Silk fibroin tea polyphenol hydrogel as well as preparation method and application thereof

Abstract

The invention discloses silk fibroin tea polyphenol hydrogel as well as a preparation method and application thereof. The silk fibroin tea polyphenol hydrogel is prepared by adopting the following method: (S10) preparing silk fibroin; (S20) preparation of silk fibroin hydrogel: irradiating the aqueous solution of the SFMA and the LAP under ultraviolet light to prepare the SFMA hydrogel; and (S30) preparing the silk fibroin tea polyphenol hydrogel. When the hydrogel is applied, TP is injected into the bladder, then the SFMA hydrogel is injected, light curing is carried out, and the silk fibroin tea polyphenol hydrogel is formed on the bladder wall. The prepared silk fibroin tea polyphenol hydrogel is suitable for being applied to treatment of hemorrhagic cystitis, is good in adhesion and has a good hemostatic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel preparation, in particular to a vegan protein and tea polyphenol hydrogel and a preparation method and application thereof. Background Art

[0002] Hemorrhagic cystitis is a more serious type of cystitis, common in women. The current treatments for hemorrhagic cystitis mainly include the following aspects: 1. Antibiotic treatment: For hemorrhagic cystitis caused by bacterial infection, antibiotics are a conventional treatment method. Doctors will select appropriate antibiotics for treatment based on the results of bacterial culture, which usually has a good effect. 2. Symptomatic treatment: Including hemostatic drugs, analgesics, etc., which help to relieve symptoms and improve the patient's quality of life. 3. Bladder lavage: For severe hemorrhagic cystitis, doctors may perform bladder lavage to clean blood clots and inflammatory substances in the bladder, which helps to relieve symptoms.

[0003] However, there are still some problems with treating hemorrhagic cystitis using the above methods: 1. Antibiotic abuse: Long-term and excessive use of antibiotics can easily lead to the development of drug resistance, making some bacteria insensitive to conventional antibiotics and increasing the difficulty of treatment. 2. Recurrent attacks in some patients: Some patients may experience recurrent attacks and require long-term regular medication or further investigation of the cause to reduce the recurrence of symptoms. 3. Limited effectiveness of symptomatic treatment: Although symptomatic treatment can relieve symptoms, it cannot cure the disease. Comprehensive treatment methods are still needed to improve treatment effectiveness.

[0004] Therefore, further research is needed in the future to investigate the etiology and treatment mechanisms of hemorrhagic cystitis and develop more personalized and effective treatment methods to improve the treatment outcomes and quality of life of patients. Summary of the Invention

[0005] One advantage of the present invention is that it provides a silk fibroin-tea polyphenol hydrogel and a preparation method and application thereof, wherein the silk fibroin-tea polyphenol hydrogel has a good hemostatic effect and is suitable for the treatment of hemorrhagic cystitis.

[0006] Another advantage of the present invention is that it provides a silk fibroin-tea polyphenol hydrogel and a preparation method and application thereof. The silk fibroin-tea polyphenol hydrogel has good adhesion and can form a close fit with bladder tissue.

[0007] Another advantage of the present invention is that it provides a filament protein tea polyphenol hydrogel and its preparation method and application. The SFMA / TP hydrogel system can react with urea in urine, thereby changing the molecular action in the system and achieving a better hemostatic effect.

[0008] Another advantage of the present invention is that it provides a silk fibroin-tea polyphenol hydrogel and a preparation method and application thereof. The silk fibroin-tea polyphenol hydrogel has good tensile and compressive properties.

[0009] Another advantage of the present invention is that it provides a silk fibroin tea polyphenol hydrogel and its preparation method and application. The hemorrhagic cystitis treated with the silk fibroin tea polyphenol hydrogel has no toxic side effects and does not recur, which is of great significance for the complete treatment of hemorrhagic cystitis.

[0010] According to one aspect of the present invention, the present invention provides a method for preparing a vegan protein tea polyphenol hydrogel, comprising the following steps:

[0011] (S10) Preparation of silk fibroin;

[0012] (S20) preparing silk fibroin hydrogel; and

[0013] (S30) Preparation of silk fibroin-tea polyphenol hydrogel.

