A polyhydroxy phenolic compound loaded silk fibroin complex, and a preparation method and application thereof

Abstract

This invention addresses the shortcomings of existing hemostatic materials in treating large wounds and severe bleeding by providing a silk fibroin complex loaded with polyhydroxyphenolic compounds and its preparation method. The complex is prepared by self-assembly of polyhydroxyphenolic compounds and silk fibroin. The silk fibroin undergoes a β-sheet conformational transformation process. The polyhydroxyphenolic compound is preferably a poorly soluble compound, most preferably ellagic acid, and the mass ratio of polyhydroxyphenolic compound to silk fibroin is <1:1, preferably 1:2 to 1:16, and more preferably 1:4. This complex exhibits significantly superior hemostatic ability compared to polyhydroxyphenolic compounds, silk fibroin, Yunnan Baiyao (a traditional Chinese medicine), tranexamic acid, and chitosan hemostatic powder. It also has a larger particle size and heavier texture than silk fibroin, allowing for rapid sedimentation at the bleeding site in the blood. This complex is non-cytotoxic, non-hemolytic, and self-degradable in vivo, demonstrating high safety. Furthermore, it possesses wound-healing, antibacterial, anti-infective, and anti-adhesion properties, making it suitable for application in the preparation of appropriate formulations.

Description

Technical Field

[0001] This invention belongs to the field of materials technology, specifically relating to a silk fibroin complex loaded with polyhydroxyphenolic compounds, its preparation method, and its application. Background Technology

[0002] Currently, the hemostatic market is dominated by absorbable hemostatic materials, including gauze, sponges, sprays, and hemostatic powders. Their main components are mostly natural polymers such as cellulose, chitosan, and fibrin. Their main advantages include short hemostatic time, biodegradability, relatively mild foreign body reaction, no need for secondary surgery, and avoidance of secondary injury to the patient. However, their high cost, varying absorption times, limited adaptability, and poor effectiveness in large traumas and severe bleeding limit their clinical application. For example, oxidized regenerated cellulose hemostatic materials contain a large number of acidic groups, reaching a pH of 1.7 after full water saturation. In an acidic environment, although they have some antibacterial effect, residual material at the wound site may cause a strong inflammatory reaction and hinder bone regeneration, even leading to granulomas and abscesses. Therefore, it is necessary to develop a fast, efficient, economical, and non-toxic absorbable hemostatic material.

[0003] Silk fibroin, a natural high-molecular-weight protein extracted from silk, possesses excellent in vivo degradability, biocompatibility, and anti-inflammatory properties. In its hemostatic mechanism, silk fibroin activates coagulation factor XII, thereby triggering an intrinsic coagulation cascade. It is a biomolecule composed of 5509 amino acids, consisting of a heavy chain (391 kDa) and a light chain (26 kDa), linked by disulfide bonds. When silk fibroin dissolved in LiBr solution or a ternary solution is added to an excess of a polar organic solvent such as alcohol, a white suspension of the protein complex immediately forms. When silk fibroin remains in solution, due to the multiple interactions between the side chains of the fibroin and salt ions and water molecules, the peptide chain exists in a random coil conformation, i.e., an α-helix. This state is also the native conformation of silk fibroin. In this state, silk fibroin lacks the ability to self-assemble with polyhydroxyphenolic compounds because the steric hindrance of the benzene rings of the amino acid residues makes it difficult for polyhydroxyphenolic compounds to integrate into the α-helix structure, thus exposing them on the outside of the silk fibroin molecule. When an antisolvent such as ethanol is used, the peptide chain is induced to rapidly fold toward a more stable β-sheet assembly. The β-sheet conformation exposes hydrophobic amino acid residues, which can enhance the encapsulation of hydrophobic drugs through hydrophobic interactions and π-π stacking, facilitating subsequent self-assembly with polyhydroxyphenolic compounds.

[0004] Currently available silk fibroin hemostatic agents cannot overcome the obstruction of the hydration layer at the blood interface, resulting in poor adhesion to bleeding wounds and failing to meet the tissue strength adhesion requirements in a wet environment. In contrast, polyhydroxyphenolic compounds can form secondary cross-linked structures with silk fibroin, thereby recruiting more platelets and preventing silk fibroin from being washed away by the blood. Furthermore, polyhydroxyphenolic compounds and silk fibroin exhibit a synergistic effect, promoting intrinsic hemostasis. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies, such as poor effectiveness against large wounds and severe bleeding, insufficient blood absorption and seepage capacity, and inability to treat irregular wounds. This invention provides a silk fibroin complex loaded with polyhydroxyphenolic compounds, its preparation method, and its applications. The technical solution is as follows:

[0006] This invention provides a silk fibroin complex loaded with polyhydroxyphenolic compounds, which is prepared by self-assembly of polyhydroxyphenolic compounds and silk fibroin; wherein the silk fibroin is treated by a β-sheet conformational transformation process, and the feed ratio of the polyhydroxyphenolic compounds to silk fibroin is <1:1 by mass, preferably 1:2 to 1:16, and most preferably 1:4.

[0007] As a further improvement, the polyhydroxyphenolic compound is selected from one or more of the following: resveratrol, quercetin, baicalin, kaempferol, lignans, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolic acid, rosin, puerarin, rhein, honeysuckle glycoside, proanthocyanidins, p-coumaric acid, vanillic acid, hesperidin, naringenin, luteolin, genistein, rhein, oleanolic acid, epigallocatechin gallate, robinin, tea polyphenols, epigallocatechin, tanshinone, gallic acid, pyrogallic acid, catechin, and tannic acid.

[0008] As a further improvement, the polyhydroxyphenolic compound is a poorly soluble polyhydroxyphenolic compound, preferably one or more of the following: resveratrol, quercetin, baicalin, kaempferol, lignans, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolic acid, rosin, puerarin, rhein, honeysuckle glycoside, proanthocyanidins, p-coumaric acid, vanillic acid, hesperidin, naringenin, luteolin, genistein, rhein, oleanolic acid, and epigallocatechin gallate, with ellagic acid being the most preferred.

[0009] Ellagic acid is the preferred ingredient, and the feeding ratio of ellagic acid to silk fibroin is preferably 1:2 to 1:16 by mass, more preferably 1:4 by mass.

[0010] The present invention also provides a method for preparing the above-mentioned silk fibroin complex, comprising two steps: conformational transformation of silk fibroin and self-assembly with polyhydroxyphenolic compounds. Step (1): Silk fibroin solution is prepared by a process of transforming silk fibroin from an α-helix to a β-sheet conformation. The conformational change can be achieved by chemical or physical methods. Chemical methods include promoting the transformation through polyols, polylactic acid, metal ions, pH, or hydroxypropyl methylcellulose. Physical methods include promoting the transformation through high temperature, hydration and pressure, ultra-low temperature placement, freeze drying, shear force, ultrasound, eddy current, laser irradiation, and high-pressure carbon dioxide treatment. Step (2): After dissolving or suspending the polyhydroxyphenolic compounds, they are mixed and stirred with the silk fibroin solution obtained in step (1) to induce self-assembly, thereby obtaining the silk fibroin complex loaded with polyhydroxyphenolic compounds. In step (1), polyols are preferably used to promote the transformation of the β-sheet conformation of silk fibroin, and ethanol is more preferably used as the polyol.

[0011] As an improvement to the preparation method, the polyhydroxyphenolic compound is a poorly soluble polyhydroxyphenolic compound. In step (2), the poorly soluble polyhydroxyphenolic compound is dissolved or suspended in an organic solvent, which is selected from methanol, ethanol, propanol, propylene glycol, glycerol, n-butanol, and isobutanol.

[0012] When the poorly soluble polyhydroxyphenolic compound is ellagic acid, the preferred organic solvent is propylene glycol. After the ellagic acid suspension is co-incubated with silk fibroin in step (2), the final concentration of propylene glycol in the resulting solution is 5-20%, and the most preferred final concentration of propylene glycol is 10%.

[0013] This invention provides the application of the above-mentioned silk fibroin complex in the preparation of formulations with hemostatic effects, comprising the above-mentioned silk fibroin complex and pharmaceutically acceptable excipients. The dosage forms of the formulation include gauze, sponge, spun fibers, sprays, powders, granules, gels, sealants, ointments, films, patches, and embolic agents.

[0014] The present invention also provides the use of the above-mentioned silk fibroin complex in the preparation of formulations having wound healing, and / or anti-infection, and / or anti-adhesion effects, comprising the above-mentioned silk fibroin complex and pharmaceutically acceptable excipients.

