Medical silk fibroin adhesive for emergency hemostasis and infection resistance and preparation method of medical silk fibroin adhesive

Abstract

The invention discloses a silk fibroin medical adhesive for emergency hemostasis and infection resistance and a preparation method thereof, and belongs to the field of biological material preparation and biomedicine application. Biomimetic marine mussel biological substances such as silk fibroin, sodium alginate and tannic acid are used as components to form the medical adhesive with strong wet adhesion performance, and based on molecular chain entanglement and cross-linking among the materials, the obtained medical adhesive has good biocompatibility and anti-infection performance; in addition, the adhesive has the in-situ spraying capacity, can be directly sprayed on a damaged part, can further derive powder and band-aid forms from the adhesive, and is more convenient to use. The raw materials used by the adhesive prepared by the invention are safe, non-toxic and wide in source, the preparation method is simple, the adhesive is expected to replace a traditional medical adhesive, and high application value is achieved under emergency hemostasis conditions such as battlefields.

Description

Technical Field

[0001] The present invention belongs to the fields of biomaterials and biomedical applications, and particularly relates to a silk fibroin medical adhesive for emergency hemostasis and anti-infection and a preparation method thereof. Background Art

[0002] Bleeding is the main cause of death in traumatic injuries, as massive blood loss often leads to serious complications, including hypotension and multiple organ dysfunction, which can cause severe disruption of the healing cascade, such as uncontrolled hemostasis and long-term inflammation, ultimately resulting in impaired healing progress and inevitable scar formation. Hemorrhage control is time-sensitive, and most deaths occur within the first hour after traumatic injury. Therefore, rapid and effective hemorrhage control is crucial for life-threatening and / or in-compressible bleeding, such as visceral or high-pressure arterial bleeding.

[0003] Currently, the main categories of hemostatic materials include powders, gauzes, hydrogels, and sponges. However, sponges and gauzes are not suitable for wounds with irregular shapes, large or in-compressible bleeding, as they cannot reach deep bleeding sites. On the other hand, conventional hemostatic powders cannot form stable crusts and cannot even be fixed in the blood. In recent years, compared with prefabricated hydrogels, in-situ spray gels have become one of the most promising solutions, which have unique advantages, including excellent portability, enhanced flexibility, and superior conformity to wounds of different sizes and irregular shapes. Unfortunately, due to the lack of hemorrhage management, moisture and breathability regulation, and wound nutrition to support scarless wound healing, current sprayable hydrogels cannot handle a wide range of wounds with complex pathological conditions. At the same time, they do not have sufficient tissue adhesion and mechanical properties, so they can neither stabilize the initial wound nor firmly adhere to and match body movements to protect the wound site. In addition, they cannot provide necessary oxygen diffusion and penetration for the wound while preventing microbial infection. All these limitations may lead to prolonged inflammatory responses, delayed skin epithelialization, and scarring.

[0004] The existing patent CN 118806979A discloses an antibacterial and hemostatic, wound-healing promoting hydrogel and its preparation method. The antibacterial and hemostatic, wound-healing promoting hydrogel is prepared by uniformly mixing gelatin, modified lignin microspheres loaded with polypeptide chelated calcium, water and glycerol, then adding ε-polylysine, and finally adding acrylamide, N,N'-methylenebisacrylamide, and ammonium persulfate, and pouring it into a mold for reaction. This multifunctional hydrogel dressing is based on a bioactive polymer with excellent mechanical properties, aiming to provide antibacterial properties and accelerate the efficiency of wound healing. However, the preparation process of this dressing is complex, the raw materials are numerous, and no relevant data on wet adhesion strength is mentioned. Another example is patent CN 106822989A, which discloses a sprayable gel for promoting wound healing and its preparation method. This sprayable gel is safe and non-irritating, has good biocompatibility, and is used for wound treatment. Its good water absorption enables it to fully absorb the exudate of the wound, and the swelling after water absorption can maintain the physiological moisture of the wound surface. When the total mass fraction is 5-10%, the viscosity of the gel shows a plastic characteristic of decreasing with the increase of the shear rate, which is particularly suitable for spraying. At this concentration, it also has good antibacterial properties, thus protecting against bacterial infection and effectively promoting wound healing. However, the sodium periodate used in this material has a stimulating effect on the skin and may be unfavorable for wound healing. Another example is patent CN118892572A, which discloses a preparation method of a Bletilla striata-silk fibroin hydrogel dressing with antibacterial, hemostatic and repair functions. This dressing is prepared by a freeze-thaw method using Bletilla striata polysaccharide, silk fibroin and PVA as raw materials. The preparation method is simple, and the obtained dressing has good hemostatic and antibacterial effects and mechanical properties, and maintains its good shape during the wound repair process and will not cause secondary damage to the wound. However, the mechanical strength of this dressing is too high to match the strength that the tissue can bear. Summary of the Invention