[0014] The step (S10) includes the following steps: (S101) placing the sliced ​​cocoons into a Na2CO3 solution and boiling to remove the sericin, washing, and drying; (S102) dissolving the dry silk in a LiBr solution, adding glyceryl methacrylate, stirring, filtering the resulting solution, and dialyzing; (S103) freeze-drying the silk fibroin glycidyl methacrylate solution, and storing the freeze-dried SFMA powder for subsequent use.

[0015] In the step (S10), 40 g of sliced ​​cocoons are placed in 1 L of 0.05 M Na2CO3 solution and boiled at 100° C. for 30 minutes to remove sericin; the silk is then washed with distilled water for multiple times, and the degummed silk is dried at room temperature. Subsequently, 20 g of dry silk is dissolved in 100 mL of 9.3 M LiBr solution at 60° C. for 1 hour; 6 mL of glycerol methacrylate is added to the mixture, and the mixture is stirred at 300 rpm at 60° C. for 3 hours. The resulting solution is filtered with gauze and dialyzed against distilled water using a 12-14 kDa dialysis tube for 4 days; finally, the silk fibroin glycidyl methacryloyl solution is freeze-dried for 48 hours, and the freeze-dried SFMA powder is stored at -80° C. for subsequent use.

[0016] In the step (S20), the photocrosslinking agent used includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 2-hydroxy-2-methylpropiophenone or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0017] In the step (S20), the aqueous solution of SFMA and LAP is irradiated under ultraviolet light to prepare SFMA hydrogel.

[0018] Wherein in the step (S20), an aqueous solution containing 10% SFMA and 0.2% LAP is irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel.

[0019] In the step (S30), the prepared SFMA hydrogel is immersed in a tea polyphenol aqueous solution for a predetermined time to obtain a silk fibroin tea polyphenol hydrogel. When used, the silk fibroin and the tea polyphenol are in situ gelled at the lesion site to form the silk fibroin tea polyphenol hydrogel.

[0020] In the step (S30), the prepared SFMA hydrogel is immersed in a 10% TP aqueous solution for 3 hours to obtain a SFMA / TP hydrogel.

[0021] The proportion of the LAP is in the range of 0.1%-0.5%, and the concentration of the tea polyphenols is in the range of 5-20%.

[0022] According to another aspect of the present invention, the present invention also provides a silk fibroin tea polyphenol hydrogel, wherein the silk fibroin tea polyphenol hydrogel is obtained by mixing the silk fibroin hydrogel and tea polyphenol under predetermined conditions.

[0023] The silk fibroin tea polyphenol hydrogel is prepared by the above-mentioned preparation method.

[0024] The silk fibroin and tea polyphenol hydrogel is suitable for adhering to the bladder wall.

[0025] The silk fibroin tea polyphenol hydrogel is suitable for treating hemorrhagic cystitis.

[0026] The silk fibroin tea polyphenol hydrogel is prepared by the preparation method according to any one of claims 1 to 8.

[0027] The silk fibroin tea polyphenol hydrogel is suitable for adhering to the mucosal layer of the bladder. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the molecular structure and organizational function of silk fibroin and silk fibroin tea polyphenol hydrogel according to one embodiment of the present invention.

[0029] Figure 2 Schematic diagram of the molecular structure and nuclear magnetic spectrum of the silk fibroin according to the above embodiment of the present invention.

[0030] Figure 3 Schematic diagram of the rheological properties of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.

[0031] Figure 4Schematic diagram of the mechanical properties of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.

[0032] Figure 5 Schematic diagram of the tensile test of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.

[0033] Figure 6 Schematic diagram of the electron microscope microstructure of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.

[0034] Figure 7 3. It is a schematic diagram of the adhesion performance test of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.

[0035] Figure 8 This is a schematic diagram of the operation process of intravesical instillation of the silk fibroin and tea polyphenols hydrogel according to the above embodiment of the present invention.

[0036] Figure 9 Schematic diagram of the dyed slice of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.

