A silk fibroin protein-based embolism agent loaded with polyhydroxy phenolic compounds and a preparation method thereof

Abstract

The present application provides a silk fibroin vascular embolism agent loaded with polyhydroxy phenolic compounds, which is prepared from a silk fibroin compound loaded with polyhydroxy phenolic compounds, wherein the silk fibroin compound loaded with polyhydroxy phenolic compounds is prepared by self-assembly of polyhydroxy phenolic compounds and silk fibroin, the silk fibroin is treated by a beta-fold conformation transformation process, and the mass ratio of polyhydroxy phenolic compounds to silk fibroin is <1:1; the vascular embolism agent is prepared by one of emulsification dispersion method, phase separation coagulation method, supercritical fluid technology, electrostatic adsorption method and dry granulation method. The vascular embolism agent has good biocompatibility and degradability, can effectively block blood flow, realize rapid hemostasis, and can be used for tumor vascular embolism treatment. Through the innovative self-assembly strategy, the mechanical properties of the silk fibroin microspheres are significantly improved. Compared with traditional hemostatic materials, the vascular embolism agent exhibits faster settling speed and higher hemostatic efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of materials, and particularly relates to a silk fibroin egg vascular embolization agent loaded with polyhydroxy phenolic compounds and a preparation method thereof. BACKGROUND

[0002] Hemostasis and tumor treatment are two important and challenging directions in the field of medicine. In the case of trauma, surgery or certain disease states, improper hemostasis can lead to serious health problems, even death. Similarly, in tumor treatment, how to effectively block the blood supply of tumors and limit their growth and metastasis is the key to improving treatment effect. Vascular embolization agents, as an effective treatment method, are widely used in hemostasis and tumor vascular embolization therapy. However, the existing embolization agents have certain limitations, such as poor biocompatibility, unstable embolization effect, and the possibility of causing inflammatory reactions. Therefore, it is of great clinical significance to research and develop new and efficient vascular embolization agents.

[0003] Traditional hemostatic methods include direct compression, suturing, using a tourniquet and other physical means, as well as the use of hemostatic drugs. Physical means may not be effective or applicable in some cases, and hemostatic drugs may have systemic side effects. In recent years, with the development of materials science, various new hemostatic materials have emerged, such as mineral-based materials, polymer-based materials, and biological-based materials. These materials have made certain progress in improving hemostatic efficiency, but still have some problems, such as slow hemostatic speed, possible infection, and toxic degradation products. Tumor vascular embolization therapy blocks tumor blood vessels to cut off the nutrient supply of tumors, thereby achieving the purpose of controlling tumor growth and metastasis. Common embolization agents include gelatin sponge, polyvinyl alcohol particles, and metal coils. These materials have achieved certain efficacy in clinical practice, but have problems such as poor biocompatibility, unstable embolization effect, and the possibility of causing inflammation. In addition, how to achieve precise embolization of tumor blood vessels and avoid damage to normal tissues is also a hot and difficult point of current research.

[0004] Silk fibroin protein has outstanding biocompatibility, biodegradability, and excellent mechanical properties, showing significant application potential in vascular embolization therapy. This natural protein is derived from silk and has low immunogenicity and good biological stability, allowing it to minimize the immune response of patients when used in the body. In addition, the degradation products of silk fibroin protein can be naturally cleared by the human body, reducing the risk of complications that may be caused by long-term implantation of materials. By fine-tuning the self-assembly process of silk fibroin protein and natural polyhydroxy phenolic compounds, a composite embolization agent with good blood clotting activity and high embolization capacity can be prepared. This self-assembly technology not only improves the mechanical properties and biological activity of the material, but also ensures the consistency of the product size and shape, thereby reducing the potential damage to the surrounding normal tissues.

[0005] Although the self-assembled embolic agent of silk fibroin and natural polyhydroxy compounds has significant advantages, it still faces technical challenges such as precise control of self-assembly conditions, optimization of material ratio, and verification of long-term biological safety and effectiveness. The present application aims to solve the limitations of the prior art by an innovative self-assembly method, and to provide a safer and more effective solution for vascular embolization. SUMMARY

[0006] The present application aims to solve the problem of multiple side effects of existing vascular embolic agents, and provides a silk fibroin vascular embolic agent loaded with polyhydroxy phenolic compounds, which is prepared from a silk fibroin complex loaded with polyhydroxy phenolic compounds. The silk fibroin complex loaded with polyhydroxy phenolic compounds is prepared by self-assembly of polyhydroxy phenolic compounds and silk fibroin, and the silk fibroin is treated by a beta-sheet conformation transformation process. The feeding ratio of polyhydroxy phenolic compounds to silk fibroin is less than 1:1 by mass. The vascular embolic agent is prepared by one of emulsification dispersion method, phase separation coagulation method, supercritical fluid technology, electrostatic adsorption method, and dry granulation method.

[0007] As a further improvement, the polyhydroxy phenolic compounds are selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignan, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolol, fisetin, puerarin, rhein, honeysuckle glycoside, proanthocyanidin, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, epigallocatechin gallate, tea polyphenols, epigallocatechin, salvianolic acid, gallic acid, pyrogallic acid, catechin, and tannic acid.

[0008] As a further improvement, the polyhydroxy phenolic compounds are poorly soluble polyhydroxy phenolic compounds, preferably selected from one or more of resveratrol, quercetin, baicalein, kaempferol, lignan, curcumin, ellagic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, rutin, myricetin, magnolol, fisetin, puerarin, rhein, honeysuckle glycoside, proanthocyanidin, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, oleanolic acid, epigallocatechin gallate; and most preferably ellagic acid.

[0009] As a further preferred solution, the feeding ratio of ellagic acid to silk fibroin is 1:2 to 1:16 by mass, and most preferably 1:4.

[0010] The application also provides a preparation method of the above-mentioned silk fibroin vascular embolic agent, which comprises three steps of silk fibroin conformation transformation, self-assembly with a polyhydroxy phenolic compound and preparation of the vascular embolic agent. Step (1): silk fibroin solution is prepared by transforming the silk fibroin from an alpha helix to a beta sheet conformation. The conformation change can be achieved by a chemical method or a physical method. The chemical method comprises promoting the transformation by a polyol, polylactic acid, a metal ion, pH or hydroxypropyl methylcellulose. The physical method comprises promoting the transformation by high temperature, hydration and pressurization, ultralow temperature placement, freeze drying, shear force, ultrasonic wave, vortex, laser irradiation and high pressure carbon dioxide treatment. Step (2): after the polyhydroxy phenolic compound is dissolved or suspended, it is mixed with the silk fibroin solution obtained in step (1) to stir and self-assemble, thereby obtaining a silk fibroin complex loaded with the polyhydroxy phenolic compound. Step (3): the silk fibroin complex loaded with the polyhydroxy phenolic compound obtained in step (2) is prepared into the silk fibroin vascular embolic agent loaded with the polyhydroxy phenolic compound by one of emulsification dispersion, phase separation coagulation, supercritical fluid technology, electrostatic adsorption and dry granulation.