[0005] The purpose of the present invention is to provide a silk fibroin medical adhesive for emergency hemostasis and anti-infection, which is used for hemostasis and promoting the healing of infected wounds. The present invention deeply biomimics mussels by introducing silk fibroin (SF) and sodium alginate (SA), and constructs a hemostatic material with universality, which exhibits the advantages of strong adhesion, multiple usage forms, good biocompatibility, excellent mechanical properties, wide application range, safety and non-toxicity, and easy operation, and is expected to be used as a medical adhesive to realize potential clinical application value under emergency hemostasis conditions such as on the battlefield.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: A silk fibroin medical adhesive for emergency hemostasis and anti-infection, and its preparation method includes the following steps: (1) Stir and mix anhydrous sodium carbonate in boiling deionized water, add silk fibers for degumming treatment; after treatment, wash and dry, dissolve in lithium bromide solution or calcium chloride / absolute ethanol / water ternary solution, then dialyze in deionized water, and then centrifuge and filter to obtain a silk fibroin solution; (2) Stir and dissolve sodium alginate in deionized water to obtain a sodium alginate solution; (3) Dissolve the biomimetic marine mussel biomaterial in deionized water, stir and mix evenly to obtain a biomimetic marine mussel biomaterial solution; (4) At room temperature, fully mix the obtained silk fibroin solution with the sodium alginate solution, and then fully mix the obtained mixture with the biomimetic marine mussel biomaterial solution to prepare a silk fibroin hydrogel with emergency hemostasis and anti-infection functions.

[0007] Further, the time of the degumming treatment in step (1) is 5 - 120 min, preferably 10 - 100 min.

[0008] Further, the temperature of the drying in step (1) is 5 - 100 °C, preferably 15 - 100 °C; the drying time is 1 - 50 h, preferably 2 - 50 h.

[0009] Further, the concentration of the lithium bromide solution in step (1) is 9.3 mol / L.

[0010] Further, in the calcium chloride / absolute ethanol / water ternary solution in step (1), the molar ratio of calcium chloride, absolute ethanol to water is 1:2:8.

[0011] Further, the mass concentration of the silk fibroin solution obtained in step (1) is 0.1 - 50.0%, preferably 1 - 15%.

[0012] Further, the temperature of the stirring in step (2) is 10 - 80 °C, preferably 20 - 70 °C.

[0013] Further, the mass concentration of the sodium alginate solution obtained in step (2) is 0.01 - 30.0%, preferably 0.1 - 15%.

[0014] Further, the biomimetic marine mussel biomaterial in step (3) is one or more of dopamine, phenolic hydroxyl chitosan, tannic acid, and gallic acid.

[0015] Further, the temperature of the stirring in step (3) is 10 - 60 °C.

[0016] Further, the mass concentration of the biomimetic marine mussel biomaterial solution obtained in step (3) is 0.1 - 80.0%, preferably 5 - 50%.

[0017] Furthermore, the volume ratio of the silk fibroin solution to the sodium alginate solution used in step (4) is 1:500 - 500:1, preferably 1:30 - 30:1; the volume ratio of the mixed solution used to the biomimetic marine mussel biomaterial solution is 1:500 - 500:1, preferably 1:30 - 30:1.

[0018] Furthermore, the mixing method of the mixed solution and the biomimetic marine mussel biomaterial solution in step (4) can be one or more of spraying, injection, and manual stirring.

