A double-protein composite hemostatic material based on fiber type protein-globulin synergistic assembly and a preparation method and application thereof

Abstract

The application discloses a kind of double protein composite hemostatic materials based on fiber class protein-globulin synergistic assembly and its preparation method and application, the double protein composite hemostatic materials mainly is by structural skeleton component and functional filling component through the physical assembly of no exogenous chemical crosslinking agent is formed;Structural skeleton component is the fiber class protein derivative obtained by fiber class protein after catechol modification;Functional filling component is the globulin derivative obtained by globulin after cationic polymer modification.The application first establishes the general "fiber class protein-globulin" double protein composite hemostatic material technical platform, and realizes uniform assembly and bidirectional synergistic effect at molecular level by the specific interaction between catechol group and cationic polymer.The preparation process of the application does not use chemical crosslinking agent throughout, and the material has good biocompatibility, and can be widely applied to arterial hemorrhage, internal hemorrhage, surgical wound bleeding and chronic refractory wound hemostasis and repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a dual-protein composite hemostatic material based on the synergistic assembly of fibrin-globulin, its preparation method and application. Background Technology

[0002] Traumatic bleeding is a leading cause of death and disability worldwide. Statistics show that approximately 30% of traumatic deaths are caused by uncontrollable bleeding, with more than half of these occurring before the patient reaches a medical facility. In clinical settings, rapid and massive bleeding from arterial rupture, internal organ damage, extensive surgical wound oozing, and war trauma poses a significant challenge to existing hemostatic materials.

[0003] Currently, commonly used hemostatic materials in clinical practice mainly include gelatin sponges, oxidized cellulose, chitosan-based materials, and zeolite-based hemostatic powders. However, these materials generally have significant limitations: gelatin sponges and oxidized cellulose have weak wet adhesion, making it difficult to maintain close contact with the wound surface under high-flow-rate blood flushing, and their coagulation initiation speed is slow; although chitosan-based materials have certain adhesion and coagulation capabilities, their mechanical properties are poor, and their degradation rate is difficult to control; although zeolite-based hemostatic powders achieve rapid hemostasis, they can cause local high-temperature burns, and residual particles may lead to inflammatory reactions and tissue adhesions.

[0004] From the perspective of the development of protein-based hemostatic materials, natural fibrous proteins generally possess excellent mechanical properties and biocompatibility, but they share common problems such as insufficient active coagulation activity and limited wet adhesion in their natural state. Globulin-based coagulation factors, while exhibiting highly efficient coagulation activity, have extremely poor adhesion to wound surfaces when used alone, are easily washed away by blood, and have poor mechanical properties, making them difficult to form independently. For example, fibrous proteins such as silk fibroin and collagen can form a three-dimensional network framework, but lack active coagulation activity in their natural state; globulins such as fibrinogen and thrombin are key components of the coagulation cascade reaction, but are difficult to retain stably on wound surfaces when used alone.

[0005] To resolve this contradiction, researchers have attempted to combine fibrin and globulin to prepare hemostatic materials. However, existing technologies have failed to overcome the following fundamental technical bottlenecks: (1) There is a lack of universal and mild protein functionalization modification methods, making it difficult to introduce complementary functional groups without destroying the biological activity of the two types of proteins; (2) The molecular structure, solubility, and surface charge of fibrin and globulin are very different, making it impossible to achieve uniform assembly at the molecular level and resulting in poor functional synergy; (3) The potential for bidirectional synergy between the two types of proteins in terms of "structure-function" has not been recognized, and they are simply superimposed as independent functional units; (4) In order to improve the stability of the composite, exogenous chemical cross-linking agents such as glutaraldehyde and genipin are often used, which not only have cytotoxicity but also seriously destroy the biological activity of the proteins.

[0006] While sulfur-fluoride exchange (SuFEx) click chemistry has been applied in protein modification and bioconjugation due to its mild reaction conditions and high site selectivity, there are no reports on its use in constructing dual-protein synergistic hemostatic materials. Therefore, developing a universal technology to achieve the specific assembly of fibrinoids and globulins at the molecular level, fully leveraging their bidirectional synergistic effect, and constructing composite hemostatic materials with strong wet adhesion, efficient active coagulation ability, and good biocompatibility is a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides a dual-protein composite hemostatic material based on the synergistic assembly of fibrinoids and globulins, along with its preparation method and applications. This invention establishes for the first time a universal "fibrinoid-globulin" dual-protein composite hemostatic material technology platform. Utilizing the high efficiency, specificity, and mildness of SuFEx click chemistry, the two types of proteins are functionalized at specific sites. Through the specific non-covalent interaction between catechol groups and cationic polymers, uniform assembly and bidirectional synergistic effects at the molecular level are achieved, ultimately yielding a composite hemostatic material with comprehensive performance exceeding that of a single protein or physical mixture.

[0008] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a dual-protein composite hemostatic material based on the synergistic assembly of fibrin-globulin.