[0037] Figure 10 This is a schematic diagram of quantitative analysis of the silk fibroin and tea polyphenol hydrogel in treating mucosal bleeding according to the above embodiment of the present invention.

[0038] Figure 11 Schematic diagram of the in vitro degradation test results of silk fibroin tea polyphenol hydrogel (SFMA / TP).

[0039] Figure 12 Schematic diagram of HE staining results after in vivo degradation of silk fibroin tea polyphenol hydrogel (SFMA / TP). DETAILED DESCRIPTION

[0040] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are for illustrative purposes only, and those skilled in the art will readily appreciate other obvious variations. The basic principles of the present invention defined in the following description may be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the present invention.

[0041] Example 1

[0042] The preparation method of silk fibroin and silk fibroin tea polyphenol hydrogel according to a preferred embodiment of the present invention is as follows:

[0043] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0044] 40 g of sliced ​​cocoons were placed in 1 L of 0.05 M Na2CO3 solution, boiled at 100 ° C for 30 minutes to remove sericin, and then washed with distilled water several times. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution and dissolved at 60 ° C for 1 hour. Then, 6 mL of glyceryl methacrylate (GMA, 424 mM) was added to the mixture and stirred at 300 rpm at 60 ° C for 3 hours. The resulting solution was filtered with gauze and dialyzed against distilled water for 4 days using a 12-14 kDa dialysis tube. Finally, the silk fibroin glycidyl methacryloyl solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80 ° C for subsequent use. The obtained SFMA was analyzed on a 500 MHz Bruker nuclear magnetic resonance spectrometer using D2O as a solvent. 1 H NMR analysis. The molecular structure and organization diagram are shown in the following figure. Figure 1 As shown, 1 The results of H NMR analysis are as follows Figure 2 As shown, Figure 2 (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the nuclear magnetic resonance spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.

[0045] (S20) Synthesis of Silk Fibroin Hydrogel

[0046] An aqueous solution containing 10% SFMA and 0.2% LAP was irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel. LAP is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, which is a photocrosslinker for the hydrogel.

[0047] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0048] The prepared SFMA hydrogel was immersed in a 10% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the bladder mucosa.

[0049] Example 2

[0050] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:

[0051] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0052] 40 g of sliced ​​cocoons were placed in 1 L of 0.05 M Na2CO3 solution, boiled at 100 ° C for 30 minutes to remove sericin, and then washed with distilled water several times. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution and dissolved at 60 ° C for 1 hour. Then, 6 mL of glyceryl methacrylate (GMA, 424 mM) was added to the mixture and stirred at 300 rpm at 60 ° C for 3 hours. The resulting solution was filtered with gauze and dialyzed against distilled water for 4 days using a 12-14 kDa dialysis tube. Finally, the silk fibroin glycidyl methacryloyl solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80 ° C for subsequent use. The obtained SFMA was analyzed on a 500 MHz Bruker nuclear magnetic resonance spectrometer using D2O as a solvent. 1 H NMR analysis. The molecular structure and organization diagram are shown in the following figure. Figure 1 As shown, 1 The results of H NMR analysis are as follows Figure 2 As shown, Figure 2 (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the nuclear magnetic resonance spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.

[0053] (S20) Synthesis of Silk Fibroin Hydrogel

[0054] An aqueous solution containing 20% ​​SFMA and 0.5% LAP was irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel. LAP is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, which is a photocrosslinker for the hydrogel.

[0055] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0056] The prepared SFMA hydrogel was immersed in a 15% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the bladder mucosa.

[0057] Example 3

[0058] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:

[0059] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0060] 40 g of sliced ​​cocoons were placed in 1 L of 0.05 M Na2CO3 solution, boiled at 100 ° C for 30 minutes to remove sericin, and then washed with distilled water several times. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution and dissolved at 60 ° C for 1 hour. Then, 6 mL of glyceryl methacrylate (GMA, 424 mM) was added to the mixture and stirred at 300 rpm at 60 ° C for 3 hours. The resulting solution was filtered with gauze and dialyzed against distilled water for 4 days using a 12-14 kDa dialysis tube. Finally, the silk fibroin glycidyl methacryloyl solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80 ° C for subsequent use. The obtained SFMA was analyzed on a 500 MHz Bruker nuclear magnetic resonance spectrometer using D2O as a solvent. 1 H NMR analysis. The molecular structure and organization diagram are shown in the following figure. Figure 1 As shown, 1 The results of H NMR analysis are as follows Figure 2 As shown, Figure 2 (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the nuclear magnetic resonance spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.