[0015] The beneficial effects of this invention are as follows:

[0016] The silk fibroin complex loaded with polyhydroxyphenolic compounds developed in this invention serves as an effective hemostatic agent. Its hemostatic and coagulation abilities are significantly superior to those of the polyhydroxyphenolic compounds and silk fibroin itself, indicating a synergistic effect between the two materials. Furthermore, this hemostatic agent exhibits significantly better hemostatic effects than several commercially available hemostatic agents, such as Yunnan Baiyao, tranexamic acid, and chitosan hemostatic powder. The silk fibroin complex provided by this invention has a larger particle size, heavier texture, more stable mechanical strength, and stronger wet tissue adhesion compared to silk fibroin. It also exhibits a faster blood sedimentation rate compared to other drugs, easily settling quickly at the bleeding site. In contrast, the powders of Yunnan Baiyao, tranexamic acid, chitosan hemostatic powder, and silk fibroin are lighter and more easily float on the surface of the blood, thus being washed away by the blood flow and affecting their hemostatic effect. This silk fibroin complex hemostatic agent is non-cytotoxic, non-hemolytic, and self-degradable in vivo, demonstrating high safety. Simultaneously, this silk fibroin complex also possesses wound healing, antibacterial, anti-infective, and anti-adhesion effects. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings:

[0018] Figure 1 Fourier transform infrared spectroscopy (FTIR).

[0019] Figures 2-4 Blood viscosity comparison chart (n=3), A in each chart: *, compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; #, compared with the β-SF-EA group, #P<0.05, ##P<0.01, ###P<0.001. Compared with the β-SF-polyhydroxyphenolic compound group, *P<0.05, **P<0.01, ***P<0.001, for example: compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 5 Comparison of clotting time (n=3), *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001.

[0021] Figure 6 Drug loading comparison chart (n=3). *, Compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001; ND indicates Not Detected.

[0022] Figures 7-8 Comparison of drug loading (n=3). *, Compared with the water group, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 9 Fourier transform infrared spectroscopy (FTIR) analysis was performed, in which EA and β-SF were physically mixed in a 1:1 (w / w) ratio.

[0024] Figure 10 Molecular docking diagram. L74 represents Leu (leucine) at position 74, and D27 represents Asp (aspartic acid) at position 27.

[0025] Figure 11 Characterization: (A) FTIR spectrum, (B) XRD, (C) TG, (D) 1H NMR.

[0026] Figure 12 Blood adsorption experiment. Where a is blank, b is β-SF, c is EA, d is Yunnan Baiyao, e is tranexamic acid, and f is β-SF-EA. Figure 13 Same as above.

[0027] Figure 13 Diagram of a tilted test tube coagulation test.

[0028] Figure 14 BCI (coagulation index) plot (n=3).

[0029] Figure 15 Rats underwent tail amputation experiment. Figure A shows the bleeding immediately after tail amputation when drug powder was applied, and Figure B shows the hemostasis time after tail amputation. *Compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 16 Time-varying images of hepatic and femoral artery hemorrhage in rats (n=3). *, Compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001.

[0031] Figure 17 Relative cell proliferation rate of LO2 and L929 cells after co-incubation with the drug for 48 h (n=6).

[0032] Figure 18 In vitro degradation of β-SF-EA (n=3).

[0033] Figure 19 Characterization of silk fibroin nanoparticle hemostatic powder loaded with ellagic acid (β-SFN-EA).

[0034] Figure 20 Time-varying images of hemorrhage from the marginal auricular artery, liver, and femoral artery in rabbits (n=6).

[0035] Figure 21 Image showing the amount of bleeding from the marginal auricular artery, liver, and femoral artery in rabbits (n=6).

[0036] Figure 22 In vivo biocompatibility: H&E and Masson trichrome staining images of surrounding tissues isolated 7, 15 and 30 days after subcutaneous implantation.

[0037] Figure 23 Wound closure rate comparison chart (n=3), *, compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; #, compared with the β-SF-EA group, #P<0.05, ##P<0.01, ###P<0.001.

[0038] Figure 24 H&E staining of wound.

[0039] Figure 25 H&E staining of rabbit femoral artery wounds.

[0040] Figure 26 Comparison of liver healing rates (n=3), *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0041] Example 1: Preparation of silk fibroin complex loaded with polyhydroxyphenolic compounds (β-SF-polyhydroxyphenolic compounds)

[0042] Step (1) Conversion of the β-sheet of silk fibroin: Silk fibroin (SF) is diluted with water 5 to 10 times and then slowly poured into an excess of organic solvent under vigorous stirring. The mixture is stirred for 2 to 4 hours to complete the conversion of the β-sheet. The liquid portion is removed by mechanical separation methods such as filtration, centrifugation, and membrane filtration. The remaining portion is washed repeatedly and then freeze-dried to obtain β-sheet silk fibroin (β-SF). The organic solvent is selected from methanol, ethanol, propylene glycol, butanediol, etc., with ethanol being preferred. The mechanical separation method is preferably high-speed centrifugation, such as centrifugation at 10,000 rpm for 15 to 20 minutes.

[0043] Fourier Transform Infrared (FTIR) spectroscopy was performed by mixing 5% of the sample with 95% KBr and grinding it into a fine powder. The powder was then scanned using a Nicolet IS5 FTIR spectrometer. Each spectrum was obtained in transmission mode (ten scans) with a resolution of 4 cm⁻¹. -1 The spectral range is 4000-500cm. -1 The FTIR spectra of the relatively high-level β-fold structure are in the 1615–1640 cm⁻¹ range. -1 1510-1525cm -1 It shows a sharp peak at 1640-1660 cm⁻¹, while the FTIR spectrum of SF in its natural state is at 1640-1660 cm⁻¹. -1 1535-1542cm-1 A sharp peak, α-SF, is observed at this point. Figure 1 The results of the measurements show that the characteristic peak of natural α-SF is 1642 cm⁻¹. -1 and 1535cm -1 The characteristic peak of SF after step (1) is 1620 cm⁻¹. -1 and 1517cm -1 It can be seen that SF has completed the conformational transition of β-folding and become β-SF.

[0044] Step (2) Self-assembly of polyhydroxyphenolic compounds and silk fibroin: The β-SF obtained in step (1) is prepared into a suspension with water and placed in a magnetic stirrer at 500-2000 rpm; the polyhydroxyphenolic compounds are dissolved or suspended in a solvent, wherein water-soluble polyhydroxyphenolic compounds are dissolved in water and poorly soluble polyhydroxyphenolic compounds are dissolved in water or organic solvents such as ethanol, propylene glycol, and butanediol. The prepared solution or suspension is added dropwise to the β-SF suspension and mixed and stirred for 12-48 hours to obtain the silk fibroin complex loaded with polyhydroxyphenolic compounds.

[0045] The polyhydroxyphenolic compounds mentioned above are selected from water-insoluble polyhydroxyphenolic compounds, including ellagic acid (EA), resveratrol (RES), quercetin (QUE), baicalein (BAE), kaempferol (KAE), lignan, curcumin (CUR), chlorogenic acid (CGA), ferulic acid (FA), caffeic acid (CFA), syringic acid (SA), sinapic acid, rutin (RUT), myricetin (MYR), and magnolol. Acids, including MA, Fisetin (FIS), Puerarin (PUE), Rhein (RHE), Lonicerin (Lon), Proanthocyanidins (PC), p-Coumaric Acid (p-CA), Vanillic Acid (VA), Hesperetin (Hes), Naringenin (NAR), Luteolin (Lut), Genistein (Gen), Emodin (Emo), Oleanolic Acid (Ola), Danshensu (DS), Epigallocatechin gallate (EGCG); and water-soluble polyhydroxyphenolic compounds, including Gallic Acid (GA) and Pyrogallic Acid (GA). Acid (PA), catechin (C), tannic acid (TA), tea polyphenols (TP), acaciin, epigallocatechin (EGC), etc.

[0046] Example 2: Preparation of silk fibroin complex loaded with polyhydroxyphenolic compounds

[0047] Unlike Example 1, in step (1), ethanol is used as the organic solvent, and the mechanical separation method is high-speed centrifugation at 10,000 rpm for 15-20 min. In step (2), equal amounts of polyhydroxyphenolic compounds and silk fibroin are added. In the preparation of the polyhydroxyphenolic compound suspension, water-soluble polyhydroxyphenolic compounds are prepared using water as the solvent, and poorly soluble polyhydroxyphenolic compounds are prepared using water or an organic solvent. The mechanical separation method is high-speed or ultra-high-speed centrifugation followed by collection of the precipitate. Step (3) is added to dry into powder: the solution obtained in step (2) is dried into powder after removing the liquid using mechanical separation methods such as filtration, centrifugation, membrane filtration, and sedimentation, or it can be dried into powder in one step using a spray dryer or other drying equipment to obtain the silk fibroin complex loaded with polyhydroxyphenolic compounds. The preferred mechanical separation method is high-speed or ultra-high-speed centrifugation followed by collection of the precipitate.