[0019] Compared with the prior art, the significant advantages of the present invention are as follows: In the marine environment, mussels exhibit excellent adhesion, being able to firmly adhere to solid surfaces such as ships, rocks, and underwater facilities, and remaining stable even under the impact of wind and waves. This adhesion is mainly due to the mussel foot proteins (Mfps) secreted by the mussel byssus, where 3,4-dihydroxy-L-phenylalanine (Dopa) or its catechol group plays a key role in the binding of Mfps to the surface. The medical hydrogel constructed by the present invention using biomimetic ideas is based on three aspects: adhesive groups, dynamic redox balance, and strong mechanical properties, and can more comprehensively and deeply simulate the adhesion of wounds. Taking tannic acid (TA) as an example, initially, TA mimics the Mfp-3 and Mfp-5 components of mussels as the basis for adhesive characteristic functional groups. Due to its large number of phenolic hydroxyl groups, it can effectively ensure the strong interfacial adhesion of the hydrogel; secondly, silk fibroin (SF) is crucial for the long-term adhesion performance of the hydrogel. The hydrophobic amino acids in it reduce the sensitivity of the phenolic hydroxyl groups in tannic acid to oxidation, thus ensuring the long-term excellent adhesion performance of the hydrogel; finally, the added sodium alginate (SA) can improve the mechanical properties of the hydrogel to mimic the role of collagen in mussels. More importantly, the obtained hydrogel can be used as a medical adhesive and further made into three sample forms: powder, anti-adhesion band-aid, and spray, so that it can be used for different types of wound bleeding, and it exhibits the advantages of strong adhesion, good biocompatibility, excellent mechanical properties, wide application range, safety and non-toxicity, and easy operation, realizing potential clinical application value under emergency hemostasis conditions such as on the battlefield. Description of the Drawings

[0020] Figure 1 It is the Fourier transform infrared spectrum of the silk fibroin hydrogel (FTS-G) prepared in Example 2.

[0021] Figure 2 It is the test result diagram of the wet adhesion performance and bursting pressure performance of the silk fibroin hydrogel prepared in Example 2.

[0022] Figure 3Synthesis flowchart (a), microscopic image of water-sprayed gel formation (b), stability diagram of the powder after hydration gelation (c), photo of powder gelation at 37 °C (d), adhesion strength diagram of the powder after gelation at 25 and 37 °C (e), adhesion strength diagram of the powder after gelation in contact with blood and PBS at 37 °C (f), powder temperature-dependent mechanism diagram (g), adhesion strength of the powder after gelation over time diagram (h) for the medical adhesive in powder form (FTS-G) prepared in Example 3.

[0023] Figure 4 Effect diagram of spraying for the medical adhesive in spray form prepared in Example 4.

[0024] Figure 5 Composition and adhesion effect diagram of the medical adhesive in band-aid form prepared in Example 5.

[0025] Figure 6 Antibacterial performance comparison diagram of the medical adhesives prepared in Examples 2 and 3 and commercial products of Comparative Examples 1 and 2. Among them, (a) is the antibacterial activity of different samples against methicillin-resistant Staphylococcus aureus (MRSA), (b) is the scanning electron microscope image of bacteria growing on the surface of different samples, (c) is the resazurin indicator color development experiment of different samples, (d) is the diameter of the inhibition zone of different samples co-cultured with MRSA for 24 h, and (e) is the absorbance of different samples in the resazurin indicator color development experiment.

[0026] Figure 7 Cell compatibility diagram of the medical adhesives prepared in Examples 2 and 3. Among them, (a) is the live / dead staining images of NIH / 3T3 cells cultured in cell culture plates (control), FTS-G (gel sample), and FTS-P (powder sample) for 1, 3, and 5 days, (b) is the hemolysis rate experiment, and (c) is the cytotoxicity test.

[0027] Figure 8 Hemostasis effect comparison diagram of the medical adhesive prepared in Example 4 and commercial products of Comparative Examples 1 and 2. Among them, (a) is the schematic diagram of hemostasis of rat liver and femoral artery, (b, c) are the flowcharts of hemostasis of rat liver and femoral artery, (d, f) are the comparison diagrams of the hemostasis amounts of rat liver and femoral artery, and (e, g) are the comparison diagrams of the hemostasis times of rat liver and femoral artery.