[0010] The dual-protein composite hemostatic material is formed by physical assembly of structural skeleton components and functional filler components, without the addition of exogenous chemical cross-linking agents during the preparation process; the physical assembly refers to the absence of covalent cross-linking between the two types of protein molecules, and the stability of the composite network is maintained by non-covalent interactions and the physical cross-linking of the fibrous proteins themselves; the covalent modification of protein side chains and functional groups does not fall under the category of exogenous chemical cross-linking.

[0011] The structural backbone component is a fibrous protein derivative obtained by modifying fibrous protein with catechol; the functional filler component is a globulin derivative obtained by modifying globulin with cationic polymer.

[0012] As a specific implementation scheme, the physical assembly refers to the synergistic effect of structural skeleton components and functional filler components through electrostatic interactions, hydrogen bonding, and cation-π interactions, combined with the physical cross-linking formed by the β-sheet conformational transformation of fibrous proteins, to jointly constitute a stable molecular-level complex network.

[0013] As a specific implementation scheme, the fibrous protein is selected from at least one of silk fibroin, collagen, gelatin, elastin, and spider silk protein; preferably, it is silk fibroin. The globulin is selected from at least one of fibrinogen, thrombin, albumin, immunoglobulin, and transferrin; preferably, it is fibrinogen.

[0014] As a specific implementation scheme, the fibrous protein being modified with catechol means that the fibrous protein is covalently linked to the catechol group via a sulfonamide bond (-SO2-NH-) generated by the SuFEx click chemical reaction; the globulin being modified with a cationic polymer means that the globulin is covalently linked to the cationic polymer via a sulfonamide bond generated by the SuFEx click chemical reaction.

[0015] As a further embodiment: the catechol group is derived from at least one of dopamine, norepinephrine, levodopa, or gallic acid, preferably dopamine; the cationic polymer is at least one of polyethyleneimine (PEI), polylysine, polyarginine, chitosan, or polyamidoamine (PAMAM); preferably a branched polyethyleneimine with a molecular weight of 500-5000 Da, more preferably a branched polyethyleneimine with a molecular weight of 1800-2000 Da; the catechol group is covalently linked to tyrosine and / or lysine residues of fibrous proteins via sulfonamide bonds; the cationic polymer is covalently linked to tyrosine and / or lysine residues of globulins via sulfonamide bonds.

[0016] In a specific implementation, the mass ratio of the structural skeleton component to the functional filler component is 1:1 to 10:1, preferably 2:1 to 5:1, and more preferably 3:1. This ratio range is crucial for achieving the best synergistic effect; a ratio that is too high or too low will weaken the synergistic effect.

[0017] As a specific implementation, the composite material is in the form of a sponge, gel, film, powder, or injectable formulation, preferably a porous sponge-like structure.

[0018] Secondly, the present invention provides a method for preparing the aforementioned dual-protein composite hemostatic material based on the synergistic assembly of fibrin-globulin, comprising the following steps:

[0019] (1) Preparation of structural framework components: The fibrous protein was dissolved in the corresponding solvent to fully expand the protein domains; a fluorosulfonyl imidazolium salt reagent was added to react with the fibrous protein to introduce -OSO2F active groups on its tyrosine residues and / or lysine residues; then a compound containing catechol groups and primary amines was added to the reaction system, and a SuFEx click chemistry reaction was carried out under weakly alkaline conditions to form sulfonamide bonds and covalently link the catechol groups to the fibrous protein; after the reaction was completed, the fibrous framework component powder was obtained by dialysis or ultrafiltration purification and freeze drying.

[0020] (2) Preparation of functional filler components: Dissolve globulin in aqueous buffer, add fluorosulfonyl imidazolium salt reagent, react with globulin to introduce -OSO2F active groups on its tyrosine residues and / or lysine residues; then add cationic polymer, carry out SuFEx click chemistry reaction under weakly alkaline conditions to form sulfonamide bonds, and covalently link the cationic polymer to globulin; after the reaction is completed, purify by dialysis or ultrafiltration and freeze dry to obtain functional filler component powder.

[0021] (3) Synergistic assembly: The functional filling component solution is slowly added dropwise to the structural framework component solution under stirring conditions. Through the synergistic effect of electrostatic interaction, hydrogen bonding and cation-π interaction, the molecular-level physical assembly is achieved to obtain a homogeneous complex solution.

[0022] (4) Conformational transformation and curing: Ultrasonic treatment is used to induce a β-sheet conformational transformation of fibrin, followed by freeze-drying to obtain the dual-protein composite hemostatic material based on the co-assembly of fibrin and globulin. Preferably, when preparing a porous sponge-like structure, a pore-forming agent can be added and gradient pre-freezing can be performed before freeze-drying.