[0048] Using poorly soluble polyhydroxyphenolic compounds as raw materials, the following complexes were prepared: ellagic acid-silk fibroin complex (β-SF-EA), resveratrol-silk fibroin complex (β-SF-RES), quercetin-silk fibroin complex (β-SF-QUE), baicalin-silk fibroin complex (β-SF-BAE), kaempferol-silk fibroin complex (β-SF-KAE), lignan-silk fibroin complex (β-SF-Lignan), curcumin-silk fibroin complex (β-SF-CUR), chlorogenic acid-silk fibroin complex (β-SF-CGA), ferulic acid-silk fibroin complex (β-SF-FA), caffeic acid-silk fibroin complex (β-SF-CFA), syringic acid-silk fibroin complex (β-SF-SA), and sinapic acid-silk fibroin complex (β-SF-Sinapic). Acid, Rutin-Silk Fiber Complex (β-SF-RUT), Myricetin-Silk Fiber Complex (β-SF-MYR), Magnoliic Acid-Silk Fiber Complex (β-SF-MA), Oxalis Yellow-Silk Fiber Complex (β-SF-FIS), Puerarin-Silk Fiber Complex (β-SF-PUE), Rhein-Silk Fiber Complex (β-SF-RHE), Lonicerin-Silk Fiber Complex (β-SF-Lon), Proanthocyanidin-Silk Fiber Complex (β-SF-PC), Coumaric Acid-Silk Fiber Complex (β-SF-p-CA), Vanillic Acid-Silk Fiber Complex (β-SF-VA), Hesperidin-Silk Fiber Complex (β-SF-Hes), Naringenin-Silk Fiber Complex (β-SF-NAR), Luteolin-Silk Fiber Complex (β-SF-Lut), Gentian Fiber The following complexes were prepared using water-soluble polyhydroxyphenolic compounds as raw materials: β-SF-Gen (a β-SF-Gen complex), β-SF-Emo (a β-SF-Emo complex), β-SF-Ola (a β-SF-Ola complex), β-SF-DS (a β-SF-DS complex), and β-SF-EGCG (a β-SF-EGCG complex). Other complexes prepared included gallic acid-silk fibroin (β-SF-GA), pyrogallic acid-silk fibroin (β-SF-PA), β-SF-C (a β-SF-C complex), β-SF-TA (a β-SF-TA complex), β-SF-Acaciin-silk fibroin (β-SF-Acaciin complex), β-SF-EGC (a β-SF-EGC complex), and β-SF-TP (a β-SF-TP complex).

[0049] Example 3: Investigation of Coagulation Effect

[0050] The obtained β-SF-polyhydroxyphenolic compounds were prepared into a 2 mg / mL test solution using physiological saline for coagulation effect assessment: Blood was collected from the abdominal aorta of New Zealand white rabbits and placed in a heparin sodium anticoagulant tube. 2 mL of the test solution was added to the tube, with physiological saline as a control. The mixture was stirred well and incubated at 37°C for 15 min. The whole blood viscosity was then measured using a fully automated blood rheometer (all operations were completed within 2 hours after blood collection).

[0051] Whole blood viscosity is the result of friction between blood cells and plasma protein molecules during blood flow, and it is the most important indicator in blood rheology. When blood viscosity increases, it indicates that blood flow is obstructed. At the same time, the deformability of red blood cells decreases and their aggregation increases, reducing the flow of blood into microvessels and capillaries, and decreasing their permeability. Figures 2-4 The results showed that the silk fibroin complex loaded with polyhydroxyphenolic compounds (β-SF-polyhydroxyphenolic compounds) significantly increased whole blood viscosity compared with the blank group (P<0.05), and was significantly better than the effect of β-SF and the original compound itself (P<0.05), indicating that the formation of the complex can significantly enhance the coagulation effect; and overall, the complex of silk fibroin and insoluble polyhydroxyphenolic compounds was better than the complex of silk fibroin and water-soluble polyhydroxyphenolic compounds.

[0052] Weigh 0.1g of the drug powder to be tested into a 1.5mL centrifuge tube. Simultaneously, measure 0.5mL of anticoagulated blood and add it to the centrifuge tubes containing the different drugs. Place the tubes vertically on the workbench and start timing. After 30 seconds, rotate the inverted centrifuge tubes and observe whether the blood flows. Repeat the operation until the anticoagulated blood can no longer flow, then stop timing and record the clotting time. Figure 5 The results showed that Figures 2-4 The results tend to be similar.

[0053] Among all the complexes, β-SF-EA exhibited the best coagulation effect. This may be related to the multiple hydrogen bonds and planar structure of EA. On the one hand, the presence of multiple hydrogen bonds increases the synergistic effect between hydrogen bonds, further improving the stability of hydrogen bonds; on the other hand, ellagic acid has a symmetrical catechol structure and a planar structure, which can form intramolecular and intermolecular hydrogen bonds, thereby enhancing the rigidity of the complex molecule. This stable molecular structure can reduce molecular dynamic changes, thus indirectly improving the stability of intermolecular hydrogen bonds.

[0054] Example 4: Preparation of polyhydroxyphenolic compound-silk fibroin complex (α-SF-polyhydroxyphenolic compound)

[0055] Unlike in Example 2, step (1) is deleted, and in step (2), β-SF is replaced with untreated SF, that is, SF with the natural α-helical conformation. In step (2), equal amounts of polyhydroxyphenolic compound and silk fibroin are added to prepare a polyhydroxyphenolic compound-α-silk fibroin complex, which is called α-SF-polyhydroxyphenolic compound.

[0056] Example 5: Preparation of polyhydroxyphenolic compound-silk fibroin complex (SH-SF-polyhydroxyphenolic compound)

[0057] Unlike Example 2, step (1) uses the following method: silk fibroin is dissolved in an aqueous solution containing tris(2-carboxyethyl)phosphonic acid hydrochloride, and subjected to a reduction reaction at room temperature for 5 minutes to obtain a 1.0-5.0 wt% reduced SF solution. The rest is the same as in Example 2, except that in step (2), equal amounts of polyhydroxyphenolic compounds and silk fibroin are added to prepare the resulting polyhydroxyphenolic compound-silk fibroin complex, referred to as SH-SF-polyhydroxyphenolic compound. This method uses the method of breaking disulfide bonds to expose hydrophobic amino acids to bind polyhydroxyphenolic compounds such as ellagic acid.

[0058] Example 6 Self-assembly performance evaluation

[0059] Ellagic acid was used as a model drug in the preparation of three ellagic acid-silk fibroin complexes according to the steps in Examples 2, 4, and 5. Water was used as the solvent for EA, resulting in β-SF-EA, α-SF-EA, and SH-SF-EA. Assembly efficiency was evaluated using drug loading and encapsulation efficiency, calculated using formulas 1) and 2), respectively. The amount of EA in the silk fibroin complex was calculated using the difference method, subtracting the amount of EA added from the amount of EA after co-incubation with SF. EA content was analyzed by high-performance liquid chromatography (HPLC). A Hedera C18 column was used, with an acetonitrile:water ratio of 20:80 (v / v) as the mobile phase, a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, an injection volume of 10 μL, and a chromatogram acquisition time of 15 min. Drug loading and encapsulation efficiency were calculated using the following formulas:

[0060] Drug loading = Amount of EA in silk fibroin complex / Total amount of SF and EA (Formula 1)

[0061] Encapsulation efficiency = Amount of EA in silk fibroin complex / Amount of EA added (Formula 2)

[0062] Depend on Figure 6The results showed that the drug loading was β-SF-EA > SH-SF-EA >> α-SF-EA. β-SF-EA had a drug loading of 9.6%, while EA was almost undetectable in α-SF-EA. This indicates that the self-assembly efficiency of α-helical silk fibroin with ellagic acid is very low, highlighting the necessity of conformational inversion of silk fibroin. SH-SF-EA uses the disruption of disulfide bonds in the silk fibroin molecule to expose its hydrophobic amino acids, thereby completing the self-assembly with ellagic acid, but the drug loading was only 4.5%, far lower than β-SF-EA (P<0.001).

[0063] Example 7: Investigation of the effect of solvent on EA drug loading

[0064] Ellagic acid was used as a model drug for polyhydroxyphenolic compounds. β-SF-EA was prepared using the steps in Example 2. In step (2), polyhydroxyphenolic compound suspensions were prepared using solvents such as water, ethanol, propylene glycol, and butanediol. If an organic solvent was used as the suspension solvent, the final concentration of the organic solvent in the solution obtained after co-incubating the polyhydroxyphenolic compound suspension with β-SF was 10%. The drug loading was investigated using the method in Example 6. Figure 7 The results showed that the addition of organic solvents effectively increased the drug loading of ellagic acid, with the order being propylene glycol > butylene glycol > ethanol > water. The highest drug loading was achieved when propylene glycol was used, reaching 19.57 ± 1.01%.

[0065] Next, the effect of propylene glycol concentration on EA drug loading was investigated. In step (2), propylene glycol was used as a solvent to prepare EA suspension. The final concentrations of propylene glycol in the solutions obtained after co-incubation with β-SF were 0%, 5%, 10%, 15%, and 20%, respectively. Figure 8 The results showed that the drug loading of the propylene glycol aqueous solution group was better than that of the water group, with the 10% propylene glycol aqueous solution group showing the best effect, with a drug loading of 19.57±1.01%.