[0011] As a further improvement, the step (1) adopts a polyol to promote the transformation of the silk fibroin beta sheet conformation. As a further improvement, the polyol is ethanol.

[0012] Beneficial effects

[0013] The embolic agent provided by the application can be rapidly solidified and shaped in a blood vessel, effectively blocks blood flow and achieves the effect of rapid hemostasis. More importantly, the embolic agent can also be combined with appropriate antitumor drugs for adjuvant therapy, thereby significantly improving the effectiveness of tumor vascular embolization therapy and having the effect of tumor adjuvant therapy. Meanwhile, the embolic agent has the advantages of simple preparation process, high production efficiency, easy industrial production and environmental protection. Through the innovative mixed polyhydroxy phenolic compound strategy, the mechanical properties of the silk fibroin microspheres are significantly improved, and the biocompatibility and degradability are maintained. In addition, the embolic agent has an ideal degradation period, can provide effective support during treatment and avoid the risks caused by long-term retention. Compared with traditional hemostatic materials, the silk fibroin complex exhibits faster settling speed and higher hemostatic efficiency, has excellent biological safety, does not cause cytotoxicity, does not induce hemolytic reaction and can be naturally degraded in the body, thereby providing a fast, efficient and safe hemostatic solution for clinical first aid and surgical operations. BRIEF DESCRIPTION OF DRAWINGS

[0014] The application will be further described below with reference to the accompanying drawings:

[0015] Figure 1 Fourier infrared spectrum analysis (FTIR)

[0016] Figures 2-4Blood 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-polyphenol 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

[0017] Figure 5 Blood coagulation time comparison chart (n=3), *, compared with the β-SF-EA group, *P<0.05, **P<0.01,

[0018] ***P<0.001

[0019] Figure 6 Drug loading amount comparison chart (n=3). *, compared with the β-SF-EA group, *P<0.05, **P<0.01,

[0020] ***P<0.001; ND represents not detected, Not Detected

[0021] Figures 7-8 Drug loading amount comparison chart (n=3). *, compared with the water group, *P<0.05, **P<0.01, ***P<0.001 Figure 9 Fourier infrared spectrum analysis (FTIR), in which EA is physically mixed with β-SF at 1:1 (w / w) Figure 10 Molecular docking chart. L74 represents Leu (leucine) at position 74, and D27 represents Asp (aspartic acid) at position 27

[0022] Figure 11 Characterization: (A) FTIR spectrum, (B) XRD, (C) TG, (D) 1H NMR

[0023] Figure 12 Blood adsorption experiment. Among them, a is blank, b is β-SF, c is EA, d is Yunnan white, e is tranexamic acid, f is β-SF-EA, Figure 13 same

[0024] Figure 13 Test tube tilt coagulation experiment chart

[0025] Figure 14 Coagulation index BCI chart (n=3)

[0026] Figure 15Rat tail cutting experiment. Among them, Figure A is the bleeding diagram of rat tail cutting immediately after the drug powder is scattered, and Figure B is the hemostasis time diagram after tail cutting; *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001. Figure 16 Rat liver and femoral artery bleeding time diagram (n=3). *, compared with the β-SF-EA group, *P<0.05, **P<0.01, ***P<0.001

[0027] Figure 17 LO2 cell, L929 cell and drug co-incubation for 48h cell relative proliferation rate diagram (n=6) Figure 18 β-SF-EA in vitro degradation diagram (n=3)

[0028] Figure 19 Swelling rate determination (n=3)

[0029] Figure 20 Degradation rate determination (n=3)

[0030] Figure 21 Rabbit kidney injury bleeding experiment (n=3)

[0031] Figure 22 Rabbit ear tumor experiment (n=3), Figure 21 、 22 Compared with the autologous thrombus group, *P<0.05, **P<0.01, ***P<0.001 DETAILED DESCRIPTION

[0032] Example 1 Preparation of silk fibroin complex loaded with polyhydroxy phenolic compound (β-SF-polyhydroxy phenolic compound)

[0033] Step (1) Transformation of silk fibroin β-sheet conformation: dilute silk fibroin (Silk Fibroin, SF) with water by 5-10 times, then slowly pour into an excess of organic solvent under vigorous stirring, stir for 2-4h to complete the transformation of β-sheet conformation, remove the liquid part by mechanical separation method such as suction filtration, centrifugation, membrane filtration, etc., and then freeze-dry after repeated washing of the remaining part, to obtain β-sheet silk fibroin (β-SF), wherein the organic solvent is selected from methanol, ethanol, propylene glycol, butylene glycol, etc., and ethanol is preferred; the mechanical separation method is preferably high-speed centrifugation, such as 10000rpm high-speed centrifugation for 15-20min.

[0034] Fourier infrared spectroscopy analysis (FTIR), mix 5% sample with 95% KBr and grind into fine powder, use FTIR spectrometer (Nicolet is5) for transmission scanning, each spectrum is obtained in transmission mode (ten scans), the resolution is 4cm -1 , and the spectral range is 4000-500cm -1The FTIR spectra of the relatively high-level β-fold structure are in the range of 1615–1640 cm⁻¹. -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.

[0035] 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.

[0036] The polyphenol compound is selected from water-insoluble polyphenol 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), Magnolol Acid (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 polyphenol compounds including Gallic Acid (GA), Pyrogallic Acid (PA), Catechin (C), Tannic Acid (TA), Tea Polyphenols (TP), Acaciin, Epigallocatechin (EGC), and the like.

[0037] Example 2 Preparation of the silk fibroin complex loaded with polyphenol compound

[0038] Different from example 1, the organic solvent in step (1) is ethanol, and the mechanical separation method is high speed centrifugation at 10000 rpm for 15-20 min. In step (2), the same amount of polyhydroxy phenolic compound and silk fibroin is used, and in the preparation of the polyhydroxy phenolic compound suspension, water-soluble polyhydroxy phenolic compound is prepared using water as solvent, and poorly soluble polyhydroxy phenolic compound is prepared using water or organic solvent as solvent, and the mechanical separation method is to take the precipitate after high speed or super high speed centrifugation. Step (3) is added, i.e. drying into powder: the solution obtained in step (2) is dried into powder after removing liquid by mechanical separation methods such as suction filtration, centrifugation, membrane filtration, and sedimentation, or using spray dryer and other drying equipment to dry into powder in one step, thereby obtaining the silk fibroin compound loaded with polyhydroxy phenolic compound; and the mechanical separation method is preferably high speed or super high speed centrifugation to take the precipitate.