[0028] Figure 9Comparison diagram of the promotion of wound healing in infected rats by the medical adhesives prepared in Example 2 and Example 3 and the commercial product in Comparative Example 3. Among them, (a) is a schematic diagram of the operation for infecting rat wounds, (b) is a photo of injecting bacterial solution into the back and stopping bleeding of wounds in different groups, (c) is a photo of full-thickness infected wounds in different groups on the 3rd, 7th, 10th, and 14th days of treatment, (d) is a simulated diagram of wound healing changes in different groups on the 0th, 3rd, 7th, 10th, and 14th days of treatment, (e) is the skin bleeding condition in different groups (the middle photo is the blood trace wiped by filter paper), (f) is the colony count in different groups on the 0th and 14th days of treatment, and (g) is the statistical situation of the wound healing rate in different groups. Detailed implementation mode

[0029] The preparation of a silk fibroin medical adhesive for emergency hemostasis and anti-infection includes the following steps: (1) Stir and mix anhydrous sodium carbonate in boiling deionized water, add silk, and perform degumming treatment for 5 - 120 min; after treatment, wash, dry at 5 - 100 °C for 1 - 50 h, then dissolve with a lithium bromide solution with a concentration of 9.3 mol / L or a ternary solution of calcium chloride / absolute ethanol / water with a molar ratio of 1:2:8, and then dialyze in deionized water and filter by centrifugation to obtain a silk fibroin solution with a mass concentration of 0.1 - 50.0%; (2) Dissolve sodium alginate in deionized water and stir to dissolve at 10 - 80 °C to obtain a sodium alginate solution with a mass concentration of 0.01 - 30.0%; (3) Dissolve the biomimetic marine mussel biomaterial in deionized water and stir and mix at 10 - 60 °C to obtain a biomimetic marine mussel biomaterial solution with a mass concentration of 0.1 - 80.0%; (4) At room temperature, fully mix the obtained silk fibroin solution and sodium alginate solution in a volume ratio of 1:500 - 500:1, and then fully mix the obtained mixture with the biomimetic marine mussel biomaterial solution in a volume ratio of 1:500 - 500:1 by spraying, injection, and / or manual stirring to prepare a silk fibroin hydrogel with the functions of emergency hemostasis and anti-infection.

[0030] Among them, the biomimetic marine mussel biomaterial in step (3) is one or more of dopamine, phenolic hydroxyl chitosan, tannic acid, and gallic acid.

[0031] Through in vivo and in vitro experiments, it is confirmed that this material not only has good cell and blood biocompatibility, but also integrates the advantages of strong wet adhesion, anti-infection, and promoting repair, and is expected to be used as a medical adhesive to realize potential clinical application value under emergency hemostasis conditions such as on the battlefield.

[0032] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0033] Example 1 (1) Boil 5 L of deionized water, add 8 g of sodium carbonate and stir well to mix. Subsequently, add 15 g of silk, and after degumming treatment for 25 min, wash the degummed silk fibroin fiber after treatment thoroughly with deionized water 8 times, and dry it in an oven at 60 °C for 6 h. Then, put the dried degummed silk fibroin fiber into a 9.3 M lithium bromide solution and dissolve it at 60 °C for 6 h. Then, put it into deionized water for dialysis for 3 days; the dialyzed solution is centrifuged and filtered twice to obtain a silk fibroin (SF) solution with a mass concentration of 8.9%. (2) Dissolve 1.0 g of sodium alginate in 50 mL of deionized water and stir overnight at 50 °C to obtain a sodium alginate (SA) solution with a mass concentration of 2%. (3) Dissolve 15 g of tannic acid (TA) in 50 mL of deionized water and stir and mix evenly at room temperature to obtain a tannic acid solution with a mass concentration of 30%. (4) At room temperature, after mixing the obtained SF solution and SA solution, add the TA solution (the mixing volume ratio is SF solution:SA solution:TA solution = 5:1:5), and stir and mix well to obtain a silk fibroin hydrogel.

