA2 because the latter had more covalent cross-linking, making the structure more compact and restricting the water absorption capacity. In addition, the HPAA1 patch also showed the fastest swelling trend in the initial stage because the phenylborate ester bond increased the water absorption rate, as shown in 3B and 3C of Figure 3 .

[0056] Rheological measurements were carried out using a Kinexus rheometer equipped with 25 mm diameter parallel plates. A dynamic amplitude sweep test was used to determine the linear viscoelastic region, and the elastic modulus (G’) and viscous modulus (G”) were recorded at a fixed frequency in the strain range of 0.1% to 10,000%. Dynamic frequency sweep measurements were performed at a constant strain of 1% and frequencies between 0.1 and 100 Hz to further characterize the rheological properties of the hydrogels.

[0057] The strain amplitude sweep test showed that the gel point modulus value of the HPAA hydrogel containing phenylborate ester bonds was significantly higher than that of the HAAA hydrogel, and the G’ and G” modulus values of the HPAA2 hydrogel containing highly substituted Ac-β-CD were even higher, as shown in Figure 3 3D and 3E in

[0058] The morphology of the hydrogels was analyzed by scanning electron microscopy (SEM). After rapid freezing in liquid nitrogen for 30 s, the sample stage with the attached sample was transferred to the sample preparation chamber. After sublimation at -90 °C for 10 min, it was gold-plated with a current of 10 mA for 60 s and then sent to the scanning electron microscope sample chamber for observation. SEM imaging was performed using a FEI Quanta 450 instrument at an acceleration voltage of 3.0 kV, and the pore size of each sample was calculated using the tools of ImageJ.

[0059] The experimental results showed that the HPAA2 hydrogel with a higher degree of substitution of Ac-β-CD exhibited an obvious dense structure and smaller pore size, which was due to more covalent cross-linking of the Ac-β-CD groups in the poly(AA / Ac-β-CD) chains, and this was related to its smaller absorption rate and lower swelling rate, as shown in Figure 3 3F and 3G in

[0060] Test Example 2

[0061] Mechanical self-healing performance of HPAA: A step oscillation test was performed on the HPAA1 hydrogel diluted 1.3 times for 5 consecutive cycles under continuous alternating strain scans (1% to 10,000%) of the rheometer. Please refer to Figure 4 4A in Figure 4 4B to 4D in

[0062] Test Example 3

[0063] Mechanical properties: The mechanical properties of the materials were evaluated at room temperature using a universal testing machine (CMT-1104, SUST, Zhuhai, China). Four specimens (30×15×2 mm) of each material were tested at a rate of 100 mm / min. Please refer to Figure 5 5A in . The fracture toughness was determined using the stress-strain curve. The force magnitude was recorded and then calculated according to the formula:

[0064] where F is the plateau force in the peeling test, and W and L are the length and width of the sample, respectively. The tensile stress-strain curves showed that the tensile strengths of the HAAA1, HAAA2, HPAA1, and HPAA2 hydrogels were 32 kPa, 54 kPa, 45 Pa, and 107 kPa, respectively, and the fracture strains were 1812%, 482%, 2334%, and 755%, respectively. Please refer to Figure 5 5B in

[0065] Compared with the control group, the HPAA1 hydrogel had better tensile elongation. This is due to the dynamic non-covalent bond characteristics of the phenylborate ester bond, which provides the hydrogel with more excellent ductility.

[0066] The data in Table 1 also showed that the HPAA1 prepared in the present invention exhibited the highest tensile properties under medium intermolecular forces by adjusting the ratio of covalent bonds and phenylborate ester bonds. In addition, to confirm the underwater tensile properties and adhesiveness of the hydrogel and to confirm its application potential as an underwater bandage, the HPAA1 hydrogel was stretched and wound around the distal limb to evaluate its performance in blocking distal blood supply. Please refer to Figure 5 5C in Figure 5 5D in

[0067] Test Example 4

[0068] Adhesion properties and detachment separation method: On a universal testing machine, lap shear tests and standard 180° peel tests were used to evaluate the tissue adhesion properties of the hydrogel. Biological tissues (i.e., pig skin, heart, stomach, liver) were cut into regular shapes (50×20×3 mm) and first washed with PBS buffer. Two substrate samples (tissue / tissue) were adhered in the middle with the hydrogel (15×15×1 mm). The lap shear test and the standard 180° peel test were carried out at room temperature with a tensile rate of 100 mm / min until failure. The stress-displacement curve in the lap shear test was recorded, and the shear stress was calculated using the formula: , where F max1is the maximum force of the lap shear test, and W and L represent the width and length of the hydrogel sample. The force / width-displacement curve in the 180° peel test was recorded, and the interfacial toughness was calculated using the formula: Interfacial toughness = F max / W, where F max is the maximum force in the 180° peel test, and W represents the width of the hydrogel sample.