[0023] As a specific implementation plan:

[0024] When the fibrous protein is silk fibroin, the solvent in step (1) is lithium bromide solution with a concentration of 6~12 M, preferably 9.3 M;

[0025] The fluorosulfonyl imidazolium salt mentioned in steps (1) and (2) is fluorosulfonyl imidazolium trifluoromethanesulfonate;

[0026] In step (1), the reaction temperature of the fluorosulfonyl imidazolium salt with the fibrous protein is 0-4 °C, and the reaction time is 1-3 hours; the pH of the SuFEx click chemistry reaction is 7.5-9.0, and the reaction time is 1-2 hours at room temperature; the mass ratio of the fibrous protein and the compound containing catechol group and primary amine is 10:1 to 2:1.

[0027] In step (2), the aqueous buffer is PBS buffer or physiological saline buffer with pH 7.4; the reaction temperature of the fluorosulfonyl imidazolium salt with globulin is 0-4 ℃, and the reaction time is 1-2 hours; the pH of the SuFEx click chemistry reaction is 7.5-9.0, and the reaction time is 1-2 hours at room temperature; the mass ratio of globulin to cationic polymer is 10:1 to 1:1.

[0028] In step (3), before the dropwise addition, the functional filler component solution and the structural framework component solution are concentrated by ultrafiltration; the physical assembly is carried out at room temperature and under stirring conditions of 200-500 rpm, and the dropwise addition rate of the functional filler component solution is 0.2-1 mL / min; the solid content of the complex solution is 3-5 wt%.

[0029] In step (4), the ultrasonic treatment is intermittent, with a power ratio of 20-50%, and the system temperature is controlled below 25°C.

[0030] The preparation method described in this invention does not use any exogenous chemical crosslinking agents throughout the entire process.

[0031] Thirdly, the present invention provides applications of the aforementioned dual-protein composite hemostatic material, which can be used in the preparation of hemostatic products, wound repair dressings, tissue adhesives, or drug carriers. Specifically, the hemostatic products are used for hemostasis of arterial bleeding, visceral bleeding, superficial traumatic bleeding, or surgical wound oozing; the wound repair dressings are used for the repair of diabetic foot ulcers, burn wounds, or chronic, non-healing wounds.

[0032] This invention is the first to discover that protein systems with a structural division of labor of "fibrous proteins as the backbone and globulins as fillers" can produce the following bidirectional synergistic effects by introducing catechol groups and cationic polymers through SuFEx click chemistry:

[0033] 1. The fibrous cytoskeleton provides a triple, universal enhancement of globulin function:

[0034] Anti-erosion enhancement: The three-dimensional network formed by fibrin protein firmly fixes the globulin inside the material through physical entanglement, fundamentally solving the common problem that globulin is easily washed away by high-flow-rate blood when used alone, enabling globulin to continuously play a coagulation role at the wound site;

[0035] Concentration enrichment enhancement: The catechol groups on the skeleton surface and the cationic polymers on the globulin surface achieve high-density enrichment of globulin on the fiber surface through electrostatic interaction, hydrogen bonding and cation-π interaction, which significantly increases the local coagulation factor concentration and accelerates coagulation initiation.

[0036] Coagulation acceleration and enhancement: The ordered conformation of fibrin (such as β-sheet) can provide a natural template for the conformational transition of globulins, accelerate the coagulation cascade reaction, and promote the rapid formation of blood clots.

[0037] 2. Globulin filling provides dual and universal reinforcement of the fibrous skeleton's properties:

[0038] Enhanced adhesion properties: The positive charge on the surface of globulin can generate strong electrostatic interaction with the negatively charged groups on the tissue surface. At the same time, it enhances the binding force between catechol groups and tissue through cation-π interaction, making the wet adhesion strength of the composite network significantly higher than that of a single fiber skeleton.

[0039] Enhanced mechanical properties: Cationic globulins act as "molecular bridges" to crosslink adjacent fibrin chains, filling the pores of the fiber network and significantly improving the tensile strength and rupture resistance of the composite network, enabling the hemostatic barrier to withstand higher blood pressure impacts.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0041] 1) A universal dual-protein composite hemostatic material technology platform has been established for the first time: This invention breaks through the limitation of specific protein combinations and is applicable to all protein systems that conform to the structural division of "fibrous skeleton + globulin filling," providing a brand-new technical route for the development of next-generation hemostatic materials. Based on this platform, a series of hemostatic materials that meet different clinical needs can be developed by replacing different fibrous proteins and globulins.

[0042] 2) Achieving true bidirectional synergistic effect: This invention reveals and utilizes for the first time the "structure-function" complementary relationship between fibrous proteins and globulins, enabling the composite material to achieve comprehensive performance exceeding that of a single protein or a simple physical mixture of the two, achieving a technical effect of 1+1>2.

[0043] 3) The modification method is universal and mild: The SuFEx click chemistry reaction is universally applicable to various proteins, and the reaction conditions are mild. It can achieve site-specific modification without destroying the higher-order structure and biological activity of the protein, thus preserving the protein's function to the greatest extent.