[0039] Adopting the insoluble polyhydroxy phenolic compounds as raw materials: ellagic acid-silk fibroin complex (β-SF-EA), resveratrol-silk fibroin complex (β-SF-RES), quercetin-silk fibroin complex (β-SF-QUE), baicalein-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), sinapic acid-silk fibroin complex (β-SF-Sinapic Acid), rutin-silk fibroin complex (β-SF-RUT), myricetin-silk fibroin complex (β-SF-MYR), magnololic acid-silk fibroin complex (β-SF-MA), fisetin-silk fibroin complex (β-SF-FIS), puerarin-silk fibroin complex (β-SF-PUE), rhein-silk fibroin complex (β-SF-RHE), lonicerin-silk fibroin complex (β-SF-Lon), proanthocyanidin-silk fibroin complex (β-SF-PC), p-coumaric acid-silk fibroin complex (β-SF-p-CA), vanillic acid-silk fibroin complex (β-SF-VA), hesperetin-silk fibroin complex (β-SF-Hes), naringenin-silk fibroin complex (β-SF-NAR), luteolin-silk fibroin complex (β-SF-Lut), genistein-silk fibroin complex (β-SF-Gen), emodin-silk fibroin complex (β-SF-Emo), oleanolic acid-silk fibroin complex (β-SF-Ola), salvianolic acid-silk fibroin complex (β-SF-DS), epigallocatechin gallate-silk fibroin complex (β-SF-EGCG); adopting the water-soluble polyhydroxy phenolic compounds as raw materials: gallic acid-silk fibroin complex (β-SF-GA), pyrogallic acid-silk fibroin complex (β-SF-PA), catechin-silk fibroin complex (β-SF-C), tannic acid-silk fibroin complex (β-SF-TA), acaciin-silk fibroin complex (β-SF-Acaciin), epigallocatechin-silk fibroin complex (β-SF-EGC), tea polyphenol-silk fibroin complex (β-SF-TP).

[0040] Example 3 Coagulation effect investigation

[0041] The obtained β-SF-polyphenol compound was prepared into a 2 mg / mL test solution with normal saline for coagulation effect investigation: blood was taken 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, normal saline was used as a control, and the mixture was mixed and incubated at 37°C for 15 min, then the whole blood viscosity was detected by a full-automatic blood rheometer (all operations were completed within 2 h after blood collection).

[0042] The whole blood viscosity is the result of friction between blood cells and plasma protein molecules when blood flows, and is the most important indicator in hemorheology. When the blood viscosity increases, it indicates that the blood flow is blocked, the deformation ability of red blood cells decreases and the aggregation increases, the flow of blood into small blood vessels and capillaries decreases, and the passing ability decreases. Figures 2-4 The results showed that the silk fibroin complex loaded with polyphenol compounds (β-SF-polyphenol compound) could significantly increase the 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 could significantly enhance the coagulation effect; and overall, the effect of the complex formed by self-assembly of silk fibroin and poorly soluble polyphenol compounds was better than that of the self-assembly complex of silk fibroin and water-soluble polyphenol compounds.

[0043] 0.1 g of the drug powder to be tested was weighed into a 1.5 mL centrifuge tube, 0.5 mL of anticoagulant was measured and added to the centrifuge tube containing different drugs, the centrifuge tube was placed vertically on the experimental workbench, and the timing was started. After 30 s, the inverted centrifuge tube was rotated, and whether the blood flowed was observed, and the operation was repeated until the anticoagulant could not flow, the timing was stopped, and the coagulation time was recorded. Figure 5 The results showed a similar trend as Figures 2-4 The results showed a similar trend as

[0044] Among all the complexes, β-SF-EA had 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, the symmetrical catechol structure and planar structure of ellagic acid can form intramolecular and intermolecular hydrogen bonds, which increases the rigidity of the complex molecules. This stable molecular structure can reduce the dynamic change of the molecules, thereby indirectly improving the stability of the intermolecular hydrogen bonds.

[0045] Example 4 Preparation of polyphenol compound-silk fibroin complex (α-SF-polyphenol compound)

[0046] The difference from Example 2 is that step (1) is deleted, and β-SF is replaced by untreated SF, i.e. SF in the natural α-helix conformation, in step (2), and an equal amount of the polyhydroxy phenolic compound and silk fibroin is fed in step (2) to prepare a polyhydroxy phenolic compound-α-silk fibroin complex, referred to as α-SF-polyhydroxy phenolic compound.

[0047] Example 5 Preparation of polyhydroxy phenolic compound-silk fibroin complex (SH-SF-polyhydroxy phenolic compound)

[0048] The difference from Example 2 is that step (1) is performed as follows: silk fibroin is dissolved in an aqueous solution containing tris(2-carboxyethyl)phosphine hydrochloride, and is left to stand at room temperature for 5 minutes to perform reduction reaction treatment, to obtain a solution of reduced SF with a concentration of 1.0-5.0 wt%. The rest is the same as in Example 2, wherein an equal amount of the polyhydroxy phenolic compound and silk fibroin is fed in step (2) to prepare the resulting polyhydroxy phenolic compound-silk fibroin complex, referred to as SH-SF-polyhydroxy phenolic compound. This method uses the method of destroying disulfide bonds to expose hydrophobic amino acids to combine the polyhydroxy phenolic compound such as ellagic acid.

[0049] Example 6 Investigation of self-assembly performance

[0050] The polyhydroxy phenolic compound uses ellagic acid as a model drug, and three ellagic acid-silk fibroin complexes are prepared according to the steps in Examples 2, 4 and 5, wherein the solvent for EA is water, i.e. β-SF-EA, α-SF-EA and SH-SF-EA. The assembly performance is evaluated by drug loading and encapsulation efficiency, which are calculated by formula 1) and formula 2), respectively, wherein the amount of EA in the silk fibroin complex is calculated by the difference method, i.e. the amount of EA fed is subtracted by the amount of free EA after co-incubation with SF. The content of EA is analyzed by high performance liquid chromatography (HPLC), using a Hedera C18 column, a mobile phase of acetonitrile: water = 20:80 (v / v), a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, a sample injection amount of 10 μL, and a chromatogram collection time of 15 min. The drug loading and encapsulation efficiency are calculated by the following formula:

[0051] Drug loading = amount of EA in silk fibroin complex / total amount of SF and EA fed in formula 1)

[0052] Encapsulation efficiency = amount of EA in silk fibroin complex / amount of EA fed in formula 2)

[0053] From the above results, it can be seen that the self-assembly performance of the three ellagic acid-silk fibroin complexes is different. Figure 6From the results, the drug loading capacity is: β-SF-EA > SH-SF-EA >> α-SF-EA, wherein the drug loading capacity of β-SF-EA reaches 9.6%, while EA in α-SF-EA is almost undetectable, indicating that the efficiency of self-assembly of α-helix structure silk fibroin with ellagic acid is very low, highlighting the necessity of conformational transition of silk fibroin; SH-SF-EA uses the destruction of disulfide bonds in silk fibroin molecules to expose its hydrophobic amino acids, thereby completing self-assembly with ellagic acid, but the drug loading capacity is only 4.5%, which is much lower than that of β-SF-EA (P < 0.001).