[0061] (S20) Synthesis of Silk Fibroin Hydrogel

[0062] An aqueous solution containing 10% SFMA and 0.35% LAP was irradiated under 405 nm ultraviolet light for 4 minutes to prepare SFMA hydrogel. LAP is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, which is a photocrosslinker for the hydrogel.

[0063] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0064] The prepared SFMA hydrogel was immersed in a 20% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, a silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the mucosal layer of the bladder.

[0065] Example 4

[0066] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:

[0067] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0068] 40 g of sliced ​​cocoons were placed in 1 L of 0.05 M Na2CO3 solution, boiled at 100 ° C for 30 minutes to remove sericin, and then washed with distilled water several times. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution and dissolved at 60 ° C for 1 hour. Then, 6 mL of glyceryl methacrylate (GMA, 424 mM) was added to the mixture and stirred at 300 rpm at 60 ° C for 3 hours. The resulting solution was filtered with gauze and dialyzed against distilled water for 4 days using a 12-14 kDa dialysis tube. Finally, the silk fibroin glycidyl methacryloyl solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80 ° C for subsequent use. The obtained SFMA was analyzed on a 500 MHz Bruker nuclear magnetic resonance spectrometer using D2O as a solvent. 1 H NMR analysis. The molecular structure and organization diagram are shown in the following figure. Figure 1 As shown, 1 The results of H NMR analysis are as follows Figure 2 As shown, Figure 2 (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the nuclear magnetic resonance spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.

[0069] (S20) Synthesis of Silk Fibroin Hydrogel

[0070] An aqueous solution containing 10% SFMA and 0.1% LAP was irradiated under 405 nm ultraviolet light for 6 minutes to prepare SFMA hydrogel. LAP is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, which is a photocrosslinker for the hydrogel.

[0071] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0072] The prepared SFMA hydrogel was immersed in a 5% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the bladder mucosa.

[0073] Example 5

[0074] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:

[0075] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0076] 40 g of sliced ​​silkworm cocoons were placed in 1 L of 0.05 M Na₂CO₃ solution and boiled at 100°C for 30 minutes to remove the sericin. The silk was then washed several times with distilled water. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution at 60°C for 1 hour. Six mL of 424 mM glycerol methacrylate (GMA) was then added to the mixture, and the mixture was stirred at 300 rpm for 3 hours at 60°C. The resulting solution was filtered through gauze and dialyzed against distilled water using 12-14 kDa dialysis tubing for 4 days. Finally, the glycidyl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80°C for subsequent use.

[0077] (S20) Synthesis of Silk Fibroin Hydrogel

[0078] An aqueous solution containing 10% SFMA and 0.2% 2-hydroxy-2-methylpropiophenone was irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel, wherein 2-hydroxy-2-methylpropiophenone was a photocrosslinking agent for the hydrogel.

[0079] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0080] The prepared SFMA hydrogel was immersed in a 10% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the bladder mucosa.

[0081] Example 6

[0082] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:

[0083] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0084] 40 g of sliced ​​silkworm cocoons were placed in 1 L of 0.05 M Na₂CO₃ solution and boiled at 100°C for 30 minutes to remove the sericin. The silk was then washed several times with distilled water. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution at 60°C for 1 hour. Six mL of 424 mM glycerol methacrylate (GMA) was then added to the mixture, and the mixture was stirred at 300 rpm for 3 hours at 60°C. The resulting solution was filtered through gauze and dialyzed against distilled water using 12-14 kDa dialysis tubing for 4 days. Finally, the glycidyl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80°C for subsequent use.