[0069] First, HPAA and HAAA were adhered to wet porcine skin to observe their adhesion properties. The results showed that the shear stress values of HAAA1, HAAA2, HPAA1, and HPAA2 were 15.42 kPa, 21.95 kPa, 159.8 kPa, and 115.9 kPa, respectively, and the interfacial toughness values were 96.94 N / m, 56.68 N / m, 750.19 N / m, and 406.23 N / m, respectively. Please refer to Figure 6 Figures 6A to 6F in it.

[0070] Comparing the HPAA patch and the borate-free HAAA patch can show the effect of phenylborate ester bonds on the adhesion properties. The results showed that HPAA1 had the strongest adhesion. Due to the presence of borate ester bonds, the adhesion strength of the HPAA patch to wet porcine skin was significantly higher than that of the HAAA patch. In addition, the adhesion strength of the HPAA1 hydrogel on wet porcine skin was significantly higher than that of HPAA2, which may be due to the higher hardness of HPAA2, reducing its adhesion performance. To prove the adhesion of the hydrogel patch underwater, the porcine skin was placed in a petri dish filled with water, and then the patch was gently placed on the porcine skin. After waiting for 2 seconds, the hydrogel tissue was lifted with forceps to observe its adhesion performance. Without applying pressure, the HPAA1 patch could adhere to the underwater porcine skin surface and stably lift nearly 100 times the porcine skin. Please refer to Figure 6 Figure 6G in it. This enhancement is due to the presence of phenylborate ester bonds. After contacting the tissue, it breaks the water film through self-cleavage, absorbs the interfacial water, and then reacts with the amino groups on the tissue surface. Please refer to Figure 6 Figure 6H in it.

[0071] In addition, the hydrogel also has separability. Two identical skin incisions were made on the back of rats, and the HPAA1 patch was used for wound treatment. Cotton balls containing normal saline or 20% glucose solution were covered on the surface of the hydrogel for the separability test, and the separability of the hydrogel was observed after 20 minutes.

[0072] The results showed that using 20% glucose solution, it could be separated painlessly and non-invasively as needed within 20 minutes without causing any obvious adverse effects on the wound. Please refer to Figure 66I in it. And the glucose solution is pH neutral, does not irritate the wound, and the sterile glucose solution is easily available in clinical practice. The on-demand detachable property of the HPAA1 patch also solves a key problem in wound management.

[0073] In addition, HPAA1 was adhered to a series of representative wet organ tissues (heart, liver, spleen, lung, kidney) and other materials (iron, rubber, plastic, glass, polytetrafluoroethylene) to observe its adhesion performance.

[0074] The experimental results showed that the hydrogel exhibited moderate adhesion to various abiotic materials such as metal, rubber, plastic, and glass, but almost no obvious adhesion to polytetrafluoroethylene. Please refer to Figure 7 7A in it. The animal organ adhesion experiments all showed that the HPAA1 patch exhibited the strongest adhesion to various wet tissues compared with the control group. Please refer to Figure 7 7B to 7H in it.

[0075] Test Example 5

[0076] Cells and animals: The L929 cell line was obtained from KeyGEN BioTECH (Nanjing, China). SD rats (female, 180 - 220 g) were from the animal center of Southern Medical University and were acclimated in the laboratory for one week before the experiment. The animals had free access to food and water. The animal experiment was approved by the Institutional Animal Care and Use Committee of Zhujiang Hospital (Ethical No. LAEC - 2024 - 209).

[0077] Biocompatibility: The in vitro biocompatibility of the HPAA1 hydrogel with L929 fibroblasts was evaluated by a live / dead cell assay. The live / dead cell staining showed that compared with the control group, almost no cell death was observed in the HPAA1 group and the HPAA2 group within 2 days. Please refer to Figure 8 8A and 8B in it.

[0078] The HPAA1 sample (5 mm in diameter, 1 mm in thickness) was implanted into the back of SD rats to evaluate the in vivo biocompatibility test. Tissue samples of the heart, liver, spleen, lung, kidney, skin, etc. were collected on the 28th day and stained with hematoxylin - eosin (HE). The results showed that after 28 days, there was mild fibrosis in the subcutaneous tissue near the implant. Further evaluation of the main organs (heart, liver, spleen, lung, kidney) of the rats showed no obvious histological reaction or pathological changes. Please refer to Figure 8 8F in it.