[0054] Example 7 Investigation of the effect of solvent on the drug loading capacity of EA

[0055] The polyphenolic compound takes ellagic acid as a model drug, and β-SF-EA is prepared by the steps in Example 2. In step (2), water, ethanol, propylene glycol, butanediol, etc. are used as solvents to prepare polyphenolic compound suspensions. If an organic solvent is used as a suspension solvent, the final concentration of the organic solvent in the solution obtained after the polyphenolic compound suspension is incubated with β-SF is 10%. The drug loading capacity of each sample is investigated by the method in Example 6. Figure 7 The results show that the addition of organic solvents effectively improves the drug loading capacity of ellagic acid, wherein propylene glycol > butanediol > ethanol > water, and the drug loading capacity is highest when propylene glycol is used, reaching 19.57 ± 1.01%.

[0056] Next, the effect of propylene glycol concentration on the drug loading capacity of EA is investigated. In step (2), propylene glycol is used as a solvent to prepare an EA suspension, and the final concentration of propylene glycol in the solution obtained after the EA suspension is incubated with β-SF is 0%, 5%, 10%, 15%, and 20%, respectively. Figure 8 The results show that the drug loading capacity of the propylene glycol aqueous solution group is better than that of the water group, and the effect of the 10% propylene glycol aqueous solution group is best, with a drug loading capacity of 19.57 ± 1.01%.

[0057] Example 8 Investigation of different EA:SF feeding ratios

[0058] The polyphenolic compound takes ellagic acid as a model drug, and β-SF-EA is prepared by the steps in Example 2. In step (2), propylene glycol is used as a solvent to prepare an EA suspension, and the final concentration of propylene glycol in the solution obtained after the EA suspension is incubated with β-SF is 10%. Meanwhile, the feeding ratio of EA to β-SF is controlled to be EA: β-SF = 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:12, and 1:16 (w / w). As shown in Table 1, the drug loading capacity and encapsulation efficiency of EA cannot be observed for EA: β-SF = 1:0.5 and 1:1, while they can be observed for other groups. Meanwhile, Fourier infrared spectroscopy analysis is performed on each sample by the method in Example 1. Figure 9), it can be seen that the EA hydroxyl peak (3471 cm -1 ) disappeared, indicating that EA and β-SF had completed hydrogen bonding at this time, and self-assembly was successful, but the EA hydroxyl peak still existed in the EA: β-SF = 1:0.5, 1:1 groups and their physical mixing groups, and the self-assembly of the two molecules could not be achieved. The above results comprehensively show that the self-assembly of β-SF and EA can only be achieved when the feed ratio EA: β-SF < 1:1 (w / w), and the EA: β-SF = 1:4 can achieve the maximum drug loading and encapsulation efficiency (Table 1), which is called the optimal feed ratio.

[0059] Further investigate the effect of different EA: β-SF feed ratios on whole blood viscosity by the method in Example 3, the results in Table 2 show that each group with EA: β-SF < 1:1 can significantly increase the whole blood viscosity at low, medium and high shear rates (P < 0.5), and is higher than the EA and β-SF groups (P < 0.5), indicating that β-SF-EA prepared by EA: β-SF < 1:1 feed ratio has significant coagulation efficacy, and EA and β-SF have synergistic effect; among them, EA: β-SF = 1:4 has the best coagulation effect.

[0060] In summary, the preparation process of β-SF-EA is to prepare SF with β-fold, the feed ratio EA: β-SF < 1:1 (w / w), and the specific steps are shown in Example 1; the optimal process is EA: β-SF = 1:4 (w / w), and the final concentration of propylene glycol in the solution obtained after incubation of the ellagic acid suspension prepared with propylene glycol as the solvent with SF is 10%. Neither the β-fold conformation transformation of SF nor the feed ratio EA: β-SF ≥ 1:1 can achieve the self-assembly of EA and β-SF. When EA: β-SF = 1:2 (w / w), the drug loading and encapsulation efficiency are both low, indicating that although the self-assembly of EA and β-SF can be achieved at this time, there is a large loss.

[0061] Table 1 Effect of β-SF and EA feed ratio on drug loading and encapsulation efficiency (n = 3)

[0062]

[0063] ND represents not detected, Not Detected.

[0064] Table 2 Effect of different EA: β-SF feed ratios on whole blood viscosity (n = 3)

[0065]

[0066] *: Compared with the physiological 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, ###P<0.001.

[0067] Example 9 Molecular docking of silk fibroin complex

[0068] Computer simulation was performed on the binding mode of ellagic acid and silk fibroin. The silk fibroin heavy chain crystal structure (PDB code: 3UA0) was obtained from the PDB bank. Due to the less previous binding mode research, we performed reasonable prediction of the binding site by the SiteMap module in 2018, and then the hydrogen was added to the silk fibroin crystal structure after removing the irrelevant ions, and the ellagic acid small molecule was converted into a 3D conformation by the LigPrep module, and output to 32 isomers, and finally 20 binding poses were output by Glide extra precision (XP). As shown in Fig. 8, two phenolic hydroxyl groups in the ellagic acid small molecule form a hydrogen bond with L74, and another hydrogen bond is formed with D27 on the other heavy chain, which indicates that another two highly symmetrical hydroxyl groups in the ellagic acid small molecule form a hydrogen bond to achieve stable binding. In addition, due to the maintenance of highly similar β-sheet secondary structure in the heavy chain of silk fibroin, the ellagic acid small molecule may not have sufficient hydrophobic interaction with two heavy chain proteins to maintain stability, and it is reasonably speculated that the ellagic acid small molecule will be at least wrapped by three to four heavy chain proteins to achieve stable binding. The molecular docking result explains the molecular mechanism of the experimental result in Example 8 that “EA: β-SF < 1:1 (w / w) can realize the self-assembly of β-SF and EA”. Figure 10

[0069] The samples measured in Examples 10-14 were prepared by the optimal process.