[0044] 4) No chemical cross-linking agents, excellent biocompatibility: The composite network is constructed entirely through physical processes, avoiding problems such as cytotoxicity, protein denaturation and inflammatory reactions caused by chemical cross-linking agents, and significantly improving the biosafety of the material.

[0045] 5) The preparation process is easy to scale up and adapt to multiple scenarios: By adjusting the amount of pore-forming agent and the protein ratio, the porosity and water absorption rate of the material can be precisely controlled, which can meet the strong adhesion and erosion resistance requirements of high-flow-rate arterial bleeding scenarios, as well as the blood adsorption and exudate management requirements of chronic wound bleeding scenarios, and is easy to scale up.

[0046] 6) Precisely adjustable grafting rate and high process flexibility: This invention allows for precise control of the grafting rate of functional groups within a certain range by adjusting the mass ratio of fluorosulfonyl imidazolium trifluoromethanesulfonate to protein. When the mass ratio of protein to activator is within the range of 10:1-2:1, the modification degree of tyrosine sites increases accordingly with the increase of activator dosage. The catechol grafting rate can be flexibly controlled within the range of 40-110 μg / mg, and the grafting rate of globulin-side cationic groups can be simultaneously controlled by the activator dosage ratio. The appropriate ratio can be selected according to the performance requirements of the target hemostasis scenario, adapting to different clinical application scenarios from venous oozing to arterial bleeding. Attached Figure Description

[0047] Figure 1 For comparison of whole blood clotting time in vitro (n=3);

[0048] Figure 2 For comparison of burst pressure performance (n=3);

[0049] Figure 3 Comparison of femoral artery hemostasis and transection experiments in rats (n=3);

[0050] Figure 4 Comparison chart of burst pressure performance for universality verification (n=3);

[0051] Figure 5 Comparative diagram of rat femoral artery transection hemostasis experiment for universal validation (n=3);

[0052] Figure 6 The graph shows the results of the cytotoxicity test (n=3). Detailed Implementation

[0053] The technical solution of the present invention is illustrated below with detailed examples. However, it is worth noting that the scope of protection of the present invention is not limited to the specific embodiments described.

[0054] If specific techniques or conditions are not explicitly described in the examples, operations can be performed according to the techniques or conditions described in relevant literature in the field, or by following the relevant product instructions. Reagents or instruments, unless otherwise specified by manufacturer, are conventional products that can be purchased through legal channels.

[0055] Those skilled in the art should understand that, without departing from the principles of this invention, the substitution of other fibrous proteins (such as collagen and gelatin) and other globulins (such as thrombin and albumin) is within the scope of protection of this invention.

[0056] Example 1: Preparation of a dual-protein composite hemostatic sponge (silk fibroin-fibrinogen combination)

[0057] 1. Preparation of structural framework components (taking silk fibroin as an example)

[0058] Weigh 1.0 g of degummed silk fibroin and add it to 50 mL of 9.3 M lithium bromide solution. Heat in a 60 °C water bath and stir for two hours until completely dissolved. After cooling the solution to room temperature, transfer it to an ice bath for pre-cooling, and purge with inert gas throughout the process.

[0059] Weigh 0.25 g of fluorosulfonyl imidazolium trifluoromethanesulfonate and prepare an activator solution. The mass ratio of degummed silk fibroin to fluorosulfonyl imidazolium trifluoromethanesulfonate is 4:1. Weigh 0.08 g of sodium bicarbonate solid and slowly add it in portions to the stirred silk fibroin solution. Then, add the activator solution dropwise in portions to the reaction system and react in an ice bath for 1.5 hours.

[0060] Weigh 0.15 g of dopamine hydrochloride and dissolve it in 1 mL of pre-deoxygenated ultrapure water. Under ice bath, nitrogen protection, and light protection, slowly add the dopamine solution dropwise to the above reaction solution at a rate of 0.5 mL / min. After the addition is complete, continue the reaction in the ice bath for 30 min, then slowly raise the temperature to room temperature (25 °C) and react for 1.5 h, controlling the pH of the system to be 7.5. The mass ratio of the degummed silk fibroin fiber to dopamine hydrochloride is 4:1.

[0061] After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 30 kDa and dialyzed against 5 mM PBS buffer (pH 7.4) at 4 °C for 48 h, with the dialysate being changed every 6 h. After dialysis, a purified silk fibroin-dopamine derivative solution (structural backbone component) was obtained.

[0062] 2. Preparation of functional filler ingredients (taking fibrinogen as an example)

[0063] Weigh 0.33 g of bovine fibrinogen and add it to 30 mL of pre-cooled 0.9% physiological saline. Stir gently at 200 rpm in an ice bath until completely dissolved (1 h). Weigh 0.1 g of fluorosulfonyl imidazolium trifluoromethanesulfonate and prepare an activator solution. The mass ratio of fibrinogen to fluorosulfonyl imidazolium trifluoromethanesulfonate is approximately 3.3:1. Weigh 0.05 g of sodium bicarbonate and dissolve it in pre-deoxygenated ultrapure water to prepare an alkaline solution. Under ice bath conditions and gentle stirring, add the alkaline solution and activator solution sequentially to the fibrinogen solution and mix thoroughly. React in an ice bath for 1.5 h.