[0066] Example 8: Investigation of different EA:SF feed ratios

[0067] Ellagic acid was used as a model drug for polyhydroxyphenolic compounds. β-SF-EA was prepared using the steps in Example 2, where step (2) used propylene glycol as a solvent to prepare the EA suspension. The final concentration of propylene glycol in the solution after co-incubation with β-SF was 10%. The feed ratio of EA to β-SF was controlled at EA:β-SF = 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:12, and 1:16 (w / w). Table 1 shows that the drug loading and encapsulation efficiency of EA were almost unobservable in EA:β-SF = 1:0.5 and 1:1, while these were observed in other groups. Fourier transform infrared spectroscopy analysis of each group of samples was performed using the method described in Example 1. Figure 9From this, we can see that the EA:β-SF ratios of 1:2, 1:4, 1:8, 1:12, and 1:16 (w / w) showed an EA hydroxyl peak (3471 cm⁻¹). -1 The disappearance of the peak indicates that EA and β-SF have completed hydrogen bonding and self-assembled successfully. However, the EA:β-SF ratios of 1:0.5 and 1:1, as well as their physical mixtures, still show EA hydroxyl peaks, indicating that self-assembly of the two molecules is not possible. These results suggest that a feed ratio of EA:β-SF < 1:1 (w / w) is required for the self-assembly of β-SF and EA. The optimal feed ratio is EA:β-SF = 1:4, which achieves the maximum drug loading and encapsulation efficiency for EA (Table 1).

[0068] The effect of different EA:β-SF ratios on whole blood viscosity was further investigated using the method described in Example 3. The results in Table 2 show that, compared with the saline group, the EA:β-SF < 1:1 groups significantly increased whole blood viscosity at low, medium, and high shear rates (P < 0.5), and were also higher than the EA and β-SF groups (P < 0.5). This indicates that the β-SF-EA prepared with an EA:β-SF < 1:1 ratio has significant coagulation efficacy, and that EA and β-SF have a synergistic effect; among them, EA:β-SF = 1:4 showed the best coagulation effect.

[0069] In summary, the preparation process of β-SF-EA involves using β-sheet SF with a feed ratio of EA:β-SF < 1:1 (w / w), as detailed in Example 1. The optimal process is EA:β-SF = 1:4 (w / w), where ellagic acid in step (2) is prepared as a suspension using propylene glycol as a solvent, and the final concentration of propylene glycol in the solution after co-incubation with SF is 10%. Self-assembly of EA and β-SF cannot be achieved without β-sheet conformational transformation of SF or a feed ratio of EA:β-SF ≥ 1:1. When EA:β-SF = 1:2 (w / w), both drug loading and encapsulation efficiency are low, indicating that although self-assembly of EA and β-SF can be achieved at this ratio, the losses are significant.

[0070] Table 1. Effect of the feed ratio of β-SF to EA on drug loading and encapsulation efficiency (n=3)

[0071]

[0072] ND indicates Not Detected.

[0073] Table 2. Effect of different EA:β-SF feed ratios on whole blood viscosity (n=3)

[0074]

[0075]

[0076] *: Compared with the saline group, *P<0.05, **P<0.01, ***P<0.001; #: Compared with the EA:β-SF = 1:4 group, #P<0.05, ##P<0.01,

[0077] ###P<0.001.

[0078] Example 9 Molecular docking of silk fibroin complex

[0079] Computer simulations were used to study the binding mode of ellagic acid and silk fibroin. The heavy chain crystal structure of silk fibroin (PDB number: 3UA0) was obtained from a PDB bank. Due to limited prior research on the binding mode, we employed computer simulations to investigate this process. In 2018, the SiteMap module performed reasonable prediction of binding sites, while the silk fibroin crystal structure was hydrogenated to remove irrelevant ions. Ellagic acid small molecules were then converted into 3D conformations via the LigPrep module, outputting up to 32 isomers. Finally, Glide extraprecision (XP) was used to output 20 binding poses. Figure 10 As shown, after the two phenolic hydroxyl groups in the ellagic acid small molecule form hydrogen bonds with L74, they still need to form hydrogen bonds with D27 on another heavy chain. This indicates that further hydrogen bonds with the other two highly symmetrical hydroxyl groups in ellagic acid are required to achieve stable binding. In addition, since the heavy chains in silk fibroin maintain a highly similar β-sheet secondary structure, the ellagic acid small molecule and the two heavy chain proteins may not have sufficient hydrophobic interactions to maintain stability. It is reasonable to speculate that the ellagic acid small molecule will bind and encapsulate at least three to four heavy chain proteins to achieve stable binding. The molecular docking results explain the molecular mechanism of the experimental result in Example 8 that "EA:β-SF < 1:1 (w / w) is required to achieve self-assembly of β-SF and EA".

[0080] The β-SF-EA samples tested in Examples 10-14 and 22-24 were prepared using the optimal process.

[0081] Example 10 Characterization of silk fibroin complex loaded with polyhydroxyphenolic compounds

[0082] FT-IR proved the self-assembly of EA with β-SF ( Figure 11 A): EA hydroxyl peak (3471cm) -1 The disappearance of ) suggests that EA and β-SF may self-assemble via intermolecular hydrogen bonds. X-ray diffraction (XRD) Figure 11In B), the characteristic peaks of EA weaken or disappear after binding with silk fibroin, indicating that this is not a simple physical mixture, but a new crystalline phase formed through interaction, confirming the FT-IR results. At the same time, a change in crystal form is observed. β-SF shows broad diffraction peaks, indicating that they are amorphous macromolecules. EA shows many strong diffraction peaks, indicating that it is in a crystalline state. When β-SF and EA self-assemble, the peak intensity of EA decreases significantly, which indicates that β-SF-EA is mainly an amorphous macromolecule.

[0083] The thermogravimetric (TG) curves were evaluated and divided into three stages ( Figure 11 C). In the first stage, from room temperature to 120°C, the weight loss was approximately 6.1%, mainly due to the evaporation and desorption of adsorbed water. In the second stage, the weight loss was between 150°C and 450°C, corresponding to the decomposition of silk fibroin molecular chains, the decomposition of amino acid residue side chains, and peptide bond breakage. Specifically, the fibroin I crystal structure (α-helix and random coil) degraded at ~250°C, and the fibroin II crystal structure (β-sheet) degraded at ~260°C. β-SF showed a major degradation peak at 260°C, forming a stable β-sheet conformation on its surface. Interestingly, the major degradation peak of β-SF-EA shifted to 280°C, indicating that the fibroin II structure was more stable. This may be due to the strong interaction between EA and the protein during self-assembly, thereby improving the stability of the complex. To determine the optimal amount of EA incorporated into β-SF, proton nuclear magnetic resonance (1H NMR) was performed. Figure 11 D): In β-SF-EA, the peak associated with EA was significantly weakened (<0.007), confirming that EA binds to β-SF through catechol hydrogen, further proving their binding.

[0084] Example 11: Investigation of Coagulation Effect

[0085] (1) Plasma sample processing

[0086] New Zealand white rabbits were acclimatized with a one-week diet and fasted the night before the experiment. 1% sodium pentobarbital was administered at a concentration of 3 mL / kg. -1 The anesthetic dose is administered intraperitoneally. After anesthesia, blood is collected via the abdominal aorta. The blood is collected using a 5mL heparin sodium anticoagulant tube. After blood collection, the blood and anticoagulant must be thoroughly mixed and kept ready for use.

[0087] (2) In vitro blood adsorption capacity test

[0088] Weigh 0.1g of the drug powder to be tested into a 1.5mL centrifuge tube. Simultaneously, measure 0.5mL of anticoagulated blood and add it to the centrifuge tubes containing the different drugs. Place the tubes vertically on the workbench and start timing. After 30 seconds, rotate the inverted centrifuge tubes and observe whether the blood flows. Repeat the operation until the anticoagulated blood stops flowing, then stop timing. Figure 12The results showed that β-SF-EA achieved complete adsorption of blood within two minutes, while other groups could not achieve the same effect. This indicates that the ability of β-SF-EA to adsorb blood is significantly better than that of the positive control Yunnan Baiyao and tranexamic acid, and also significantly better than β-SF and EA monomers, suggesting that β-SF and EA can have a synergistic effect.

[0089] (3) Test tube tilting coagulation test

[0090] Accurately weigh 5.0 mg of the test drug powder into a 10 mL test tube, spreading the powder as evenly as possible at the bottom of the tube. Add 1 mL of anticoagulated blood to each test tube, then add 0.2 mol·L⁻¹ of the solution. -1 Add 25 μL of CaCl2 solution and mix thoroughly immediately, then start timing immediately. Let stand for 1 minute, then tilt the test tube every 30 seconds until the blood clots and stops flowing, then stop timing. Figure 13 The study observed that the complete clotting time of the blood in the β-SF-EA group (2 min) was significantly shorter than that in the Yunnan Baiyao group (5 min), the tranexamic acid group (4 min 30 s), β-SF (5 min), and EA (>5 min), indicating that β-SF and EA can synergistically enhance each other's effects. This experiment also demonstrated that β-SF-EA has a faster clotting ability compared to other drugs.