[0070] Example 10 Characterization of silk fibroin complex loaded with polyphenolic compounds

[0071] FT-IR proved the self-assembly of EA and β-SF Figure 11 A): The disappearance of the EA hydroxyl peak (3471 cm -1 ) indicates that EA and β-SF may be self-assembled by intermolecular hydrogen bonds. X-ray diffraction (XRD) Figure 11 ​B) The characteristic peaks of EA were weakened or disappeared after combining with silk fibroin, indicating that it was not a simple physical mixture, but a new crystal phase formed by interaction, which confirmed the FT-IR results; at the same time, the change of crystal form was seen, β-SF showed a broad diffraction peak, indicating that they were amorphous macromolecules; EA showed many strong diffraction peaks, indicating that it was in a crystalline state; after self-assembly of β-SF and EA, the EA peak intensity decreased significantly, which could be judged that β-SF-EA was mainly amorphous macromolecule.

[0072] Thermogravimetric (TG) curves were evaluated and divided into three stages Figure 11 C). The first stage, from room temperature to 120℃, the weight loss was about 6.1%, mainly due to the evaporation and desorption of adsorbed water; the second stage, the temperature was between 150℃ and 450℃, corresponding to the decomposition of silk fibroin molecular chain, the decomposition of amino acid residue side chain and the breaking of peptide bond, among which the silk I crystal structure (α-helix and random coil) degraded at 250℃, the silk II crystal structure (β-sheet) degraded at 260℃; the main degradation peak of β-SF appeared at 260℃, the surface formed a stable β-fold conformation; interestingly, the main degradation peak of β-SF-EA moved to 280℃, indicating that the silk II crystal structure was more stable, which might be due to the strong interaction between EA and protein during self-assembly, thus improving the stability of the complex. To determine the optimal amount of EA incorporated into β-SF, proton nuclear magnetic resonance (1H NMR) Figure 11 D) : In β-SF-EA, the peak related to EA was significantly weakened (<0.007), confirming that EA was combined with β-SF through catechol hydrogen, further proving their combination.

[0073] Example 11 Investigation of coagulation effect

[0074] (1) Blood sample processing

[0075] New Zealand white rabbits were adaptively fed for one week, and fasted the night before the experiment. 1% sodium pentobarbital was injected intraperitoneally at a dose of 3mL·kg -1 of anesthetic, and blood was taken from the abdominal aorta after anesthesia, and collected in a 5mL heparin sodium anticoagulant tube. After blood collection, the blood and anticoagulant were mixed evenly, and were ready for use.

[0076] (2) In vitro adsorption of blood capacity experiment

[0077] 0.1g of the drug powder to be tested was weighed into a 1.5mL centrifuge tube, and 0.5mL of anticoagulated blood was measured and added to the centrifuge tube containing different drugs, which was placed vertically on the experimental workbench and the timing was started. After 30s, the inverted centrifuge tube was rotated, and whether the blood flowed was observed, and the operation was repeated until the anticoagulated blood could not flow, and the timing was stopped. Figure 12As shown in Table 2, the β-SF-EA achieved complete adsorption of blood within two minutes, while the other groups did not achieve this result, indicating that the ability of β-SF-EA to adsorb blood was significantly better than the positive control Yunnan Baiyao, tranexamic acid, and significantly better than β-SF and EA alone, indicating that β-SF and EA can synergistically enhance each other.

[0078] (3) Test tube tilt coagulation experiment

[0079] Precisely weigh 5.0 mg of the drug powder to be tested into a 10 mL test tube, and spread the powder as much as possible on the bottom of the test tube. Add 1 mL of anticoagulated blood into the test tube, then add 0.2 mol·L -1 CaCl2 solution 25 μL, and immediately repeat mixing, and immediately start timing. First stand for 1 min, tilt the test tube every 30 s, until the blood coagulates and does not flow, then stop timing. Figure 13 As shown in Table 2, the β-SF-EA achieved complete adsorption of blood within two minutes, while the other groups did not achieve this result, indicating that the ability of β-SF-EA to adsorb blood was significantly better than the positive control Yunnan Baiyao, tranexamic acid, and significantly better than β-SF and EA alone, indicating that β-SF and EA can synergistically enhance each other. This experiment observed that β-SF-EA had faster blood coagulation ability than other drugs.

[0080] (4) Blood rheology determination

[0081] Whole blood viscosity is the result of friction between blood cells and plasma protein molecules when blood is flowing, and is the most important indicator in blood rheology. When blood viscosity increases, it indicates that blood flow is blocked, and the deformability of red blood cells decreases and aggregation increases, reducing blood flow into small blood vessels and capillaries, and reducing the ability to pass. The method in Example 3 was used to determine the whole blood viscosity of blood treated with β-SF-EA at 1·s -1 , 30·s -1 , 100·s -1 , 150·s -1 , 200·s -1 shear rate, and the results in Table 3 show that β-SF-EA significantly increased the viscosity of whole blood at any shear rate (P<0.05), and was significantly better than the commercially available chitosan hemostatic powder group (P<0.05). The control group of commercially available chitosan hemostatic powder was purchased from Sikes Biological Technology Co., Ltd. Instant composite microporous polysaccharide hemostatic powder, and its main active ingredient is chitosan.

[0082] Table 3 Comparison of whole blood viscosity of β-SF-EA and chitosan (n=3)

[0083]

[0084] *: Compared with the blank 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, ###P < 0.001.

[0085] (4) Coagulation index BCI

[0086] Prepare β-SF-EA of different mass (10, 20, 30, 40, 50 mg) and place them in several 50 mL centrifuge tubes. Add 0.1 mL anticoagulant to the samples, and then immediately add 0.02 mL of 0.2 mol / L CaCl2 solution. After 5 min, add 25 mL of deionized water to the beaker and centrifuge at 300 rpm for 5 min. Then take out the solution and measure the Abs value at a wavelength of 540 nm using an enzyme marker. Set up a control: add 0.1 mL of anticoagulant to the beaker, and then add 25 mL of deionized water. The Abs value measured at the same wavelength is assumed to be 100 as a reference value. Then the coagulation index BCI is: BCI = 100 x A 样品 / A 对照 The BCI index is an indicator of the coagulation effect, and the smaller the index, the better the coagulation effect. Figure 14 It is shown that increasing the dosage (increasing the concentration in the same volume) will greatly reduce the coagulation index (BCI), which indicates that the coagulation effect is positively correlated with the dosage.

[0087] Example 12: Hemostatic effect evaluation

[0088] Male SD rats (about 250.0 g) were used to evaluate the hemostatic ability of β-SF-EA.

[0089] Rat tail amputation hemostasis experiment: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. A 6 cm section was taken from the tail of the rat, and the rat tail was cut with surgical scissors. After bleeding, 20 mg of sample powder (β-SF-EA, Yunnan Baiyao, tranexamic acid) was sprinkled on the wound, and the time was counted from the start of bleeding.