[0034] Example 2 (1) Boil 5 L of deionized water, add 8 g of sodium carbonate and stir well to mix. Subsequently, add 15 g of silk, and after degumming treatment for 25 min, wash the degummed silk fibroin fiber after treatment thoroughly with deionized water 8 times, and dry it in an oven at 60 °C for 6 h. Then, put the dried degummed silk fibroin fiber into a 9.3 M lithium bromide solution and dissolve it at 60 °C for 6 h. Then, put it into deionized water for dialysis for 3 days; the dialyzed solution is centrifuged and filtered twice to obtain a silk fibroin (SF) solution with a mass concentration of 8.9%. (2) Dissolve 0.5 g of sodium alginate in 50 mL of deionized water and stir overnight at 50 °C to obtain a sodium alginate (SA) solution with a mass concentration of 1%. (3) Dissolve 15 g of tannic acid (TA) in 50 mL of deionized water and stir and mix evenly at room temperature to obtain a tannic acid solution with a mass concentration of 30%. (4) At room temperature, after mixing the obtained SF solution and SA solution, add TA solution (the mixing volume ratio is SF solution: SA solution: TA solution = 5:1:5), stir well and mix, then silk fibroin hydrogel can be obtained.

[0035] Example 3 (1) Boil 5 L of deionized water, add 8 g of sodium carbonate and stir well, then add 15 g of silk. After degumming for 25 min, wash the degummed silk fibroin fibers thoroughly with deionized water 8 times, dry in an oven at 60 °C for 6 h, then put the dried degummed silk fibroin fibers into 9.3 M lithium bromide solution, dissolve at 60 °C for 6 h, and then dialyze it in deionized water for 3 days; the dialyzed solution is centrifuged and filtered twice to obtain a silk fibroin (SF) solution with a mass concentration of 8.9%; (2) Dissolve 0.5 g of sodium alginate in 50 mL of deionized water, stir overnight at 50 °C to obtain a sodium alginate (SA) solution with a mass concentration of 1%; (3) Dissolve 15 g of tannic acid (TA) in 50 mL of deionized water, stir and mix evenly at room temperature to obtain a tannic acid solution with a mass concentration of 30%; (4) At room temperature, after mixing the obtained SF solution and SA solution, add TA solution (the mixing volume ratio is SF solution: SA solution: TA solution = 5:1:5), stir well and mix, then conduct freeze-drying, and then grind the dried hydrogel into powder with a mortar to obtain a medical adhesive in powder form.

[0036] Example 4 (1) Boil 5 L of deionized water, add 8 g of sodium carbonate and stir well, then add 15 g of silk. After degumming for 25 min, wash the degummed silk fibroin fibers thoroughly with deionized water 8 times, dry in an oven at 60 °C for 6 h, then put the dried degummed silk fibroin fibers into 9.3 M lithium bromide solution, dissolve at 60 °C for 6 h, and then dialyze it in deionized water for 3 days; the dialyzed solution is centrifuged and filtered twice to obtain a silk fibroin (SF) solution with a mass concentration of 8.9%; (2) Dissolve 0.5 g of sodium alginate in 50 mL of deionized water, stir overnight at 50 °C to obtain a sodium alginate (SA) solution with a mass concentration of 1%; (3) Dissolve 15 g of tannic acid (TA) in 50 mL of deionized water, stir and mix evenly at room temperature to obtain a tannic acid solution with a mass concentration of 30%; (4) At room temperature, take SF solution, SA solution, and TA solution in a volume ratio of 5:1:5, then mix the SF solution and SA solution and put them into a spray pot, and put the TA solution into the spray pot alone to obtain a medical adhesive in spray form.

[0037] Example 5 (1) 5 L of deionized water was boiled, 8 g of sodium carbonate was added and stirred, and then 15 g of silk was added. After degumming for 25 min, the degummed silk fibroin fibers were washed 8 times with deionized water and dried in an oven at 60 °C for 6 h. The dried degummed silk fibroin fibers were then placed in a 9.3 M lithium bromide solution and dissolved at 60 °C for 6 h. They were then dialyzed in deionized water for 3 days. The dialyzed solution was centrifuged twice to obtain a silk fibroin (SF) solution with a mass concentration of 8.9%. (2) Dissolve 0.5 g of sodium alginate in 50 mL of deionized water and stir overnight at 50 °C to obtain a sodium alginate (SA) solution with a mass concentration of 1%; (3) Dissolve 15 g of tannic acid (TA) in 50 mL of deionized water and stir at room temperature to obtain a tannic acid solution with a mass concentration of 30%; (4) At room temperature, the obtained SF solution and SA solution were mixed, and then TA solution was added (mixing volume ratio of SF solution: SA solution: TA solution = 5:1:5). The mixture was stirred thoroughly to obtain silk fibroin hydrogel.