[0091] (4) Blood rheology measurement

[0092] Whole blood viscosity is the result of friction between blood cells and plasma protein molecules during blood flow and is the most important indicator in blood rheology. Increased blood viscosity indicates impaired blood flow, decreased deformability of red blood cells, and increased aggregation, leading to reduced blood flow into microvessels and capillaries, and decreased throughput. The method described in Example 3 was used to determine the viscosity of β-SF-EA after its application to blood, within 1 second. -1 30·s -1 100·s -1 150·s -1 200·s -1 Table 3 shows that, regardless of the shear rate, β-SF-EA significantly increased the viscosity of whole blood (P<0.05), and was significantly superior to the commercially available chitosan hemostatic powder group (P<0.05). The commercially available chitosan hemostatic powder in the control group was purchased from Cycares Biotechnology Co., Ltd., and its main active ingredient is chitosan.

[0093] Table 3 Comparison of whole blood viscosity between β-SF-EA and chitosan (n=3)

[0094]

[0095] *: Compared with the control group, *P<0.05, **P<0.01, ***P<0.001; #: Compared with the commercially available chitosan hemostatic powder group, #P<0.05, ##P<0.01,

[0096] ###P<0.001.

[0097] (4) Coagulation index BCI

[0098] Prepare β-SF-EA at different masses (10, 20, 30, 40, 50 mg) and place them flat in several 50 mL centrifuge tubes. Gently add 0.1 mL of anticoagulated blood to the sample, followed immediately by 0.02 mL of 0.2 mol / L CaCl2 solution. After 5 minutes, gently add 25 mL of deionized water to the beaker. Centrifuge at 300 rpm for 5 minutes. Then, remove the solution and measure its Abs value using a microplate reader at a wavelength of 540 nm. A control is set up: 0.1 mL of anticoagulated blood is added to a beaker, followed by 25 mL of deionized water. The Abs value measured at the same wavelength is assumed to be 100 as a reference value. Therefore, the coagulation index BCI is: BCI = 100 × A 样品 / A 对照 The BCI index is an indicator of coagulation effectiveness; the lower the index, the better the coagulation effect. Figure 14 This indicates that increasing the dosage (increasing the concentration in the same volume) significantly reduces the coagulation index (BCI), suggesting a positive correlation between coagulation effect and dosage.

[0099] Example 12: Investigation of hemostatic effect

[0100] The hemostatic ability of β-SF-EA was evaluated using male SD rats (approximately 250.0g).

[0101] Tail amputation hemostasis experiment in rats: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. A 6cm section was measured from the tail end of the rat, and the tail was cut off with surgical scissors. Immediately after bleeding, 20mg of sample powder (β-SF-EA, Yunnan Baiyao, tranexamic acid) was sprinkled on the wound, and the time was started from the beginning of bleeding.

[0102] Hemostasis experiment on rat liver wounds: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were fixed supine on a dissection table, and a longitudinal incision was made along the midline of the abdomen to access the peritoneal cavity and expose the right lobe of the liver. Peritoneal fluid was absorbed with clean gauze. A parallel wound approximately 2.0 cm long and 0.5 cm deep was made on the right lobe of the liver using a scalpel. After bleeding occurred, the wound was immediately absorbed with pre-weighed cotton. Then, 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan Baiyao, tranexamic acid) was sprinkled onto the wound surface, and timing was started. The wound was first compressed with cotton for 30 seconds to observe for bleeding. If bleeding continued, cotton was compressed again for 30 seconds, and the observation was repeated until no obvious bloodstains appeared on the cotton surface, indicating successful hemostasis. The timing was then stopped, and the hemostasis time was recorded.

[0103] Hemostasis experiment on rat thigh muscle trauma: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were fixed supine on a dissection table, and the leg hair was shaved using a rat shaver. After disinfection with alcohol, a muscle wound approximately 3 cm long and 1 cm deep was made using a scalpel. Bleeding was timed from the start of the wound. Five seconds after bleeding, 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan Baiyao, tranexamic acid) was applied, and the hemostatic effect was observed and the hemostasis time recorded.

[0104] Figure 15 The image shows a rat tail amputation experiment. When β-SF-EA was placed on the wound, bleeding stopped rapidly, with almost no blood left on the filter paper. It significantly promoted hemostasis within 30 seconds; bleeding in the control group lasted more than five minutes, which was not statistically significant and is not shown in Figure B. As controls, the tranexamic acid group and Yunnan Baiyao group required more than four times the hemostasis time, and the amount of bleeding was significantly higher than that in the β-SF-EA group (P<0.05).

[0105] Figure 16 The graph shows the time of hemorrhage in the liver and femoral artery of rats. The femoral artery bleeding in the control group lasted for more than five minutes, which was not statistically significant and is not shown in the graph. Figure 16 The results indicate that β-SF-EA can significantly shorten the hemostasis time in both models, and its hemostatic efficacy is significantly better than Yunnan Baiyao (P<0.05) and tranexamic acid (P<0.05), and also significantly better than EA (P<0.05) and the β-SF group (P<0.05). This suggests that silk fibroin and ellagic acid have a synergistic effect. These results are consistent with the blood rheology and test tube tilt coagulation results in Example 11. In both rat hemorrhage models, β-SF-EA was observed to have a faster sedimentation rate in the blood compared to other drugs, and it easily settled to the bleeding site. In contrast, Yunnan Baiyao, tranexamic acid, and β-SF powder are lighter and more likely to float on the surface of the whole blood and be washed away by the blood flow, thus affecting their hemostatic effect.

[0106] Example 13 Safety Assessment

[0107] (1) Cell resuscitation

[0108] Remove the frozen LO2 and L929 cells and gently agitate them in a 37°C water bath until completely thawed. In a clean bench, transfer the cell suspension from the cryovials to a cell culture flask, add 4 mL of DMEM medium containing 10% fetal bovine serum, and gently pipette to evenly disperse the cells. Then, incubate the flask in a 5% CO2, 37°C cell culture incubator. After 6 hours of culture, sterilize the flask with alcohol, place it in a clean bench, aspirate the old medium, rinse, add 4 mL of fresh medium, and continue culturing in the cell culture incubator.

[0109] (2) CCK8 quantification method

[0110] Dissolve 0.2 mg of β-SF-EA and EA in 1 mL of DMSO and establish gradient concentrations.

[0111] Cells in logarithmic growth cycles were washed with PBS solution, and then trypsin digestion solution was added to suspend the cells. Cell density was calculated using a cell counting chamber, and cells were then divided into groups of 1 × 10⁶ cells per well. 5 Cells were seeded at 1 / mL per cell culture medium into 96-well plates and cultured for 24 hours. After cell adhesion, the culture medium was aspirated, and the cells were washed twice with PBS. 100 μL of each of the following solutions were added to the cell-seedled 96-well plates: experimental group (β-SF-EA and EA extracts), blank group (DMEM medium containing 10% fetal bovine serum), and positive group (paclitaxel extract). Six replicates were prepared for each group. The plates were then incubated at 37°C with 5% CO2 for 48 hours. The culture medium was aspirated, the cells were washed twice with PBS, and 100 μL of CCK8 solution (90 μL DMEM medium + 10 μL CCK8) was added. The plates were incubated in the dark for 30 minutes. The absorbance (OD) at 450 nm was then measured using a microplate reader. Six replicates were performed at each time point. The relative growth rate (RGR) was calculated using the following formula: Relative growth rate (RGR)% = OD 实验组 / OD 空白组 *100%.

[0112] The cytotoxicity level is determined according to the cytotoxicity grades in Table 4, with the positive control not lower than grade 3. A cell grade of 0-1 is considered acceptable.

[0113] Table 4. Grading Criteria for Cell Proliferation Response

[0114]

[0115] We studied the toxicity of β-SF-EA and EA on L929 cells and LO2 cells by CCK8 assay. Figure 17 As shown, the relative proliferation rate of all groups of β-SF-EA was higher than 85%. According to the toxicity grading standard of ISO10993-1, it indicated that the cytotoxicity of β-SF-EA was grade 0-1. At the same time, the addition of silk fibroin effectively improved the biocompatibility of ellagic acid. To sum up, β-SF-EA did not affect cell proliferation and had good cell compatibility.

[0116] (2) Hemolysis test

[0117] Dilute 8 mL of fresh rabbit blood with 10 mL of normal saline. Put 20 mg of β-SF-EA into a 50 mL centrifuge tube, add 10 mL of normal saline, incubate in a water bath at 37 °C for 10 min, then add 0.2 mL of diluted rabbit blood, shake gently, centrifuge at 1000 rpm for 5 min after 60 min of water bath, take the supernatant, and measure the absorbance at 540 nm. The positive control group uses 10 mL of distilled water plus 0.2 mL of rabbit blood, and the negative control group uses 10 mL of normal saline plus 0.2 mL of rabbit blood, with the same operation method. Each group has three parallels. The hemolysis rate is calculated by the following formula: Hemolysis rate (%) = (Absorbance of test sample - Absorbance of negative control) / (Absorbance of positive control - Absorbance of negative control) * 100%.

[0118] When different materials come into direct contact with blood, it is possible to cause hemolysis due to the rupture of red blood cells. The hemolysis test evaluates the hemolysis of materials by checking the hemoglobin concentration. After calculation, the hemolysis rate of β-SF-EA was 1.13 ± 0.02% (Table 5). According to the provisions of GB / T4233.2, a hemolysis rate lower than 5% is qualified. Therefore, the prepared β-SF-EA meets the international standard.