[0064] Weigh 0.1 g of branched-chain polyethyleneimine (PEI, molecular weight 1800 Da) and dissolve it in 5 mL of pre-chilled 5 mM PBS buffer (pH 7.4). Add the PEI solution dropwise to the reaction solution at a rate of 0.3 mL / min. After reacting in an ice bath for 30 min, slowly raise the temperature to room temperature (25 °C) and react for 1.5 h, maintaining the pH of the system at 7.5. The mass ratio of bovine fibrinogen to branched-chain polyethyleneimine is 3:1.

[0065] After the reaction was completed, the reaction solution was placed into a dialysis bag with a molecular weight cutoff of 30 kDa and dialyzed against 5 mM PBS buffer (pH 7.4) at 4 °C for 48 h, with the dialysate being changed every 6 h. After dialysis, a purified fibrinogen-PEI derivative solution (functional filler component) was obtained.

[0066] 3. Preparation of a dual-protein composite hemostatic sponge (silk fibroin-fibrinogen combination)

[0067] The two protein solutions were concentrated separately using ultrafiltration centrifuge tubes with a molecular weight cutoff of 30 kDa at 4 °C and 3000 rpm (15 min). The structural framework component was concentrated to 5 wt%, and the functional filler component to approximately 2.5 wt%. At room temperature and with stirring at 300 rpm, the concentrated functional filler component was slowly added dropwise to the concentrated structural framework component at a rate of 0.5 mL / min, with a mass ratio of 3:1 (structural framework: functional filler). After the addition was complete, stirring was continued for 30 min to obtain a homogeneous and transparent complex solution with a total protein solids content of approximately 4 wt%.

[0068] The complex solution was transferred to an ice bath and subjected to intermittent ultrasonic treatment using a probe-type ultrasonic instrument: 40% power, 3 seconds of operation followed by a 5-second pause, for a total treatment time of 1 minute. The temperature of the system was controlled throughout the process, not exceeding 25 °C. This induced a β-sheet conformational change in silk fibroin, resulting in a homogeneous and elastic semi-solid composite gel. The composite gel was poured into a custom-made silicone mold and pre-frozen at -80 °C for 4 hours. Subsequently, it was transferred to a freeze dryer and freeze-dried at -55 °C under a vacuum of ≤10 Pa for 24 hours, yielding a porous, sponge-like dual-protein composite hemostatic material.

[0069] Example 2: Synergistic Effect Verification Experiment

[0070] To verify the synergistic effect of the composite hemostatic material of the present invention, the following four groups of samples were set up for comparative testing:

[0071] Group A: The synergistically assembled composite hemostatic material prepared in Example 1 of this invention.

[0072] Group B: Individual structural framework components (silk fibroin-dopamine derivatives), prepared under the same conditions as in Example 1;

[0073] Group C: Individual functional filler components (fibrinogen-PEI derivatives), prepared under the same conditions as in Example 1;

[0074] Group D: A physical mixture of unmodified natural silk fibroin and natural fibroinogen, in the same amounts as in Example 1;

[0075] Group E: Commercially available gelatin sponge.

[0076] 1. Comparison of in vitro whole blood clotting time

[0077] The test was conducted according to ISO 10993-4:2017 standard: Fresh New Zealand rabbit whole blood was collected and anticoagulated with 3.2% sodium citrate (volume ratio 9:1). Materials from each group were cut into 5mm diameter discs and placed in 24-well plates. 200 μL of anticoagulated whole blood and 10 μL of 0.2 M CaCl2 solution were added to each well to initiate clotting. The plates were incubated at 37 ℃, and the blood flow was observed by tilting the plates every 5 seconds. The complete clotting time was recorded. Each experiment was repeated three times. Data are expressed as mean ± standard deviation and statistically analyzed using one-way ANOVA.

[0078] The results are as follows Figure 1As shown, the in vitro whole blood clotting time of group A in this invention was 27.7 ± 2.5 s, significantly shorter than other control groups (P<0.01), demonstrating extremely strong active coagulation ability. Compared with group C, the coagulation rate of group A increased by 2.8 times, proving that the silk fibroin backbone has a coagulation-enhancing effect on fibrinogen. The three-dimensional network of silk fibroin anchors and enriches fibrinogen on the fiber surface, preventing it from being diluted by blood and accelerating the initiation of the coagulation cascade reaction. Compared with group B, the coagulation rate of group A increased by 3.4 times, proving that cationic fibrinogen endows the material with highly efficient active coagulation ability, making up for the deficiency of insufficient coagulation activity of natural silk fibroin. Compared with group D, it is proven that the two unmodified proteins cannot achieve effective assembly at the molecular level, resulting in poor functional synergy. The complementary functional groups introduced by the SuFEx site-specific modification in this invention are the key to achieving synergistic effects between the two. The coagulation rate of group A increased by 3.6 times compared with the commercially available gelatin sponge group E. The above results demonstrate that the synergistic assembly design of the present invention, consisting of "catechol-modified fibrous protein backbone + cationic globulin functional filler", can produce a synergistic coagulation effect of 1+1>2, providing core technical support for rapid hemostasis.