[0085] (S20) Synthesis of Silk Fibroin Hydrogel

[0086] An aqueous solution containing 20% ​​SFMA and 0.5% 2-hydroxy-2-methylpropiophenone was irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel, wherein 2-hydroxy-2-methylpropiophenone was a photocrosslinking agent for the hydrogel.

[0087] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0088] The prepared SFMA hydrogel was immersed in a 20% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, a silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the mucosal layer of the bladder.

[0089] Example 7

[0090] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0091] 40 g of sliced ​​silkworm cocoons were placed in 1 L of 0.05 M Na₂CO₃ solution and boiled at 100°C for 30 minutes to remove the sericin. The silk was then washed several times with distilled water. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution at 60°C for 1 hour. Six mL of 424 mM glycerol methacrylate (GMA) was then added to the mixture, and the mixture was stirred at 300 rpm for 3 hours at 60°C. The resulting solution was filtered through gauze and dialyzed against distilled water using 12-14 kDa dialysis tubing for 4 days. Finally, the glycidyl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80°C for subsequent use.

[0092] (S20) Synthesis of Silk Fibroin Hydrogel

[0093] An aqueous solution containing 15% SFMA and 0.4% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel, wherein 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was a photocrosslinker for the hydrogel.

[0094] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0095] The prepared SFMA hydrogel was immersed in a 10% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the bladder mucosa.

[0096] Example 8

[0097] (S10) Synthesis of Silk Fibroin Acid (SFMA)

[0098] 40 g of sliced ​​silkworm cocoons were placed in 1 L of 0.05 M Na₂CO₃ solution and boiled at 100°C for 30 minutes to remove the sericin. The silk was then washed several times with distilled water. The degummed silk was dried at room temperature. Subsequently, 20 g of dry silk was dissolved in 100 mL of 9.3 M LiBr solution at 60°C for 1 hour. Six mL of 424 mM glycerol methacrylate (GMA) was then added to the mixture, and the mixture was stirred at 300 rpm for 3 hours at 60°C. The resulting solution was filtered through gauze and dialyzed against distilled water using 12-14 kDa dialysis tubing for 4 days. Finally, the glycidyl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80°C for subsequent use.

[0099] (S20) Synthesis of Silk Fibroin Hydrogel

[0100] An aqueous solution containing 5% SFMA and 0.2% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was irradiated under 405 nm ultraviolet light for 5 minutes to prepare SFMA hydrogel, wherein 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was a photocrosslinker for the hydrogel.

[0101] (S30) Synthesis of Silk Fibroin-Tea Polyphenol Hydrogel (SFMA / TP)

[0102] The prepared SFMA hydrogel was immersed in a 15% TP aqueous solution for 3 hours and photocured to obtain an SFMA / TP hydrogel for in vitro testing. It is worth mentioning that during the application process, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed by in situ gelation at the lesion site. That is, during application, TP was first injected into the bladder, followed by the SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) hydrogel was formed on the surface of the bladder wall to adhere to the bladder mucosa.

[0103] The silk fibroin tea polyphenol hydrogel (SFMA / TP) prepared by the above method is suitable for the treatment of hemorrhagic cystitis. Figure 1 It can be seen that the SFMA / TP hydrogel system can react with urea in urine, causing changes in the molecular interactions in the system.

[0104] First, an animal model was established to observe the effect of silk fibroin tea polyphenol hydrogel (SFMA / TP) in the treatment of hemorrhagic cystitis.

[0105] All animal-related procedures were performed in accordance with national and international regulations for animal experimentation. The present invention used Sprague-Dawley rats to establish a chemically induced hemorrhagic cystitis model. Cyclophosphamide (CYP) was injected intraperitoneally at a dose of 150 mg / kg, and the model was successfully established 24 hours later. Specifically, after intraperitoneal injection of 150 mg / kg into the rats, we observed gross hematuria, bladder bleeding, and submucosal hemorrhage by HE staining.