[0038] (5) Dissolve 2.96 g of polylactic acid (PLA) particles in 15 mL of dichloromethane (DCM) and 10 mL of N, N -dimethylformamide (DMF) mixed solution to prepare spinning solution; then the obtained spinning was stretched into filaments in the presence of a high voltage electric field with positive and negative electrodes of 8 kV and -8 kV, so that a PLA film with a thickness of 0.2 mm was collected on the surface of the roller release paper.

[0039] (6) Physically bonding the silk fibroin hydrogel obtained in step (4) to the PLA film obtained in step (5), and covering the other side of the hydrogel that is not bonded with a PTFE film, and then cutting it into a desired shape to obtain a medical adhesive in the form of a band-aid.

[0040] Figure 1 This is the Fourier transform infrared spectrum of the silk fibroin hydrogel (FTS-G) prepared in Example 2. As can be seen from the figure, 1706, 1610, 1534 and 1446 cm -1 The peak at 1609 cm -1The peak at [location] belongs to the stretching vibration of C=O on the carboxyl group in SA. The peak at 1660 cm -1 in SF is attributed to the stretching and bending modes of amide I (C=O) and amide II (N-H), and it redshifts to 1531 cm in FTS -1 , which is due to the transformation of SF from an α-helix structure to a β-sheet structure (a). In addition, the broad peak appearing at 3000 - 3600 cm -1 is due to the phenol hydroxyl group (Ar-OH) that remains intact after the chemical reaction, which is the key to the strong wet adhesion of the hydrogel (b). The FTIR results confirm that SA, SF, and TA have successfully combined to form a composite material.

[0041] Figure 2 Figure showing the test results of the wet adhesion performance and burst pressure performance of FTS-G prepared in Example 2. A texture analyzer was used to test the bonding strength of the obtained hydrogel to wet plastic, pig skin, and wood. As can be seen from the figure, the bonding strengths are 197.1 ± 19.2 kPa, 363.4 ± 18.0 kPa, and 708.3 ± 73.6 kPa respectively (a). These strong adhesions are mainly due to the covalent and non-covalent bonds between FTS and various substrates, such as hydrogen bonds, coordination bonds, π-π interactions, and metal chelation. At the same time, the burst pressure performance of the obtained hydrogel was tested. Specifically, a square hole with a side length of 10.0 mm was formed on a hose with a diameter of 15.0 mm, and then a pig skin or sausage casing (with a through hole with a diameter of 4.0 mm in the middle) with a side length of 20.0 mm was fixed on the hose with strong glue. Then, a circular hydrogel with a diameter of 10.0 mm was attached to the prefabricated through hole on the pig skin or sausage casing, and then it was fixed on a pressure gauge. During the experiment, PBS was pushed into the hose at a rate of 10.0 mL min −1 . The maximum pressure that causes the hydrogel to rupture at the defect was determined as the rupture pressure. As can be seen from the figure, the rupture pressures borne by FTS-G attached to pig skin or sausage casing are 180.0 ± 7.5 mmHg and 165.0 ± 7.5 mmHg respectively, and these values exceed the normal human arterial blood pressure (~120 mmHg), indicating that FTS-G has a good effect in preventing pressure-induced arterial bleeding.