[0079] Test Example 6

[0080] Degradation behavior of hydrogel: The hydrogel was degraded in vitro and in vivo. The HPAA1 sample at the swelling equilibrium state was placed in 10 mL of PBS (pH = 7.4) (in vitro experiment) or on the back of SD rats (in vivo experiment). The hydrogel samples were harvested and dried on days 0, 7, 14, 21, and 28 in each group.

[0081] The results showed that the HPAA hydrogel gradually degraded over time in vitro. When placed in PBS at 37 °C, the weight of the hydrogel decreased by more than 40% after 28 days.

[0082] In addition, the in vivo experiment showed a degradation pattern similar to that observed in vitro, with a weight loss of more than 40% within 28 days. Please refer to Figure 8 Figures 8C to 8E.

[0083] Test Example 7

[0084] Evaluation of in vivo wound closure: A skin injury model was constructed by making 4 incisions of 2 cm on the back of rats. The wounds were closed with sutures, cyanoacrylate (biological tissue glue, Beijing Ji Tian Biotechnology), and HPAA1. Another group was the blank group. Please refer to Figure 9 Figure 9A. Wound images were taken on days 0, 1, 3, 7, 10, and 14.

[0085] The results showed that compared with the control group, the HPAA1 patch exhibited stronger mechanical strength and could adhere to the skin surface to close the incision even under external forces such as pulling and twisting. From day 7, the patch was dissociated with glucose solution until the end of the observation. There was no wound tearing phenomenon similar to that in the cyanoacrylate treatment group in the HPAA1 group. Please refer to Figure 9 Figure 9B.

[0086] The wound skin tissues were collected on days 7 and 14 respectively, and subjected to HE and Masson's trichrome staining. The images were taken with a 3DHISTECH panoramic scanner. ImageJ software was used to evaluate re-epithelialization and collagen formation.

[0087] The results showed that compared with the control group, the wounds treated with HPAA1 had significantly smaller scars and less inflammation on days 7 and 14. Collagen deposition in the wounds was evaluated by Masson staining. The HPAA1 treatment group showed denser and more regular collagen fibers, similar to the reticular pattern of healthy dermis. On day 14, the HPAA1 group also showed better structural healing, such as a thinner epithelial layer and more hair follicles and subcutaneous glands. Please refer to Figure 10 Figures 10A to 10C.

[0088] IF staining was used to detect the expression of CD31 and α-SMA, and IF staining images were acquired using 3DHISTECH panoramic scanning. Compared with other groups, the CD31 vascular density in the wounds treated with HPAA1 was lower on days 7 and 14. Due to the tensile resistance of the patch, the wound area of the HPAA1-treated wounds was [description missing in the original, so this part is incomplete], and there was no granulation tissue formation. Please refer to Figure 10 in 10D and 10E of

[0089] In addition, in the HPAA1-treated wounds, the expression of α-SMA was significantly decreased on day 7 and then significantly increased on day 14. Please refer to Figure 10 in 10D and 10F of

[0090] In summary, the regulated expression of CD31 and α-SMA in the HPAA1 treatment group more effectively inhibited scar formation and promoted the restoration of normal skin structure.

[0091] Test Example 8

[0092] Hemostatic performance in vivo: The hemostatic performance of the hydrogel was evaluated using a rat liver incision model, a tail amputation model, and a cardiac puncture model.

[0093] The rat liver incision model was constructed by making a 5-mm wound on the liver, and then the wound was covered with an HPAA patch (8×8×0.2 mm). Please refer to Figure 11 in 11A of Figure 11 The HPAA1 patch adhered directly to the wound within 1 - 2 s after contact without the need to apply pressure. Please refer to Figure 11 in 11B of Figure 11 Compared with the control group, the HPAA1 treatment group had a shorter hemostasis time and stronger hemostatic ability. Please refer to Figure 11 in 11C of

[0094] The rat tail amputation model was constructed by cutting the middle part of the mouse tail with surgical scissors, and then an HPAA1 patch (8×8×0.2 mm) was attached to the wound. Please refer to Figure 11 in 11F of Compared with the control group, the HPAA1 patch quickly wrapped the amputated tail and significantly reduced bleeding.Figure 11 in 11G. The quantitative results showed that the total blood loss in the HPAA1 group (about 0.05 g) was much lower than that in the sponge group (about 0.3 g) and the blank group (about 0.9 g). Please refer to Figure 11 in 11H.

[0095] In addition, a rat heart puncture wound model was constructed by the method of a heart puncture wound (2 mm in diameter). The HPAA1 patch (8×8×0.2 mm) was immediately adhered to the bleeding site. Please refer to Figure 11 in 11I. The results showed that applying the HPAA1 patch to the epicardial surface of the beating rat heart could quickly stop bleeding. Please refer to Figure 11 in 11J.