[0106] Intravesical injection of SMFA / TP hydrogel for the treatment of hemorrhagic cystitis: After anesthetizing the rats, a midline incision about 1 cm long was made in the lower abdomen. A urinary catheter was inserted to drain excess urine from the bladder. Subsequently, 0.2 ml of 10% tea polyphenols (TP) was injected, and the bladder was massaged to ensure that the tea polyphenols (TP) were in complete contact with the bladder wall. Subsequently, 0.2 ml of 10% SFMA was injected to expand and thin the bladder. The bladder was massaged again and then exposed to ultraviolet light to solidify the hydrogel on the bladder wall. Finally, the catheter was removed and the abdominal cavity and skin were sutured. Modeling of hemorrhagic cystitis with silk fibroin tea polyphenols hydrogel (SFMA / TP) is as follows Figure 8 As shown in (a).

[0107] Rheological properties of silk fibroin tea polyphenol hydrogel (SFMA / TP) Figure 3 As shown by Figure 3 It can be seen that the storage modulus and loss modulus also increased after the reaction of SFMA hydrogel and tea polyphenols (TP), and the silk fibroin tea polyphenols hydrogel (SFMA / TP) showed stronger mechanical properties and adhesion.

[0108] The mechanical properties of silk fibroin tea polyphenol hydrogel (SFMA / TP) are as follows Figure 4 As shown, from Figure 4 It can be seen that the silk fibroin tea polyphenol hydrogel (SFMA / TP) generated by the reaction of SFMA hydrogel and tea polyphenols (TP) has significantly improved the compression, cyclic compression and stretching properties of the hydrogel. This enables the silk fibroin tea polyphenol hydrogel (SFMA / TP) to better adapt to the mechanical effects of bladder contraction and relaxation when used in hemorrhagic cystitis. When the bladder contracts and relaxes, the silk fibroin tea polyphenol hydrogel (SFMA / TP) can still have good adhesion properties with the bladder tissue.

[0109] The tensile test of silk fibroin tea polyphenol hydrogel (SFMA / TP) is as follows Figure 5 As shown, from Figure 5 The test results show that silk fibroin tea polyphenol hydrogel (SFMA / TP) can be stretched several times and has good tensile properties.

[0110] The electron microscopy image of silk fibroin tea polyphenol hydrogel (SFMA / TP) is as follows Figure 6 As shown in Figure 6, compared with silk fibroin-tea polyphenols hydrogel (SFMA / TP), silk fibroin-tea polyphenols hydrogel (SFMA / TP) exhibits a more regular and dense microstructure, indicating that silk fibroin-tea polyphenols hydrogel (SFMA / TP) has good mechanical properties such as relaxation and contraction.

[0111] Adhesion performance test of silk fibroin tea polyphenol hydrogel (SFMA / TP) and bladder tissue Figure 7 As shown, from Figure 7 It can be seen that after the silk fibroin tea polyphenol hydrogel (SFMA / TP) interacts with the bladder tissue, the 180-degree glass experiment on the glass slide is as follows Figure 7 As shown in (a) and (b), it can be seen that the adhesion performance of the silk fibroin tea polyphenol hydrogel (SFMA / TP) generated after the reaction of SFMA hydrogel and tea polyphenols is also improved.

[0112] In addition, the present invention also tested the adhesion of silk fibroin tea polyphenols hydrogel (SFMA / TP) to plastic, metal, rubber, and glass slides and its underwater adhesion ability, indicating that silk fibroin tea polyphenols hydrogel (SFMA / TP) has good adhesion ability.

[0113] The present invention further tested the adhesion ability of silk fibroin tea polyphenol hydrogel (SFMA / TP) in artificial urine. Figure 7 As shown in (d), it can be seen that silk fibroin tea polyphenol hydrogel (SFMA / TP) can closely fit with the bladder wall and can adhere tightly when the bladder is deformed.

[0114] The hydrogel was adhered to the bladder tissue and freeze-dried for electron microscopy. The electron microscopic image of the adhesion between the hydrogel and the bladder tissue is shown in the figure below. Figure 7 As shown in (e): The results show that the hydrogel tissue and bladder tissue are tightly attached together, confirming that the hydrogel has good adhesion properties.