[0079] 2. Comparison of Explosive Pressure

[0080] A self-made burst pressure testing device was used to evaluate the instantaneous compressive strength of the material: a circular material with a diameter of 10 mm was sealed and fixed in the test chamber, and PBS buffer was pumped in at a rate of 2 mL / min. The maximum pressure at which the material ruptured was recorded. Each experiment was repeated 3 times.

[0081] like Figure 2 The results show that the burst pressure of Group A in this invention can reach 195.0±5.7 mmHg, which is higher than all other control groups (P<0.01) and exceeds the upper limit of normal systolic blood pressure (139 mmHg), effectively resisting arterial blood pressure impact and meeting the mechanical requirements for arterial bleeding cessation. Compared with Group B, the burst pressure of Group A increased by about 68.5%, proving that cationic fibrinogen, as a "molecular bridge," can crosslink adjacent silk fibroin fibers through multiple non-covalent interactions, filling the pores of the fiber network and improving the overall mechanical strength and rupture resistance of the composite network. The burst pressure of Group C, which is filled with a single function, is extremely low and cannot form a continuous structure with mechanical support, further proving that the silk fibroin skeleton is a necessary basis for the mechanical properties of materials. Compared with Group D, the burst pressure of Group A increased by about 81.7%, indicating that the two types of unmodified proteins can only achieve simple blending and cannot form an effective crosslinking and synergistic network at the molecular level; by introducing complementary functional groups through SuFEx site-specific modification and achieving synergistic assembly, the mechanical synergistic effect can be fully utilized. Compared with commercially available gelatin sponge group E, group A has a burst pressure of approximately 3.1 times, demonstrating that the material of this invention has a more prominent application advantage in high-pressure arterial bleeding scenarios.

[0082] 4. Comparison of femoral artery transection hemostasis experiments in rats

[0083] The hemostatic effect of four groups of samples was tested using a rat femoral artery transection bleeding model. The results showed that the hemostasis time and blood loss in group A of this invention were significantly lower than those in the other groups, fully demonstrating the comprehensive performance improvement brought about by the synergistic assembly of fibrin and globulin.

[0084] Figure 3 The results showed that the hemostasis time in Group A of this invention was 52.0 ± 3.7 s, shorter than all other control groups (P < 0.01), with small fluctuations within the group and stable hemostasis effect, proving that the material can quickly form a stable hemostatic barrier in hypertensive arterial bleeding scenarios. Compared with single components, Group B, with its single structural framework, had a hemostasis time as long as 223.0 s, with significant individual differences, indicating that relying solely on the three-dimensional network of silk fibroin and the adhesion of catechol cannot quickly initiate the coagulation cascade reaction, resulting in low hemostasis efficiency. Group C had a hemostasis time exceeding 380 s, almost failing to achieve effective hemostasis, because it was rapidly washed away by the high-speed arterial blood flow, failing to form a sustained coagulation effect at the wound surface. Both results confirm that the anti-erosion support of the framework and the active coagulation effect of the functional components are indispensable.

[0085] Compared with group D, the hemostasis time of group A was shortened by 82.1%, indicating that simple physical mixing cannot achieve molecular-level synergy between the two types of proteins, and the functional components cannot be effectively enriched and fixed, only exerting a limited synergistic effect; while the present invention, through complementary modification and synergistic assembly, ultimately produces an in vivo hemostasis effect of 1+1>2.

[0086] Compared to commercially available gelatin sponge group E, group A showed approximately 2.1 times faster hemostasis, demonstrating significantly superior overall hemostatic performance compared to commonly used clinical hemostatic products. Combined with burst pressure test results, it is evident that the material's simultaneous high wet adhesion strength and high rupture resistance are the core guarantees for rapid and stable hemostasis under high arterial pressure, making it exceptionally valuable in clinical scenarios such as trauma emergency care and intraoperative hemostasis.

[0087] Example 3: Platform Universality Validation Experiment (Collagen-Thrombin Combination)

[0088] To verify the versatility of the technical platform of this invention, collagen was used as a fibrous skeleton and thrombin was used as a globulin filler. The composite hemostatic material was prepared according to the same method as in Example 1, and its hemostatic performance was tested.

[0089] Figure 4 The burst pressure of the collagen-thrombin co-assembly group (Group A') reached 148.7 mmHg. Figure 5The results showed that the hemostasis time in the rat femoral artery was 71.3 s, which was superior to the other three control groups (P<0.01). Among them, group B' was the collagen skeleton group alone, group C' was the thrombin function group alone, and group D' was the unmodified physical mixture group.