[0119] Table 5 Hemolysis rate (n = 3)

[0120]

[0121] Example 14 Investigation of degradation performance

[0122] Weigh a certain mass of β-SF-EA and place it in a PBS solution containing 0.1 μg / mL protease XIV, incubate at 37 °C, and use the PBS solution without protease XIV as the blank sample. Incubate the samples (n = 3) in PBS solution and PBS solution containing 0.1 μg / mL protease XIV for 5, 10, 15, 20, 25, 30 d. All degraded samples replace the fresh solution at a fixed time every day. Dry the degradation products at 60 °C, weigh the samples to a constant weight, and the remaining mass retention rate R M is calculated by the following formula: R M / % = M dt / Mi *100%, of which M i For the initial mass, M dt The mass after t days is in mg.

[0123] As a hemostatic agent used both in vivo and in vitro, it must possess good biodegradability. Ideally, in the early stages of wound healing, the hemostatic powder should firmly bind tissues together to prevent cracking. Slow degradation is preferable, with the drug gradually degrading as the wound heals, and the remaining drug components being completely degraded after wound healing, either absorbed by the body or excreted through metabolism. The degradation effect of β-SF-EA at 37℃ is as follows... Figure 18 As shown, β-SF-EA degrades slowly in PBS, with a remaining mass of 90.11 ± 3.51% at day 30. In PBS solution containing protease XIV, the degradation effect of β-SF-EA can be roughly divided into two stages: the first stage (0-15 days), during which the degradation rate is slow, with a remaining mass of 67.83 ± 2.25% at day 15; and the second stage (15-30 days), where the degradation rate begins to increase significantly, with a remaining mass of 24.50 ± 0.70% at day 30, indicating that the majority of the degradation has occurred, demonstrating its good degradability.

[0124] Example 15 Preparation of silk fibroin nanoparticles loaded with polyhydroxyphenolic compounds (β-SFN-polyhydroxyphenolic compounds)

[0125] Based on Example 1 or Example 2, silk fibroin complexes loaded with polyhydroxyphenolic compounds are nano-sized using chemical methods (desolvation, salting out, microemulsion, etc.), instrumental methods (electrospraying, electric field and supercritical fluid technology, etc.), and other methods (polymer blending, pH modification, nanoimprinting), to prepare silk fibroin nanoparticles loaded with polyhydroxyphenolic compounds (β-SFN-polyhydroxyphenolic compounds). These nanoparticles can then be prepared into dosage forms with hemostatic effects, including gauze, sponges, spun fibers, sprays, powders, granules, gels, sealants, ointments, films, patches, and embolic agents.

[0126] Example 16 Preparation of hemostatic powder of silk fibroin nanoparticles loaded with ellagic acid

[0127] Silk fibroin was diluted 5-10 times with water and slowly poured into an excess of organic solvent under vigorous stirring. The mixture was stirred for 2-4 hours to complete the β-sheet conformation change. The liquid portion was removed by filtration, high-speed centrifugation (10,000 rpm for 15-20 minutes), and dialysis. The remaining portion was repeatedly washed and then freeze-dried to obtain β-sheet silk fibroin. The obtained β-SF was prepared as a suspension with water and placed in a magnetic stirrer at 500-2000 rpm. Polyphenols were suspended in solvents such as water, ethanol, propylene glycol, and butylene glycol. The polyphenol suspension was added dropwise to the β-SF suspension, and the mixture was stirred for 12-48 hours. The resulting solution was separated by mechanical methods such as filtration, centrifugation, and membrane filtration to remove the liquid portion. The remaining portion was freeze-dried. The freeze-dried solid was ground and crushed, then sieved to obtain a hemostatic powder.

[0128] Example 17 Preparation of a hemostatic spray containing silk fibroin nanoparticles loaded with ellagic acid

[0129] Silk fibroin was diluted 5-10 times with water and slowly poured into an excess of organic solvent under vigorous stirring. The mixture was stirred for 2-4 hours to complete the β-sheet conformation change. The liquid portion was removed by vacuum filtration, high-speed centrifugation (10,000 rpm for 15-20 minutes), and dialysis. The remaining portion was repeatedly washed and then freeze-dried to obtain β-sheet silk fibroin. The obtained β-SF was prepared into a suspension with water and placed in a magnetic stirrer at 500-2000 rpm. Polyphenols were suspended in solvents such as water, ethanol, propylene glycol, and butylene glycol. The polyphenol suspension was added dropwise to the β-SF suspension, and the mixture was stirred for 12-48 hours. The resulting solution was separated by mechanical methods such as vacuum filtration, centrifugation, and membrane filtration to remove the liquid portion. The solution was then dissolved in water to prepare a 30% (w / w) suspension, which was injected into a pressure-resistant, lightweight handheld spray bottle. The bottle was shaken for 10 seconds to mix evenly and form a hemostatic spray.

[0130] Example 18 Preparation of hemostatic granules made from silk fibroin nanoparticles loaded with ellagic acid

[0131] Silk fibroin was diluted 5-10 times with water and slowly poured into an excess of organic solvent under vigorous stirring. The mixture was stirred for 2-4 hours to complete the β-sheet conformation change. The liquid portion was removed by filtration, high-speed centrifugation (10,000 rpm for 15-20 minutes), and dialysis. The remaining portion was repeatedly washed and then freeze-dried to obtain β-sheet silk fibroin. The obtained β-SF was prepared as a suspension with water and placed in a magnetic stirrer at 500-2000 rpm. Polyphenols were suspended in solvents such as water, ethanol, propylene glycol, and butylene glycol. The polyphenol suspension was added dropwise to the β-SF suspension, and the mixture was stirred for 12-48 hours. The resulting solution was separated by mechanical methods such as filtration, centrifugation, and membrane filtration to remove the liquid portion. The remaining portion was freeze-dried to obtain silk fibroin aggregate particles (β-SF-polyphenol). Finally, the freeze-dried particles were ground and sieved as needed to obtain hemostatic granules of the desired particle size.

[0132] Examples 19-22 use EA as a model drug to prepare β-SFN-EA powder using the method in Example 16: a silk fibroin complex (β-SF-EA) is prepared under optimal conditions, and then nano-processed to prepare β-SFN-EA. The optimal conditions are EA:β-SF = 1:4 (w / w). Ellagic acid in step (2) is used as a solvent to prepare a suspension. After co-incubation with SF, the final concentration of propylene glycol in the resulting solution is 10%. β-SFN without EA is used as a control.

[0133] Example 19 Characterization of silk fibroin nanoparticle hemostatic powder loaded with ellagic acid (β-SFN-EA)

[0134] Figure 19 Image A shows the macroscopic morphology of silkworm cocoons, β-SFN, and β-SFN-EA, examined using a TESCAN MAIA 3GMU field emission scanning electron microscope. Figure 19Scanning electron microscopy (SEM) images in section B show that both β-SF and β-SF-EA have spherical morphologies, with an increased volume and particle size observed in β-SF-EA. Particle size was measured using dynamic light scattering (DLS) with a NANO ZS90 microscope. Table 6 shows that the average particle size of β-SFN-EA (201.18 ± 2.17 nm) is greater than that of β-SFN (170.33 ± 0.15 nm). Both groups of samples exhibited relatively high zeta potentials (< -20 mV), indicating that β-SFN-EA tends to remain stable in aqueous solution due to electrostatic repulsion. The polydispersity index (PdI) of the β-SFN group was < 0.1, indicating uniform particle size distribution, while that of the β-SFN-EA group increased to 0.39 ± 0.05, indicating a decrease in particle size uniformity after preparation. Furthermore, β-SFN was observed to be lightweight and easily floated in solution, while the prepared β-SFN-EA became heavier and easily precipitated.

[0135] Table 6. Particle size, PdI, and zeta potential (n=3)

[0136]

[0137] Example 20: Investigation of Hemostatic Effect

[0138] New Zealand white rabbits (female, 2.5-3.0 kg) were fixed to a metal plate for surgery and anesthetized with an intraperitoneal injection of 3% sodium pentobarbital. The positive control was commercially available chitosan hemostatic powder, specifically the transient composite microporous polysaccharide hemostatic powder purchased from Saikesaisi Biotechnology Co., Ltd., whose main active ingredient is chitosan.

[0139] Auricular artery hemostasis model: The fur on the back of the rabbit's ear was shaved, and the auricular artery was severed 7 cm from the tip of the ear. Blood immediately gushed from the wound. After 5 seconds of free bleeding, the surface blood was gently wiped away with a sterile cotton ball, and 150 mg of β-SFN-EA was immediately sprinkled on the bleeding site. A pre-weighed cotton ball was then gently placed vertically on the wound, and the timing was immediately started. Every 30 seconds, the cotton ball was gently removed to observe whether bleeding continued. If bleeding continued, the cotton ball was placed down and observed every 30 seconds until bleeding stopped. The bleeding time was recorded, and the gushing blood was absorbed with a cotton ball. The control group was treated with 150 mg of commercially available chitosan hemostatic powder, while the blank group was treated with only cotton balls, with other steps being the same as above. After hemostasis was completed, the cotton balls were weighed, and the blood loss was calculated using the differential weight method. Surviving rabbits were euthanized. Each sample was repeated 6 times, and the average value was taken.