[0096] In summary, the HPAA1 patch effectively sealed the bleeding site and enabled the wound to pass through the bleeding period. Then, the wound achieved hemostasis through natural coagulation.

[0097] Test Example 9

[0098] To evaluate the sealing performance of HPAA1 on damaged lung lobes, HPAA1 was used to treat the laceration (1 cm) of porcine lung lobes to observe the air leakage of the lung lobes in a water bath. The results showed that the damaged lung lobes could be quickly adhered and repaired by the HPAA1 patch (3×1.5×0.5 cm) within 1 - 2 seconds. The sealed porcine lung lobes were subjected to continuous inflation - deflation cycles, and the seal of the HPAA patch could last for more than 6 hours. Please refer to Figure 12 in 12A.

[0099] To evaluate the sealing performance of HPAA1 on damaged hearts, HPAA1 was used to treat the puncture wound (1 cm) of porcine hearts to observe the fluid leakage of the hearts. The results showed that the damaged hearts could be quickly adhered and repaired by the HPAA1 patch (3×1.5×0.5 cm) within 1 - 2 seconds, and the sealed porcine hearts could be preserved at room temperature for more than 6 hours. Please refer to Figure 12 in 12B.

[0100] To evaluate the sealing performance of HPAA1 on damaged stomachs, HPAA1 was used to treat the circular perforation wound (1 cm in diameter) of porcine stomachs to observe the fluid leakage in the stomachs. The hole could be quickly adhered and repaired by the HPAA1 patch (3×1.5×0.5 cm) within 1 - 2 seconds, and the sealed porcine stomachs could be preserved at room temperature for 6 h. Please refer to Figure 12 in 12C. These in vitro models demonstrated the potential of the HPAA1 patch to immediately seal organ air and fluid leakage.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the present invention rather than limiting the technical solutions described in the present invention; those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a hydrogel hemostatic patch with underwater adhesion, characterized in that: The preparation method of the patch comprises: mixing hyaluronic acid modified with o-aminophenylboronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water to prepare a HPAA hydrogel precursor solution; adding the hydrogel precursor solution into a mold and irradiating the mold with ultraviolet light to obtain a hydrogel precursor solution; The preparation method of the o-aminophenylboronic acid modified hyaluronic acid comprises: dissolving hyaluronic acid in deionized water, sequentially adding N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for mixed reaction, then adding 2-aminophenylborate hydrochloride to obtain a mixed solution, adjusting the pH of the mixed solution to 4-5 for reaction, then adjusting the pH to 7.5-8.5 to stop the reaction, then dialyzing, freezing and freezing, and then placing in a freeze dryer for freeze drying to obtain the obtained solution; The preparation method of acetylated β-cyclodextrin comprises: dissolving β-cyclodextrin and triethylamine in anhydrous N,N-dimethylformamide, stirring and reacting at 250-350 r / min for 0.8-1.2 h at 0-1° C. and under nitrogen protection, then adding acryloyl chloride and stirring and reacting at 250-350 r / min for 10-14 h at room temperature, filtering and precipitating, washing, redissolving in deionized water, freezing and freezing, and then putting into a freeze dryer for freeze drying to obtain a sponge-like product.

2. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The weight ratio of the o-aminophenylboronic acid modified hyaluronic acid, acetylated β-cyclodextrin, acrylic acid, α-ketoglutaric acid and water is (13-17): (9-11): (140-160): (0.5-1.5): (300-350).

3. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The wavelength of the ultraviolet light is 360-370 nm, and the irradiation time is 9-11 min.

4. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The weight ratios of the hyaluronic acid, deionized water, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 2-aminophenylborate hydrochloride are (8-12):(180-220):(6-8):(10-14):(4-6).

5. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The pH is adjusted by adding 0.8-1.2 M sodium hydroxide aqueous solution. When the pH is 4-5, the reaction needs to be stirred at room temperature for 11-13 hours.

6. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The dialysis step comprises: using a dialysis bag with a molecular weight cut-off MWCO: 3000-4000Da, dialysis with a sodium chloride aqueous solution containing a mass concentration of 0.8-1% for 46-50 hours, and then further dialysis with deionized water for 22-26 hours.

7. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The weight ratio of the β-cyclodextrin, triethylamine, anhydrous N,N-dimethylformamide and acryloyl chloride is (8-12): (6-8): (55-66): (4-8).

8. The method for preparing a hydrogel hemostatic patch with underwater adhesion according to claim 1, characterized in that: The method for washing the precipitate is to use ethyl acetate for precipitation, add ethyl acetate in an amount of more than 50 times the volume of the precipitate, and filter and wash for more than three times.

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