[0090] Compared with the collagen backbone group alone (Group B'), the synergistic group showed a 61.6% increase in burst pressure and a 65.0% reduction in hemostasis time, demonstrating that cationic thrombin can enhance the mechanical properties of the collagen network through molecular bridging, while compensating for the deficiency of collagen's active coagulation activity. Compared with the thrombin function group alone (Group C'), the synergistic group showed a 79.9% reduction in hemostasis time, demonstrating that the three-dimensional collagen backbone can effectively anchor thrombin, preventing it from being rapidly flushed away by arterial blood flow and achieving long-term coagulation function. Compared with the unmodified physical mixture group (Group D'), the synergistic group showed a 74.3% increase in burst pressure and a 72.1% reduction in hemostasis time, demonstrating that after introducing complementary functional groups through SuFEx site-specific modification, the two types of proteins can achieve molecular-level synergistic assembly, with performance far superior to simple physical superposition.

[0091] The above results demonstrate that the "fibrin-globulin" synergistic assembly mechanism of the present invention has universal applicability and can be extended to various combinations of fibrin and globulin. Among them, the silk fibroin-fibrinogen combination in Example 1 exhibits superior mechanical strength and hemostatic efficiency, and is the optimal embodiment of the present invention.

[0092] Example 4 Biosafety Evaluation

[0093] 1. Cytotoxicity test

[0094] Cytotoxicity of the material was evaluated using mouse fibroblast L929 cells and quantitative analysis was performed using the CCK assay. 100 μL of each of the following solutions (experimental group: extracts from Examples 1 and 3; blank group: DMEM medium containing 10% fetal bovine serum; positive group: paclitaxel extract) was added to 96-well plates seeded with cells, with three replicates per group. The absorbance (OD) at 450 nm was measured using a microplate reader. The relative growth rate (RGR) was calculated using the following formula: Relative growth rate (RGR) % = OD 实验组 / OD 空白组 *100%.

[0095] The cytotoxicity level is determined based on the cytotoxicity grade, with the positive control not lower than grade 3. A cell grade of 0-1 is considered acceptable.

[0096] Grading Standards for Epicellular Proliferation Response

[0097]

[0098] The results are as follows Figure 6 As shown, the relative proliferation rates of Examples 1 and 3 were both higher than 85%. According to the toxicity classification standard of ISO 10993-1, the cytotoxicity was grade 0-1, which did not affect cell proliferation and showed good cell compatibility.

[0099] 2. Hemolysis test

[0100] For Examples 1 and 3, prepare 10 mL suspensions, add rabbit blood, incubate in a water bath for 60 min, centrifuge at 1000 rpm for 5 min, collect the supernatant, and measure the absorbance at 540 nm. The positive control group used 10 mL distilled water with 0.2 mL rabbit blood, and the negative control group used 10 mL physiological saline with 0.2 mL rabbit blood; the procedures were identical. Each group was divided into triplicate.

[0101] The hemolysis rate is calculated using the following formula: Hemolysis rate (%) = (Absorbance of test sample - Absorbance of negative control) / (Absorbance of positive control - Absorbance of negative control) * 100%.

[0102] Table of hemolysis rate (n=3)

[0103]

[0104] Different materials, when in direct contact with blood, may cause red blood cell rupture and hemolysis. A hemolysis test evaluates the hemolysis potential by examining hemoglobin concentration. The hemolysis rates in Examples 1 and 3 are both acceptable (GB / T4233.2 specifies a hemolysis rate of less than 5%). Therefore, the prepared composite hemostatic material meets international standards.

[0105] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A dual-protein composite hemostatic material based on the synergistic assembly of fibrin-globulin, characterized in that, The dual-protein composite hemostatic material is mainly formed by the physical assembly of structural skeleton components and functional filler components without external chemical cross-linking agents. The structural backbone component is a fibrous protein derivative obtained by modifying fibrous protein with catechol; the functional filler component is a globulin derivative obtained by modifying globulin with cationic polymer.

2. The dual-protein composite hemostatic material based on the synergistic assembly of fibrin and globulin according to claim 1, characterized in that, The physical assembly refers to the synergistic effect of structural backbone components and functional filler components through electrostatic interactions, hydrogen bonding, and cation-π interactions, combined with the physical cross-linking formed by the β-sheet conformational transformation of fibrous proteins, to jointly constitute a stable molecular-level complex network.

3. The dual-protein composite hemostatic material based on the synergistic assembly of fibrin and globulin according to claim 1, characterized in that, The fibrous protein is selected from at least one of silk fibroin, collagen, gelatin, elastin, and spider silk protein; preferably silk fibroin. The globulin is selected from at least one of fibrinogen, thrombin, albumin, immunoglobulin, and transferrin; preferably fibrinogen.