[0042] Figure 3Performance characterization diagram of the medical adhesive in powder form (FTS-P) prepared in Example 3. As shown in the figure, the pre-prepared FTS was freeze-dried and ground to obtain SF / TA / SA hemostatic powder; FTS-P underwent a self-fusion process after hydration and rapidly transformed into a gel within 5 s (a). This self-fusion assembly process was further observed under an inverted microscope, and it was found that FTS-P changed from fine powder to bulk gel after hydration (b). To evaluate the bonding stability of FTS-P after it was transformed into a gel form, the powder was evenly placed on a silicone mold. After hydration, mechanical vibration was applied to the mold with forceps, and the results showed that FTS-P did not detach even under vibration conditions after hydration, indicating that the formed hydrogel had excellent adhesion performance (c). In addition, it was also found in the study that the adhesion strength of the powder after gelation would change with temperature. At physiological temperature, its adhesion strength could reach 185.3 ± 13.7 kPa, and similar adhesion strengths could be achieved whether blood or PBS was used for gelation (d-f). The mechanism of the change in the adhesion strength of the hydrogel with temperature was further explored. On the one hand, it was mainly because as the temperature increased, the softened hydrogel fit better to the tissue interface; on the other hand, the increase in temperature exposed more functional groups of the hydrogel, increasing the area of binding to the tissue interface (g). Finally, the change in the adhesion strength of the powder after gelation at physiological temperature with time was also explored. The adhesion strength could reach 100 kPa 5 minutes after the powder was gelated, and it could ultimately reach nearly 200 kPa as time extended (h).

[0043] Figure 4 Effect diagram of spraying the medical adhesive in spray form prepared in Example 4. As can be seen from the figure, its spray had good gel-forming properties and uniform gel formation.

[0044] Figure 5 Composition and adhesion effect diagram of the medical adhesive in the form of a band-aid prepared in Example 5. As can be seen from the figure, it could firmly adhere to a petri dish at physiological temperature and in a wet state.

[0045] Taking commercial hemostatic sponges (Kuai Kang), hemostatic tapes (XUELIAN), and medical antibacterial dressings (3M) as Comparative Examples 1-3 respectively, further experiments were carried out.

[0046] Antibacterial performance is another major issue for hemostatic materials because bacterial infection can lead to persistent inflammation, increased bleeding, and delayed wound healing. To evaluate the antibacterial ability of the medical adhesives prepared in Examples 2 and 3, a series of antibacterial tests were carried out, and the results are shown in Figure 6 . From Figure 6It can be seen that different samples were placed on agar plates coated with methicillin-resistant Staphylococcus aureus (MRSA) and co-cultured at 37 °C. No inhibition zones were detected in the culture dishes of the commercial products in Comparative Examples 1 and 2, while obvious inhibition zones were shown in the culture dishes using the medical adhesives of Examples 2 and 3. Moreover, the MRSA in Comparative Examples 1 and 2 had good morphology, while the MRSA in Examples 2 and 3 showed atrophy, with broken cell membranes and outflow of contents, confirming that the composite products prepared in Examples 2 and 3 had good antibacterial activity (a, b, d). Further, a resazurin indicator color development experiment was carried out to verify its antibacterial ability (in the bacterial proliferation color development experiment, the indicator reacts with the reducing substances produced by MRSA, changing its color from purple to pink or even colorless). As can be seen from the figure, after 24 hours of bacterial co-culture, the color in the wells with the medical adhesives of Examples 2 and 3 remained purple, while the color in the wells with the commercial products of Comparative Examples 1 and 2 turned pink, confirming that the medical adhesives obtained in Examples 2 and 3 effectively inhibited the proliferation of MRSA (c). And the relatively low absorbance values of the medical adhesives of Examples 2 and 3 at 600 nm also quantitatively confirmed this result (d).

[0047] Cytotoxicity is another important prerequisite for the clinical application of hemostatic dressings. Therefore, the LIVE / DEAD staining of mouse embryonic fibroblasts (NIH 3T3) and the Cell Counting Kit-8 (CCK-8) experiment were used to study the cytocompatibility of the medical adhesives prepared in Examples 2 and 3, and the results are shown in Figure 7 . As Figure 7 shown, the NIH3T3 cells treated with the medical adhesives of Examples 2 and 3 presented a healthy spindle-like morphology, just like the control group (a), indicating good cytocompatibility. And through the quantitative analysis of cell proliferation behavior, it can be known that the NIH 3T3 cells cultured in each group showed a good cell proliferation trend (c). In addition, an in vitro hemolysis test was used to evaluate the blood compatibility of the medical adhesives prepared in Examples 2 and 3. The results showed that the supernatant of TritonX-100 was bright red, indicating complete hemolysis. In sharp contrast, the samples treated with the medical adhesives of Examples 2 and 3 and PBS buffer were light pink, and the hemolysis rates were all below 5% (b), fully indicating that the synthesized FTS-G and FTS-P meet the safety standards for clinical use.