[0090] Rat liver wound hemostasis experiment: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthetized rats were fixed on the dissection table in a supine position, a longitudinal incision was made along the abdominal midline to expose the right lobe of the liver. The abdominal cavity was dried with clean gauze, and a long 2.0 cm, deep 0.5 cm parallel wound was made on the right lobe of the liver with a blade. The wound was immediately dried with pre-weighed cotton, and 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan white medicine, tranexamic acid) was respectively sprinkled on the surface of the wound, and the timing started. The wound was first pressed with cotton for 30 s, and whether it continued to bleed was observed. If the bleeding continued, the wound was pressed with cotton for another 30 s, and then observed. This process was repeated until there was no obvious blood on the surface of the cotton, which was considered successful hemostasis, and the timing was stopped and the hemostasis time was recorded.

[0091] Rat thigh muscle wound hemostasis experiment: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and the anesthetized rats were fixed on the dissection table in a supine position. The leg hair was shaved with a rat hair clipper. After the inner side of the thigh was disinfected with alcohol, a muscle wound about 3 cm long and 1 cm deep was made with a scalpel. The bleeding started timing, and 20 mg of sample powder (EA, β-SF, β-SF-EA, Yunnan white medicine, tranexamic acid) was respectively sprinkled on the surface of the wound after 5 s of bleeding, and the hemostasis effect was observed, and the hemostasis time was recorded.

[0092] Figure 15 The rat tail cutting experiment figure shows that when β-SF-EA is placed on the wound, the blood loss stops quickly, and almost no blood is left on the filter paper. It can significantly promote hemostasis within 30 seconds; the blank group bleeds for more than five minutes, which loses statistical significance and is not listed in Figure B. As a control, the tranexamic acid group and the Yunnan white medicine group need more than 4 times the hemostasis time, and the bleeding amount is significantly higher than that of the β-SF-EA group (P<0.05).

[0093] Figure 16 The rat liver and femoral artery bleeding time figure shows that the femoral artery bleeding of the blank group exceeds five minutes, which loses statistical significance and is not listed in the figure. Figure 16 It is shown that β-SF-EA can significantly shorten the hemostasis time of the two models, and the hemostasis effect is significantly better than that of Yunnan white medicine (P<0.05) and tranexamic acid (P<0.05), and also significantly better than that of EA (P<0.05) and β-SF group (P<0.05), indicating that silk fibroin and ellagic acid have a synergistic effect. The above results are consistent with the blood rheology and test tube tilt coagulation results in Example 11. In both rat bleeding models, it is observed that β-SF-EA has a faster blood sedimentation speed than other drugs, and is easy to quickly settle in the bleeding port, while Yunnan white medicine, tranexamic acid and β-SF powder are lighter in texture and more likely to float on the surface of whole blood and be washed away by blood flow, thereby affecting their hemostasis effect.

[0094] Example 13 Safety Investigation

[0095] (1) Cell recovery

[0096] The frozen L02 cells and L929 cells were taken out and gently shaken in a 37°C water bath to completely melt. In the clean bench, the cell suspension in the frozen tube was transferred to a cell culture flask, and 4 mL of DMEM medium containing 10% fetal bovine serum was added to the flask, and the cells were gently blown with a dropper to evenly disperse in the medium, and then placed in a 5% CO2, 37°C cell incubator for culture. After 6h of culture, the culture flask was alcohol sterilized and placed in a clean bench, the old culture medium was aspirated and washed with 4 mL of culture medium, and then placed in a cell incubator for continuous culture.

[0097] (2) CCK8 quantitative method

[0098] β-SF-EA and EA were taken 0.2 mg respectively and dissolved in 1 mL of DMSO, and gradient concentrations were set up respectively.

[0099] The logarithmic growth period cells were taken, washed with PBS solution, and then treated with trypsin digestion solution to make the cells into a suspended state. After calculating the cell density with a cell counting plate, 1x10 5 After the cells were inoculated in the 96-well plate at 1x10 实验组 / OD 空白组 *100%. According to the cell toxicity grade in Table 4, the cell toxicity level was determined, and the positive control should not be lower than grade 3. The cell grade number was 0-1, which was qualified.

[0100] Table 4 Cell proliferation degree reaction grading standard

[0101]

[0102] We studied the toxicity of β-SF-EA and EA on L929 cells and LO2 cells by CCK8 experiment. Figure 17 As shown in Table 4, the relative growth rates of all groups of β-SF-EA were higher than 85%, according to the toxicity classification standard of ISO 10993-1, indicating that the cytotoxicity of β-SF-EA was 0-1 grade. At the same time, the addition of silk fibroin effectively improved the biocompatibility of ellagic acid. In summary, β-SF-EA did not affect cell proliferation and had good cell compatibility.

[0103] (2) Hemolysis test

[0104] 8 mL of fresh rabbit blood was diluted with 10 mL of normal saline. 20 mg of β-SF-EA was added to a 50 mL centrifuge tube, 10 mL of normal saline was added, and it was incubated in a 37°C water bath for 10 min. Then 0.2 mL of diluted rabbit blood was added, shaken gently, and incubated in a 37°C water bath for 60 min. After centrifugation at 1000 rpm for 5 min, the supernatant was taken and the absorbance was measured at 540 nm. The positive control group used 10 mL of distilled water and 0.2 mL of rabbit blood, and the negative control group used 10 mL of normal saline and 0.2 mL of rabbit blood, with the same operation method. Each group was performed in triplicate. The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = (absorbance of test sample - absorbance of negative control) / (absorbance of positive control - absorbance of negative control) * 100%.

[0105] Different materials may cause red blood cells to rupture and produce hemolysis when they come into direct contact with blood. Hemolysis test is used to evaluate the hemolysis of materials by checking the concentration of hemoglobin. According to the calculation, the hemolysis rate of β-SF-EA was 1.13 ± 0.02% (Table 5), which was lower than 5% according to GB / T4233.2, so the prepared β-SF-EA met the international standard.

[0106] Table 5 Hemolysis rate (n = 3)

[0107]

[0108] Example 14 Degradation performance test

[0109] A certain amount of β-SF-EA was weighed and placed in PBS solution containing 0.1 μ / mL protease XIV, and PBS solution without protease XIV was used as blank sample. The samples (n = 3) were incubated in PBS solution and PBS solution containing 0.1 μ / mL protease XIV for 5, 10, 15, 20, 25 and 30 days, and all degradation samples were replaced with fresh solution every day at a fixed time. The degradation products were dried at 60°C, and the sample was weighed to a constant weight, and the remaining mass retention rate R M was calculated according to the following formula: R M / %=M dt / Mi * 100%, wherein, M i M0 is the initial mass, M dt M is the mass remaining after t days.