4. The dual-protein composite hemostatic material based on the synergistic assembly of fibrin and globulin according to claim 1, characterized in that, The fibrous protein being modified with catechol refers to the fibrous protein being covalently linked to a catechol group via a sulfonamide bond (-SO2-NH-) generated by a SuFEx click chemical reaction; The globulin being modified with a cationic polymer refers to the globulin being covalently linked to the cationic polymer via sulfonamide bonds generated through a SuFEx click chemical reaction.

5. The dual-protein composite hemostatic material based on the synergistic assembly of fibrin and globulin according to claim 4, characterized in that, The catechol group is derived from at least one of dopamine, norepinephrine, levodopa or gallic acid, preferably dopamine; The cationic polymer is at least one of polyethyleneimine (PEI), polylysine, polyarginine, chitosan, or polyamide-amine (PAMAM); preferably a branched polyethyleneimine with a molecular weight of 500-5000 Da, more preferably a branched polyethyleneimine with a molecular weight of 1800-2000 Da. The catechol group is covalently linked to the tyrosine and / or lysine residues of the fibrous protein via sulfonamide bonds; the cationic polymer is covalently linked to the tyrosine and / or lysine residues of the globulin via sulfonamide bonds.

6. The dual-protein composite hemostatic material based on the synergistic assembly of fibrin and globulin according to claim 1, characterized in that, The mass ratio of the structural skeleton component to the functional filler component is 1:1 to 10:1, preferably 2:1 to 5:1, and more preferably 3:

1.

7. The dual-protein composite hemostatic material based on the synergistic assembly of fibrin and globulin according to claim 1, characterized in that, The composite material is in the form of a sponge, gel, film, powder, or injectable formulation, preferably a porous sponge-like structure.

8. The method for preparing the dual-protein composite hemostatic material based on the synergistic assembly of fibrin-globulin according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Preparation of structural framework components: Fluorosulfonyl imidazolium salt reagent was added to the fibrous protein solution to react with the fibrous protein; then a compound containing catechol group and primary amine was added to the reaction system to carry out SuFEx click chemistry reaction. After the reaction was completed, the structural framework components were purified to obtain the structural framework components. (2) Preparation of functional filler components: Fluorosulfonyl imidazolium salt reagent was added to the aqueous buffer of globulin to react with globulin; then cationic polymer was added to carry out SuFEx click chemistry reaction. After the reaction was completed, the functional filler components were purified to obtain the functional filler components. (3) Synergistic assembly: The functional filling component solution is slowly added dropwise to the structural framework component solution under stirring conditions. The molecular-level physical assembly is carried out through non-covalent interaction to obtain a homogeneous complex solution. (4) Conformational transformation and curing: Ultrasonic treatment is used to induce β-sheet conformational transformation of fibrin to form a composite gel; then freeze-drying is performed to obtain the dual-protein composite hemostatic material based on the co-assembly of fibrin and globulin.

9. The preparation method according to claim 8, characterized in that, In step (1), the solvent of the fibrous protein solution is lithium bromide, with a concentration of 6-12M, preferably 9.3M; The fluorosulfonyl imidazolium salt mentioned in steps (1) and (2) is fluorosulfonyl imidazolium trifluoromethanesulfonate; In step (1), the reaction temperature of the fluorosulfonyl imidazolium salt with the fibrous protein is 0-4 ℃, and the reaction time is 1-3 hours; the pH of the SuFEx click chemistry reaction is 7.5-9.0, and the reaction time is 1-2 hours at room temperature; the mass ratio of the fibrous protein to the fluorosulfonyl imidazolium trifluoromethanesulfonate is 10:1 to 2:1, and the mass ratio of the fibrous protein to the compound containing catechol groups and primary amines is 10:1 to 2:

1. In step (2), the aqueous buffer is PBS buffer or physiological saline buffer with pH 7.4; the reaction temperature of the fluorosulfonyl imidazolium salt with globulin is 0~4 ℃, and the reaction time is 1-3 hours; the pH of the SuFEx click chemistry reaction is 7.5-9.0, and the reaction time is 1-2 hours at room temperature; the mass ratio of globulin to fluorosulfonyl imidazolium trifluoromethanesulfonate is 10:1 to 2:1, and the mass ratio of globulin to cationic polymer is 10:1 to 1:1; In step (3), before the dropwise addition, the functional filler component solution and the structural framework component solution are concentrated by ultrafiltration; the physical assembly is carried out at room temperature and under stirring conditions of 200-500 rpm, and the dropwise addition rate of the functional filler component solution is 0.2-1 mL / min; the solid content of the complex solution is 3-5 wt%. In step (4), the ultrasonic treatment is intermittent ultrasonic with a power ratio of 20-50% and the system temperature is controlled below 25°C; a pore-forming agent can be added and gradient pre-freezing can be performed before freeze drying.

10. The use of the dual-protein composite hemostatic material based on the co-assembly of fibrin-globulin as described in any one of claims 1-7 in the preparation of hemostatic products, wound repair dressings, tissue adhesives or drug carriers.

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