[0048] To evaluate the hemostatic ability of the medical adhesive prepared in Example 4, a rat liver transection model and a femoral artery bleeding model were established for in vivo evaluation, and hemostasis was performed by spraying. The results are shown in Figure 8As can be seen from the figure, in the rat liver transection model, compared with the blank group, the gauze group and Comparative Example 1, the use of the medical adhesive of Example 4 had the best hemostatic effect, with a blood loss of 54.8 ± 15.0 mg and a hemostasis time of 6.7 ± 1.5 s (d, e). This good hemostatic effect was further verified in the rat femoral artery bleeding model (c, f, g).

[0049] Bacterial infection is an important factor that must be considered in wound hemostasis and repair. After injecting MRSA subcutaneously into rats for 24 h, a circular wound defect with a diameter of 1 cm was formed at the site of bacterial injection, causing severe infection and bleeding. Then, various samples were applied to these infected wounds to investigate the effects of different samples on the healing of infected wounds. The results are shown in Figure 9 . As shown in the figure, compared with the blank group, the hemostatic effects of the medical adhesives prepared in Examples 2 and 3 were significantly improved and slightly better than that of Comparative Example 3 (b, e). Wound healing is another important step in tissue trauma treatment. It is worth noting that the treatment groups using the medical adhesives of Examples 2 and 3 showed the best wound healing performance during the treatment (c, d, g). Subsequently, bacterial cultures were performed on the wound secretions of different groups on Day 0 and Day 14, and the results showed that the treatment groups using the medical adhesives of Example 2 and Example 3 both showed excellent antibacterial properties (f).

[0050] The above descriptions of the embodiments are only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0051] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection, characterized in that: The following steps are involved: (1) Stirring anhydrous sodium carbonate in boiling deionized water, adding the mixture to silk for degumming; washing and drying the silk after degumming, dissolving the silk with lithium bromide solution or calcium chloride / anhydrous ethanol / water ternary solution, dialyzing the solution in deionized water, and filtering the solution by centrifugation to obtain a silk fibroin solution; (2) stirring and dissolving sodium alginate in deionized water to obtain a sodium alginate solution; (3) dissolving the bionic marine mussel biological material in deionized water, stirring and mixing evenly to obtain a bionic marine mussel biological material solution; (4) The obtained silk fibroin solution and the sodium alginate solution are fully stirred and mixed at room temperature, and then the obtained mixture is fully mixed with the biomimetic marine mussel biological material solution to prepare a silk fibroin hydrogel with emergency hemostasis and anti-infection functions.

2. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The degumming treatment time in step (1) is 5-120 min.

3. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The concentration of the lithium bromide solution in step (1) is 9.3 mol / L; the molar ratio of calcium chloride, anhydrous ethanol and water in the calcium chloride / anhydrous ethanol / water ternary solution is 1:2:

8.

4. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The mass concentration of the silk fibroin solution obtained in step (1) is 0.1-50.0%.

5. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The stirring temperature in step (2) is 10-80°C and the stirring time is 5-72 h.

6. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The mass concentration of the sodium alginate solution obtained in step (2) is 0.01-30.0%.

7. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The bionic marine mussel biological material in step (3) is one or more of dopamine, phenolic hydroxy chitosan, tannic acid, and gallic acid.

8. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The mass concentration of the bionic marine mussel biological material solution obtained in step (3) is 0.1-80.0%.

9. The method for preparing a silk fibroin medical adhesive for emergency hemostasis and anti-infection according to claim 1, characterized in that: The volume ratio of the silk fibroin solution to the sodium alginate solution used in step (4) is 1:500-500:1; the volume ratio of the mixed solution to the biomimetic marine mussel biological material solution is 1:500-500:

1.

10. A silk fibroin medical adhesive for emergency hemostasis and anti-infection prepared by the method of claim 1.

Images