[0110] As a hemostatic agent applied in vivo and in vitro, it is required to have good biodegradability. Ideally, in the early stage of wound healing, the hemostatic powder can firmly bond the tissues together to avoid cracking, and preferably degrades slowly, as the wound heals, the drug gradually degrades, and finally the residual drug components can be completely degraded after the wound heals, absorbed by the body or excreted outside the body. The degradation effect of β-SF-EA at 37°C is shown in Figure 18 It can be found that the degradation rate of β-SF-EA in PBS is slow, and the remaining mass is 90.11±3.51% at 30d. In the PBS solution containing protease XIV, the degradation effect of β-SF-EA can be divided into two stages: the first stage (0-15d), the degradation rate is slow, and the remaining mass is 67.83±2.25% at 15d; the second stage (15-30d), the degradation rate increases significantly, and the remaining mass is 24.50±0.70% at 30d, which degrades most of it, indicating that it has good degradation.

[0111] Example 15 Preparation of silk fibroin vascular embolization agent loaded with polyphenolic compounds

[0112] On the basis of Example 1 or 2, step (3) is added: the silk fibroin complex loaded with polyphenolic compounds obtained in step (2) is prepared by one of the following methods: emulsion dispersion method, phase separation coagulation method, supercritical fluid technology, electrostatic adsorption method, dry granulation method. Some of the following examples show the preparation method:

[0113] (1) Emulsion dispersion method: a common method for preparing vascular embolization agents, by mixing the drug with the polymer solution, and then using emulsion technology to form microspheres or particles. For example, polylactic acid (PLA) and poly-lactic acid-poly-ether copolymer microspheres can be prepared by this method.

[0114] (2) Phase separation coagulation method: similar to the emulsion dispersion method, but mainly through physical phase separation to form microspheres, which can control the particle size and release characteristics of the microspheres.

[0115] (3) Supercritical fluid technology: using supercritical carbon dioxide to atomize the drug and polymer solution to form microspheres, this technology reduces the use of surfactants, and is suitable for the preparation of biodegradable materials.

[0116] (4) Electrostatic adsorption method: the drug (e.g. bleomycin) is loaded onto the drug-loaded microspheres with anionic groups through electrostatic interaction. This method is simple and easy to realize industrial production.

[0117] (5) Dry granulation method: the powdery raw materials are compressed into granules through physical methods (e.g. compression, extrusion), which can be used to prepare vascular embolization agents with different properties.

[0118] Preparation of the silk fibroin vascular embolization agent loaded with polyphenolic compounds in Example 16

[0119] Different from Example 15, the emulsion dispersion method was used to prepare the vascular embolization agent, and the specific steps were as follows:

[0120] S1) In a four-necked flask, paraffin and Span 80 were added and stirred uniformly, and the silk fibroin complex loaded with polyphenolic compounds obtained in step (2) and super liquid iodinated oil were added dropwise, the flask was capped, and the stirring was continued to prepare a suspension;

[0121] S2) The suspension was added to a centrifuge tube and centrifuged, and then anhydrous ethanol was added, followed by washing with petroleum ether and anhydrous ethanol. After washing, centrifugation was performed;

[0122] S3) Ethanol was added overnight, and after removing the ethanol, drying was performed. The dried product was ground into a uniform powder to obtain the vascular embolization agent.

[0123] Examples 17-19 used tannic acid as a model drug of polyphenolic compounds, and the vascular embolization agent prepared by the method in Example 16 was subjected to experiments.

[0124] Example 17 Measurement of the physical properties of the vascular embolization agent

[0125] (1) Swelling rate The swelling rate of the vascular embolization agent was measured by calculating the change in diameter before and after the swelling experiment. To evaluate the effect of pH and temperature on the swelling rate of the microspheres, the samples were immersed in PBS with different pH values (1.0, 5.8 and 7.4) at 37°C, or in PBS with a pH value of 7.4 at a speed of 100 rpm at different temperatures (4°C, 37°C and 60°C). The swollen microspheres were taken out at certain time points to measure their diameters. The swelling rate of the microspheres was calculated according to the following formula: Swelling rate = (D t -D0) / D0*100%, where D0 is the initial diameter of the microspheres, and D t is the diameter of the microspheres after swelling. The results are shown in Table 1. Figure 19As shown, the swelling tendency of the microspheres showed a rapid swelling trend in the first 10 min and reached the maximum swelling rate at 20 min in the environment with pH values of 1.0, 5.8 and 7.4, and the swelling rate of the microspheres increased significantly with the increase of pH value. The swelling behavior of the microspheres at 4℃, 21℃ and 37℃ showed a rapid swelling in the first 10 min and reached the maximum swelling degree at 20 min, indicating that the environmental temperature had a great influence on the swelling rate of the microspheres.

[0126] (2) In vitro degradation The in vitro degradation performance of the β-SF-EA vascular embolization agent was determined by the method in Example 14. The results are shown in Table 2. Figure 20 As shown, the mass loss of the microspheres increased with the increase of incubation time. The microspheres degraded slowly in the first 7 days with a degradation rate of 4.1%, and the degradation rate accelerated in the last 2 weeks. On the 21st day, the weight loss rate reached 20.8%. According to this degradation trend, it can be predicted that the microspheres have good degradability.

[0127] Example 18 Investigation of hemostatic effect

[0128] Rabbit kidney injury bleeding model intervention embolization hemostasis. Experimental grouping: 24 New Zealand white rabbits (4.0-4.4 kg) were randomly divided into vascular embolization agent experimental group, autologous thrombus control group, and blank control group (normal saline), 6 in each group. Among them, the autologous thrombus control group was prepared according to the following method: a 5mL syringe pre-filled with 500U thrombin was used to extract 5mL of whole blood through the arterial sheath, and the thrombin and whole blood were immediately mixed uniformly to form an autologous thrombus. The blank control group was directly injected into the target artery without embolization material through the catheter.

[0129] After anesthesia, the anesthetized rabbits were placed in a rabbit board in supine position, and the limbs and front upper incisors were fixed with gauze strips. The right hind limb root inside was the surgical field, and the skin was prepared and disinfected after the operation. After subcutaneous local anesthesia with rupivacaine mesylate, the right femoral artery was exposed and cut open, and a 4F arterial sheath was inserted. Heparin calcium was given at a dose of 100U / kg. Then the laparotomy kidney injury bleeding model and renal artery intervention embolization hemostasis were performed, and the steps were as follows:

[0130] ①Open the abdomen and find the right kidney, dissociate the tissue and fat capsule around the kidney, and expose the kidney. ②Inject iodixanol into the right renal artery through a 4F guide catheter and obtain the image of normal renal artery distribution. ③Make a 1.5 cm long and 0.6 cm deep wound in the renal parenchyma to simulate the rupture and bleeding of the right kidney after injury. After modeling, re-angiography is performed to obtain the image of kidney injury and bleeding. ④Place the tip of the 4F guide catheter at the end of the right renal artery, and according to the results of the contrast agent affecting the activity of thrombin, use a special "sandwich" delivery method to inject 1 mL of embolic material into the embolic site. ("Sandwich" delivery method: first fill the catheter with contrast agent, then push a certain amount of thrombus into the catheter, and then inject contrast agent). ⑤Observe the bleeding of the renal parenchyma wound after the release of the embolic agent, and perform renal arteriography to determine whether the embolization hemostasis is successful; if it is not completely hemostatic, inject embolic agent into the embolic target site through the catheter again until complete hemostasis. Record the total amount of embolic agent used during the operation and the time from angiography to complete hemostasis of the renal parenchyma wound. ⑥After the operation, remove the arterial sheath and ligate the right posterior femoral artery, suture the skin and subcutaneous tissue, and send the animals back to the animal house for observation.