[0140] Liver hemostasis model: After anesthetizing rabbits, the fur on their abdomen was shaved, and the rabbits were placed abdomen-up. The abdomen was disinfected with 75% medical alcohol, and the abdominal cavity was incised. The left medial lobe of the liver was pulled out of the abdominal cavity and placed on sterilized gauze. A wound 3.0 cm long and 0.5 cm deep was made on the liver, allowing free bleeding for 5 seconds. Immediately afterward, 50 mg of β-SFN-EA was sprinkled on the bleeding site, and a pre-weighed cotton ball was gently placed vertically on it. Timing was immediately started, and the cotton ball was gently removed every 30 seconds to observe if bleeding continued. If bleeding continued, the above procedure was repeated until bleeding stopped. The bleeding time was recorded, and the gushing blood was absorbed using a cotton ball. The control group was treated with 50 mg of commercially available chitosan hemostatic powder, while the blank group was treated only with cotton balls. After hemostasis was achieved, the cotton balls were weighed, and the blood loss was calculated using the differential weight method. Surviving rabbits were euthanized. Each sample was tested 6 times, and the average value was taken.

[0141] Femoral artery hemostasis model: Rabbits were anesthetized, placed abdomen-up, and had their hind legs shaved. The hind legs were disinfected with 75% medical alcohol. The skin and soft tissue were dissected with a scalpel to expose the femoral artery. The femoral artery was directly severed. After 5 seconds of free bleeding, the surface blood was gently wiped away with a sterile cotton ball, and 150 mg of β-SFN-EA was immediately sprinkled on the bleeding site. A pre-weighed cotton ball was gently placed vertically on top, and the timing was immediately started. Every 30 seconds, the cotton ball was gently removed to observe if bleeding continued. If bleeding continued, the cotton ball was left hanging for another 30 seconds to observe until bleeding stopped. The bleeding time was recorded, and the gushing blood was absorbed using a cotton ball. The control group was treated with 150 mg of commercially available chitosan hemostatic powder, while the blank group was treated only with cotton balls. After hemostasis, the cotton balls were weighed, and the blood loss was calculated using the differential weight method. Surviving rabbits were euthanized. Each sample was tested six times, and the average value was taken.

[0142] Figure 20 and Figure 21 These are comparison charts showing the bleeding time and amount in rabbits.

[0143] A rabbit ear has an artery located on its back, roughly the size of the aorta in an adult's arm. Conventional hemostatic materials struggle to stop the bleeding, making it an excellent model for testing the hemostatic performance of biological materials. In the marginal ear artery experiment, the β-SFN-EA group showed significantly better results than the chitosan group in both bleeding time and bleeding volume (P<0.05). Regarding bleeding time, the average hemostasis time in the chitosan group was 209.0 s, while it was 111.5 s in the β-SFN-EA group, a reduction of 87.44%. In terms of bleeding volume, the average bleeding volume in the chitosan group was 6.69 g, while it was only 2.24 g in the β-SFN-EA group, a reduction of 198.66%. This demonstrates that β-SFN-EA has a more significant hemostatic effect.

[0144] The liver has the richest blood supply of all internal organs, and liver hemorrhage is a particularly challenging issue during surgery. Therefore, using a rabbit liver hemorrhage model to simulate internal organ bleeding in humans is of significant importance. We created a 3.0 cm long and 0.5 cm deep wound on a rabbit liver to induce massive bleeding. β-SFN-EA exhibited a very significant absorption capacity for the gushing blood, which was not achieved by commercially available chitosan hemostatic powder, showing a significant difference (P<0.05). Interestingly, β-SFN-EA not only adhered to the wound to prevent secondary bleeding but also acted as a protective barrier to isolate the injured organ from surrounding tissues and prevent postoperative adhesions. Regarding bleeding time, the chitosan group had an average bleeding time of 123.2 s, while the β-SFN-EA group had 76.0 s, a reduction of 61.84%. In terms of blood loss, the chitosan group had an average blood loss of 0.35 g, while the β-SFN-EA group had an average blood loss of 0.07 g, a reduction of 400%.

[0145] Among bleeding events in the human extremities, bleeding from the femoral artery is the fastest and carries the highest risk. The femoral artery in the rabbit's groin is similar in size to the calf artery in an adult, making the rabbit femoral artery bleeding model representative for simulating massive bleeding in the human extremities. We created a lethal femoral artery injury model in rabbit surgery. The control group could not achieve visible hemostasis and even required manual pressure to achieve true hemostasis, while the β-SFN-EA group achieved automatic hemostasis without any external force, significantly superior to the chitosan group (P<0.05). Regarding bleeding time, the chitosan group averaged 246.0 s, while the β-SFN-EA group averaged 92.0 s, a reduction of 167.39%. Regarding blood loss, the chitosan group averaged 5.67 g, while the β-SFN-EA group averaged 1.64 g, a reduction of 245.73%. In conclusion, the rabbit massive hemorrhage model demonstrates that β-SFN-EA has a significant and rapid hemostatic effect.

[0146] Meanwhile, in three rabbit hemorrhage models, it was observed that β-SFN-EA had a faster sedimentation rate in the blood compared to other drugs, and easily settled to the bleeding site. In contrast, commercially available chitosan powder is lighter and more likely to float on the surface of the whole blood and be washed away by the blood flow, thus affecting its hemostatic effect.

[0147] Example 21 In vivo biocompatibility study

[0148] Biocompatibility in vivo was assessed by subcutaneous implantation in the back of SD rats at 7, 15, and 30 days, followed by histopathological examination. Figure 22H&E staining showed that acute inflammation appeared at the granule / tissue interface after 7 days, inflammation and collagen deposition decreased after 15 days, and no residual material or obvious tissue reaction was observed by 30 days, indicating initial inflammation followed by fibroblast proliferation and accelerated healing. β-SFN-EA was completely absorbed after 30 days.

[0149] Example 22: Examination of Tissue Healing Effect

[0150] (1) Full-thickness skin defect experiment in mice

[0151] The effect of a single application of β-SF-EA on subsequent skin wound healing was evaluated using a full-thickness skin defect model. C57B mice were randomly divided into a control group, a β-SF-EA group, a chitosan group, and a Johnson & Johnson SwiftVein group. Mice were anesthetized using an air anesthesia machine, their back hair was removed, and the back area was disinfected with iodine and alcohol. Subsequently, a healing pad was sutured to the back of the mouse, and a circular skin defect (d = 8 mm) was created within the pad on the back of each mouse using a skin punch. On day 0, each group received 10 mg of the drug; the control group received no treatment. All wounds were secured with breathable medical dressings to prevent drug movement. Wounds were photographed with a digital camera and dressings were changed on days 0, 3, 7, and 14. Wound size was measured using ImageJ software. Wound healing rate (%) = ((A o -A t ) / A o )*100% of which A o It is the initial wound area, A t This section shows the wound area at different time points (days 3, 7, and 14). To assess the healing process, four mice in each group were randomly euthanized on days 3, 7, and 14, and skin and muscle tissue samples were collected from the wound site and fixed in 4% paraformaldehyde for 24 hours. The harvested skin was then embedded in paraffin and stained with H&E and Masson trichrome. Healing was evaluated by analyzing indicators such as epithelial thickness, integrity, crusting, neovascularization, inflammatory cells, and collagen fiber deposition. The density of collagen deposition was quantified using ImageJ software. Immunofluorescence detection of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was used to investigate the anti-inflammatory and angiogenic effects of β-SFN-EA on the wound area. Immunohistochemical staining was used to detect the expression of IL-6, tumor necrosis factor-α (TNF-α), and platelet endothelial cell adhesion molecule-1 (CD31) in the skin wound. Positive markers were analyzed using ImageJ software.

[0152] like Figure 23As shown, the calculated wound closure rate on days 0, 3, 7, and 14 directly reflects the treatment effect. After 3 days of treatment, the wound area in each group decreased to some extent. Compared with the β-SF-EA group, the blank group had the lowest wound closure rate, indicating the worst healing effect. However, compared with the chitosan group and Johnson & Johnson's styrofoam group, the β-SF-EA group had the highest wound closure rate. On day 7, the healing rate of the β-SF-EA group remained the highest among all groups, indicating the best treatment effect. H&E stained sections after 7 days of healing showed (…). Figure 24 Compared to the control group, the chitosan group showed the generation of a large number of fibroblasts, but did not completely fill the defective tissue. New capillaries appeared sporadically in the granulation tissue, indicating a certain repair effect. At the same time, a large number of inflammatory cells appeared around the original blood vessels, resulting in an irregular overall section morphology. Compared to the control group, the Johnson & Johnson Quick-Repair group showed obvious tissue damage with inflammatory cell infiltration in the sections, but no large number of fibroblasts or granulation tissue were observed, indicating no significant repair ability. Compared to the control group, the β-SF-EA group not only generated a large number of fibroblasts and granulation tissue, but also had densely packed new capillaries, which was beneficial for wound repair. In summary, the Johnson & Johnson Quick-Repair group showed the weakest repair effect, chitosan slightly stronger but with severe inflammatory cell infiltration and incomplete tissue repair, and the β-SF-EA group showed the best repair, with mild inflammation and a large number of fibroblasts and granulation tissue, which were beneficial for repair. At day 15, the number of fibroblasts and granulation tissue in the chitosan group increased further, and the increase in newly formed capillaries was conducive to the wound repair process. However, the overall section was still not very regular, and the inflammation was reduced compared to day 7. In contrast, obvious defects could be seen in the Johnson & Johnson gauze group, indicating that Johnson & Johnson gauze had no repair effect. The number of fibroblasts and granulation tissue in the β-SF-EA group was reduced compared to day 7, the original tissue recovered to some extent, and the inflammation was weakened.