[0115] The operation process and results of hydrogel intravesical instillation Figure 8 As shown in (b) and (c):

[0116] Unlike humans, rats have a thinner urethra, making non-invasive intravesical drug delivery through the urethra and bladder difficult. To address this issue, we used a 3F (diameter = 1 mm) catheter for catheterization and intravesical drug delivery, simulating the clinical method of intravesical instillation and reducing damage to the bladder. We selected female rats with a shorter and straighter urethra as the animal model and successfully completed a series of operations, including catheterization and intravesical injection of hydrogel.

[0117] After the rats were anesthetized, a midline incision of about 1 cm was made in the lower abdomen. A urinary catheter was inserted to drain excess urine from the bladder. 0.2 ml of 10% TP was then injected, and the bladder was massaged to ensure sufficient contact between TP and the bladder wall. 0.2 ml of 10% SFMA was then injected to expand and thin the bladder. The bladder was massaged again before being exposed to ultraviolet light. Then, light curing was performed to form SFMA / TP hydrogel on the surface of the bladder wall. Finally, the urinary catheter was removed, and the abdominal cavity and skin were sutured. Figure 8 (c) The full bladder can be observed after instillation. Ultrasound images show the process of bladder instillation of hydrogel in real time.

[0118] Figure 9 Shown are the HE staining results of the silk fibroin tea polyphenol hydrogel (SFMA / TP) group and the control group rats at specific time points. Figure 9 It can be seen that in the silk fibroin tea polyphenol hydrogel (SFMA / TP) group, the silk fibroin tea polyphenol hydrogel (SFMA / TP) tissue can be seen adhering to the mucosal layer of the bladder, and a significant reduction in submucosal bleeding can be seen.

[0119] The quantitative analysis results of submucosal bleeding in the experimental and control groups of silk fibroin tea polyphenol hydrogel (SFMA / TP) in the treatment of cystitis are as follows Figure 10 As shown, Figure 10 Figure 4 shows the quantitative analysis of submucosal bleeding in the hydrogel group and the control group. The results showed that the silk fibroin tea polyphenol hydrogel (SFMA / TP) group had a significant hemostatic effect on the first and third days after treatment.

[0120] Reference Attachment Figure 11 Schematic diagram of the in vitro degradation results of silk fibroin tea polyphenol hydrogel (SFMA / TP), Figure 11 It can be seen that silk fibroin tea polyphenol hydrogel (SFMA / TP) has good degradation ability in the body and will not cause toxic side effects to the human body.

[0121] Reference Attachment Figure 12 This is a schematic diagram of the HE staining results of silk fibroin tea polyphenol hydrogel (SFMA / TP) cleaving and excreting into the bladder. Figure 12 The arrow in the figure indicates the hydrogel, which fully demonstrates the biodegradability and safety of silk fibroin tea polyphenols hydrogel (SFMA / TP) in the bladder, which is an important characteristic for ensuring urinary tract patency.

[0122] The above results show that silk fibroin tea polyphenol hydrogel (SFMA / TP) can react with urea in urine, changing the molecular interaction in the system. Silk fibroin tea polyphenol hydrogel (SFMA / TP) has good adhesion, tensile and compressive properties, and can adhere tightly to the bladder when it is deformed. Therefore, it can stop bleeding by adhering the silk fibroin tea polyphenol hydrogel (SFMA / TP) to the bladder tissue. And through the experimental group and the control group, it can be found that silk fibroin tea polyphenol hydrogel (SFMA / TP) has a significant hemostatic effect in the treatment of hemorrhagic cystitis.

[0123] Therefore, the silk fibroin tea polyphenol hydrogel (SFMA / TP) provided by the present invention can be used to treat hemorrhagic cystitis.

[0124] Those skilled in the art will appreciate that the embodiments of the present invention described above and shown in the accompanying drawings are intended to be illustrative only and are not intended to limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. Any variations or modifications may be made to the embodiments of the present invention without departing from the principles described.

Claims

1. A method for preparing a vegan protein tea polyphenol hydrogel, characterized in that: The following steps are involved: (S10) Preparation of silk fibroin; (S20) preparing silk fibroin hydrogel; and (S30) Preparation of silk fibroin-tea polyphenol hydrogel.