[0131] Figure 21 The results showed that the average time from completion of embolic agent delivery to complete hemostasis of the renal parenchyma was 1.5±1 min, the vascular embolic agent dosage was 0.53±0.16 mL (0.4 mL-0.8 mL), and the survival rate of animals 1 week after operation was 100%. The average time from embolization to complete hemostasis in the autologous thrombus control group was 1.8±1.5 min, the average thrombus dosage was 0.90±0.35 mL (0.4 mL-1.4 mL), and the mortality rate of experimental animals within 6 hours after operation was 50%. The interventional embolization hemostasis in the blank control group was not successful.

[0132] Example 19 Evaluation of the effect of application in adjuvant therapy of tumors

[0133] Establishment of the VX2 rabbit ear interventional animal model: ① The low-temperature frozen VX2 tumor strain was recovered in a 37℃ water bath, injected into the deep muscle of the rabbit thigh, observed for two weeks, and the tumor strain was allowed to grow satisfactorily in the rabbit hind limb. The animal was sacrificed and the tumor was cut into 0.5mm in diameter and placed in culture solution. ② Adult female New Zealand white rabbits, weighing 2.5-3.0 kg. The experimental rabbits were kept separately in the rabbit cage for at least 5 days. After intravenous injection of 3% sodium pentobarbital (1 mL / kg) at the ear margin, the hair on the auricle was shaved with scissors. The tumor tissue was derived from the non-necrotic, well-vascularized VX2 tumor tissue in the hind limb of the rabbit. Fresh tumor tissue was cut into 1x1x3mm strips, and the tumor tissue was used within 2h. A 18-gauge coaxial needle was used for subcutaneous implantation. The size of the rabbit ear tumor was recorded regularly with a caliper. ③ VX2 rabbit ear interventional animal model embolization experiment: rabbits were anesthetized with intravenous injection of 3% sodium pentobarbital (1 mL / kg) at the ear margin. Then, the ear was shaved and disinfected, and the skin was cut parallel and slightly lateral to the distal end of the central artery of the ear. The tumor artery can be found under the illumination of a flashlight. Local 2% lidocaine solution was dropped to prevent arterial spasm. A 21G needle with an external catheter was inserted into the distal end of the central artery of the ear, and the needle and catheter were fixed with elastic tape to prevent accidental displacement. Finally, 0.5mL of vascular embolic agent was injected into the rabbit ear artery, and 0.5mL of normal saline (pH=7) was injected into the rabbit ear artery of the control group. The injection process and embolization effect were observed by X-ray. The rabbits were sacrificed 1h after injection. The size of the rabbit ear tumor was recorded regularly with a caliper. The rabbit ear tumor was removed and CT scanning was performed. The results are shown in Figure 22 As shown in the results, the liver tumors in each group showed an increasing trend, but the tumor growth rate was significantly inhibited in the embolization treatment group, and the tumor necrosis was more obvious in the embolization treatment group, with most showing central area sheet necrosis, and individual rabbits with peripheral tumor or small sheet necrosis in the left lobe of the liver.

Claims

1. A silk fibroin vascular embolism agent loaded with a polyphenol compound, characterized in that: A vascular embolization agent is prepared from a fibroin complex loaded with a polyphenol compound, the fibroin complex loaded with the polyphenol compound being prepared by self-assembly of the polyphenol compound and the fibroin, wherein the fibroin is treated by a beta-sheet conformation transformation process, the feeding ratio of the polyphenol compound to the fibroin is <1:1 in mass ratio, and the polyphenol compound is a poorly soluble polyphenol compound; the vascular embolization agent is prepared by one of emulsion dispersion method, phase separation coacervation method, supercritical fluid technology, electrostatic adsorption method, and dry granulation method.

2. The silk fibroin vascular embolism agent as claimed in claim 1, wherein: The poorly soluble polyphenol compound is selected from one or more of the following: resveratrol, quercetin, baicalein, kaempferol, lignan, curcumin, ellagic acid, ferulic acid, caffeic acid, syringic acid, sinapic acid, myricetin, magnolignan, fisetin, rhein, proanthocyanidin, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, and epigallocatechin gallate.

3. The silk fibroin vascular embolizing agent as described in claim 2, characterized in that: The poorly soluble polyphenol compound is ellagic acid.

4. The silk fibroin vascular embolism agent of claim 3, wherein the silk fibroin is a silk fibroin protein having a molecular weight of 25 to 45 kDa. The feeding ratio of the ellagic acid to the fibroin is 1:2 to 1:16 in mass ratio.

5. The silk fibroin vaso-occlusive device of claim 4, wherein the silk fibroin is a recombinant silk fibroin. The feeding ratio of the ellagic acid to the fibroin is 1:4 in mass ratio.

6. A preparation method of the fibroin vascular embolization agent according to any one of claims 1 to 5, comprising three steps of fibroin conformation transformation, self-assembly with a polyphenol compound, and preparation of a vascular embolization agent: step (1): a fibroin solution is prepared by a transformation process from an alpha-helix to a beta-sheet conformation of the fibroin, the conformation change can be achieved by a chemical method or a physical method, the chemical method comprises promoting the transformation by alcohol, polylactic acid, metal ions, pH, or hydroxypropyl methyl cellulose, and the physical method comprises promoting the transformation by high temperature, hydration pressurization, ultralow temperature placement, freeze drying, shear force, ultrasonic wave, vortex, laser irradiation, and high pressure carbon dioxide treatment; step (2): after the polyphenol compound is dissolved or suspended, it is mixed with the fibroin solution obtained in step (1) and stirred to achieve self-assembly, thereby obtaining a fibroin complex loaded with the polyphenol compound; and step (3): the fibroin complex loaded with the polyphenol compound obtained in step (2) is prepared into a fibroin vascular embolization agent loaded with the polyphenol compound by one of emulsion dispersion method, phase separation coacervation method, supercritical fluid technology, electrostatic adsorption method, and dry granulation method.

7. The method of claim 6, wherein: The step (1) adopts alcohol to promote the transformation of the beta-sheet conformation of the fibroin.

8. The method of claim 7, wherein: The alcohol is ethanol.

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