[0153] (2) Rabbit femoral artery vascular tissue healing experiment

[0154] New Zealand white rabbits were weighed and anesthetized, fixed in an antler-up position, and their hind legs were shaved. The hind legs were disinfected with 75% medical alcohol. The skin and tissue were dissected with a scalpel to expose the femoral artery. A non-penetrating arterial gap was created at the femoral artery using a (0.7*25mm) medical puncture needle. The gushing blood was absorbed using sterile cotton balls, and β-SF-EA was rapidly administered. After bleeding stopped, the medication was removed from the muscle, and the muscle and skin tissues were sutured. In the control group, no cotton balls were used to absorb blood; the rabbits were placed in their cages to allow for natural blood loss and recovery. After death, the blood was cleaned, the rabbits were weighed, the amount of blood loss was calculated, and vascular specimens were collected and stained with H&E to observe the extent of arterial tissue loss. The rabbits were euthanized after 30 days, and femoral artery tissue was collected for H&E staining to observe the recovery of the arterial layer.

[0155] Depend on Figure 25The results showed that by day 30, the blood vessels in the β-SF-EA group were almost completely repaired. Although the blood vessels contracted and narrowed, blood flow was still possible, indicating normal blood supply. The animals' lower limbs showed normal movement. It can be concluded that β-SF-EA has a significant effect on promoting vascular tissue repair while being used for hemostasis of non-penetrating wounds of the femoral artery in rabbits.

[0156] (3) Liver tissue healing experiment

[0157] SD rats were anesthetized via intraperitoneal injection of 1% sodium pentobarbital. Anesthetized rats were fixed supine on a dissection table, and a longitudinal incision was made along the midline of the abdomen to access the abdominal cavity and expose the right lobe of the liver. Peritoneal fluid was absorbed with clean gauze. A parallel wound approximately 2.0 cm long and 0.5 cm deep was made in the right lobe of the liver using a scalpel. Bleeding was immediately absorbed with pre-weighed cotton, and 20 mg of the sample (β-SF-EA, chitosan, and Johnson & Johnson's styrax) was administered to the wound surface. The wound was first compressed with cotton for 30 seconds to observe for bleeding. If bleeding continued, compression was repeated for another 30 seconds, and the observation was repeated until hemostasis was achieved. The abdominal cavity was then sutured, and the rats were fitted with collars to prevent biting and housed individually. To assess the healing process, four rats from each group were randomly euthanized on days 3, 7, and 14. Liver tissue samples were collected from the wound site and fixed in 4% paraformaldehyde for 24 hours. The harvested skin was then embedded in paraffin and stained with H&E staining and Masson's trichrome staining to evaluate the healing process, with particular attention paid to changes in liver fibrosis.

[0158] like Figure 26 As shown, the liver wound healing rate on days 0, 3, 7, and 14 directly reflects the healing effect of each group. After 3 days of treatment, the wound area in each group decreased to some extent. Compared with the chitosan group and Johnson & Johnson's styrofoam group, the β-SF-EA group had the highest wound closure rate and the best healing effect. On days 7 and 14, the healing rate of the β-SF-EA group was still the highest among all groups, indicating the best treatment effect. Repair was basically completed by day 30.

[0159] Example 23: In vivo anti-adhesion performance investigation

[0160] The in vivo anti-adhesion effect of β-SF-EA was evaluated using a rat lateral wall defect-cecal abrasion model. SD rats were anesthetized with sodium pentobarbital (50 mg / kg) and their abdominal hair was shaved. A 5 cm incision was then made along the midline of the abdominal wall using surgical scissors. The cecum was dissected, and its serosa was gently rubbed with sterile surgical gauze until pinpoint bleeding occurred. A 1 cm × 2 cm peritoneal defect was created on the corresponding lateral side of the abdominal wall using a scalpel. In the β-SF-EA group, 1 mL of β-SF-EA sample was placed on the injured abdominal wall and cecum. In the negative control group, 1 mL of sterile saline was sprayed onto the wound surface. In the positive control group, the abdominal wall defect was covered with commercially available polylactic acid anti-adhesion film. Four rats in each group were euthanized at 7 and 14 days, and the peritoneum was opened and adhesions were examined. The adhesions formed between the cecum and the abdominal wall were scored using a standard scoring system (Table 7).

[0161] The negative control group exhibited severe and irremovable adhesions on days 7 and 14, with reduced adhesion between the abdominal wall and cecum. This may be due to the fact that commercially available polylactic acid (PLA) anti-adhesion films are applied as solid sheets, failing to completely cover the wounded surface—a deficiency of such traditional anti-adhesion barrier materials. On days 7 and 14, the β-SF-EA group showed no signs of adhesion. Furthermore, on day 14, the wounded abdominal wall and cecum in the β-SF-EA group returned to normal. In Table 7, the β-SF-EA group scored the lowest among the three groups, indicating that β-SF-EA has a better anti-adhesion effect.

[0162] Table 7 shows the scores of each group on the standard adhesion rating scale.

[0163]

[0164] Note: Control group is the blank group, Film is commercially available polylactic acid anti-stick film, compared with the blank group, *P<0.05, **P<0.05.

Claims

1. A silk fibroin complex loaded with ellagic acid, characterized in that: Ellagic acid and silk fibroin are prepared by self-assembly; the silk fibroin is treated by β-sheet conformational transformation process, and the mass ratio of ellagic acid to silk fibroin is ≤1:

2.

2. The silk fibroin complex as described in claim 1, characterized in that: The mass ratio of ellagic acid to silk fibroin is 1:2 to 1:

16.

3. The silk fibroin complex as described in claim 2, characterized in that: The mass ratio of ellagic acid to silk fibroin is 1:

4.

4. A method for preparing the silk fibroin complex according to any one of claims 1 to 3, comprising two steps: conformational transformation of silk fibroin and self-assembly with ellagic acid: Step (1): Silk fibroin is transformed from α-helix to β-sheet conformation to obtain a silk fibroin solution. The conformational change can be carried out by chemical or physical methods. The chemical method includes promoting the transformation by polyol, polylactic acid, metal ions, pH or hydroxypropyl methylcellulose. The physical method includes promoting the transformation by high temperature, hydration pressure, ultra-low temperature placement, freeze drying, shear force, ultrasound, eddy current, laser irradiation, high pressure carbon dioxide treatment; Step (2): Ellagic acid is dissolved or suspended and then mixed with the silk fibroin solution obtained in step (1) to cause self-assembly, wherein the mass ratio of ellagic acid to silk fibroin is ≤1:2, thereby obtaining the silk fibroin complex loaded with ellagic acid.

5. The preparation method according to claim 4, characterized in that: The mass ratio of ellagic acid to silk fibroin in step (2) is 1:2 to 1:

16.

6. The preparation method according to claim 5, characterized in that: The mass ratio of ellagic acid to silk fibroin in step (2) is 1:

4.

7. The preparation method according to any one of claims 4 to 6, characterized in that: In step (2), ellagic acid is dissolved or suspended in an organic solvent, which is selected from methanol, ethanol, propanol, propylene glycol, glycerol, n-butanol, and isobutanol.

8. The preparation method according to claim 7, characterized in that: The organic solvent is propylene glycol. After co-incubating the ellagic acid suspension with silk fibroin in step (2), the final concentration of propylene glycol in the resulting solution is 5-20%.

9. The preparation method according to claim 8, characterized in that: The final concentration of propylene glycol is 10%.

10. A silk fibroin complex loaded with ellagic acid prepared by any one of the preparation methods of claims 4 to 9.

11. The use of the silk fibroin complex according to any one of claims 1 to 3 and 10 in the preparation of a formulation with hemostatic effect, characterized in that: It comprises the silk fibroin complex according to any one of claims 1 to 3, 10 and a pharmaceutically acceptable excipient.

12. The application as described in claim 11, characterized in that: The hemostatic preparations include gauze, sponge, spun fibers, sprays, powders, granules, gels, sealants, ointments, films, patches, and embolic agents.

13. The use of the silk fibroin complex according to any one of claims 1 to 3, 10 in the preparation of formulations having wound healing, and / or anti-infection, and / or anti-adhesion effects, characterized in that: It comprises the silk fibroin complex according to any one of claims 1 to 3, 10 and a pharmaceutically acceptable excipient.

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