2. The method for preparing silk fibroin tea polyphenol hydrogel according to claim 1, wherein the step (S10) comprises the following steps: (S101) placing the sliced ​​silk cocoons into a Na2CO3 solution and boiling to remove the sericin, washing, and drying; (S102) dissolving the dry silk in a LiBr solution, adding methacrylic acid glycerol, stirring, filtering the resulting solution, and dialyzing; (S103) freeze-drying the methacrylic acid glycerol solution, and storing the freeze-dried SFMA powder for subsequent use.

3. The method for preparing silk fibroin-tea polyphenol hydrogel according to claim 2, wherein in the step (S10), 40 g of sliced ​​cocoons are placed in 1 L of 0.05 M Na2CO3 solution and boiled at 100°C for 30 minutes to remove sericin; the silk is then washed with distilled water multiple times, and the degummed silk is dried at room temperature. Subsequently, 20 g of dry silk is dissolved in 100 mL of 9.3 M LiBr solution at 60°C for 1 hour; 6 mL of methacrylate is added to the mixture, and the mixture is stirred at 300 rpm at 60°C for 3 hours. The resulting solution is filtered with gauze and dialyzed against distilled water for 4 days using a 12-14 kDa dialysis tube; finally, the silk fibroin glycidyl methacryloyl solution is freeze-dried for 48 hours, and the freeze-dried SFMA powder is stored at -80°C for subsequent use.

4. The method for preparing the silk fibroin-tea polyphenol hydrogel according to any one of claims 1 to 3, wherein in the step (S20), the photocrosslinking agent used comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 2-hydroxy-2-methylpropiophenone or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

5. The method for preparing silk fibroin-tea polyphenol hydrogel according to claim 2, wherein in the step (S20), the aqueous solution of SFMA and LAP is irradiated under ultraviolet light to prepare the SFMA hydrogel.

6. The method for preparing silk fibroin-tea polyphenol hydrogel according to claim 3, wherein in the step (S20), an aqueous solution containing 10% SFMA and 0.2% LAP is irradiated under 405 nm ultraviolet light for 5 minutes to prepare the SFMA hydrogel.

7. The method for preparing silk fibroin-tea polyphenol hydrogel according to claim 5, wherein in the step (S30), the prepared SFMA hydrogel is immersed in a 10% TP aqueous solution for 3 hours to obtain the SFMA / TP hydrogel.

8. The method for preparing the silk fibroin-tea polyphenol hydrogel according to claim 6, wherein in the step (S30), when used, the silk fibroin and the tea polyphenols are in situ gelled at the lesion site to form the silk fibroin-tea polyphenol hydrogel. 9 . The method for preparing silk fibroin-tea polyphenol hydrogel according to claim 7 , wherein the ratio of LAP is in the range of 0.1%-0.5%, and the concentration of tea polyphenol is in the range of 5-20%.

10. A vegan protein tea polyphenol hydrogel, characterized in that: The silk fibroin tea polyphenol hydrogel is obtained by mixing and reacting the silk fibroin hydrogel and tea polyphenol under predetermined conditions. 11 . The silk fibroin-tea polyphenol hydrogel according to claim 10 , wherein the silk fibroin-tea polyphenol hydrogel is prepared by the preparation method according to any one of claims 1 to 8 . 12 . The silk fibroin-tea polyphenols hydrogel according to claim 11 , wherein the silk fibroin-tea polyphenols hydrogel is suitable for conforming to the bladder wall.

13. The application of a vegan protein tea polyphenol hydrogel, characterized in that: The silk fibroin tea polyphenol hydrogel is suitable for treating hemorrhagic cystitis.

14. Use of the silk fibroin-tea polyphenol hydrogel according to claim 13, wherein the silk fibroin-tea polyphenol hydrogel is prepared by the preparation method according to any one of claims 1 to 8.

15. The use of the silk fibroin-tea polyphenols hydrogel according to claim 14, wherein the silk fibroin-tea polyphenols hydrogel is suitable for adhering to the mucosal layer of the bladder.

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