Silk fibroin tea polyphenol hydrogel, preparation method therefor, and use thereof
By preparing silk fibroin tea polyphenol hydrogels and utilizing their interaction with urea in urine and photocuring technology, the treatment challenges of hemorrhagic cystitis have been solved, achieving personalized and effective hemostasis and adhesion, and avoiding drug abuse and recurrence.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- NINGBO FIRST HOSPITAL
- Filing Date
- 2024-10-15
- Publication Date
- 2026-04-23
AI Technical Summary
Existing treatments for hemorrhagic cystitis suffer from problems such as antibiotic overuse, increased drug resistance, limited symptomatic treatment effectiveness, and frequent relapses, necessitating the development of more personalized and effective treatment methods.
A silk fibroin-tea polyphenol hydrogel was prepared by mixing and reacting silk fibroin with tea polyphenols to form a hydrogel. The hydrogel was used to achieve hemostasis by utilizing its adhesiveness and interaction with urea in urine. The hydrogel was then photocured to form a tight bond on the bladder wall surface.
Silk fibroin and tea polyphenol hydrogel have good adhesion and hemostatic effects, can adhere closely to bladder tissue, significantly reduce submucosal bleeding, and have no toxic side effects, providing a thorough treatment for hemorrhagic cystitis.
Smart Images
Figure CN2024125022_23042026_PF_FP_ABST
Abstract
Description
Silk fibroin tea polyphenol hydrogel, its preparation method and application Technical Field
[0001] This invention relates to the field of hydrogel preparation technology, and more particularly to fibroin-tea polyphenol hydrogel, its preparation method and application. Background Technology
[0002] Hemorrhagic cystitis is a relatively serious type of cystitis, commonly seen in women. Current treatments for hemorrhagic cystitis mainly include the following: 1. Antibiotic therapy: For hemorrhagic cystitis caused by bacterial infection, antibiotics are the standard treatment. Doctors will select appropriate antibiotics based on bacterial culture results, and this usually has good efficacy. 2. Symptomatic treatment: This includes hemostatic drugs and analgesics, which help alleviate symptoms and improve the patient's quality of life. 3. Bladder irrigation: For severe hemorrhagic cystitis, doctors may perform bladder irrigation to clean blood clots and inflammatory substances from the bladder, helping to relieve symptoms.
[0003] However, treating hemorrhagic cystitis using the above methods still presents some problems: 1. Antibiotic abuse: Long-term, excessive use of antibiotics can easily lead to drug resistance, causing some bacteria to lose sensitivity to conventional antibiotics, increasing the difficulty of treatment. 2. Recurrent episodes in some patients: Some patients may experience recurrent episodes, requiring long-term regular medication or further investigation of the cause to reduce symptom recurrence. 3. Limited effectiveness of symptomatic treatment: While symptomatic treatment can relieve symptoms, it cannot cure the disease; a comprehensive treatment approach is still needed to improve the overall treatment effect.
[0004] Therefore, further research is needed on the etiology and treatment mechanisms of hemorrhagic cystitis to develop more personalized and effective treatment methods in order to improve patients' treatment outcomes and quality of life.
[0005] Summary of the Invention
[0006] One advantage of this invention is that it provides a silk fibroin tea polyphenol hydrogel, its preparation method and application, wherein the silk fibroin tea polyphenol hydrogel has a good hemostatic effect and is suitable for the treatment of hemorrhagic cystitis.
[0007] Another advantage of this invention is that it provides a silk fibroin tea polyphenol hydrogel, its preparation method and application. The silk fibroin tea polyphenol hydrogel has good adhesion and can form a tight fit with bladder tissue.
[0008] Another advantage of this invention is that it provides a fibroin-tea polyphenol hydrogel, its preparation method and application. The SFMA / TP hydrogel system can interact with urea in urine, thereby changing the molecular interactions in the system and achieving a better hemostatic effect.
[0009] Another advantage of this invention is that it provides a silk fibroin tea polyphenol hydrogel, its preparation method and application, which has good tensile and compressive properties.
[0010] Another advantage of this invention is that it provides a silk fibroin tea polyphenol hydrogel, its preparation method and application. The hemorrhagic cystitis treated by 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.
[0011] According to one aspect of the present invention, a method for preparing a fibroin-tea polyphenol hydrogel is provided, comprising the following steps:
[0012] (S10) Preparation of silk fibroin;
[0013] (S20) Preparation of silk fibroin hydrogel; and
[0014] (S30) Preparation of silk fibroin tea polyphenol hydrogel.
[0015] The step (S10) includes the following steps: (S101) Boiling the sliced silkworm cocoons in Na2CO3 solution to remove sericin, washing, and drying; (S102) Dissolving the dried silk in LiBr solution, adding glyceryl methacrylate, stirring, filtering the resulting solution, and dialyzing; (S103) Freezing-drying the glyceryl methacrylate solution of silk fibroin, and storing the freeze-dried SFMA powder for subsequent use.
[0016] In step (S10), 40g of sliced silkworm cocoons are placed in 1L of 0.05M Na2CO3 solution and boiled at 100℃ for 30 minutes to remove sericin. Then, the silk is washed several times with distilled water, and the degummed silk is dried at room temperature. Subsequently, 20g of dried silk is dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. 6mL of glyceryl methacrylate is added to the mixture, and the mixture is stirred at 300rpm for 3 hours at 60℃. The resulting solution is filtered through gauze and dialyzed against distilled water for 4 days using a 12-14kDa dialysis tube. Finally, the glyceryl methacrylate solution is freeze-dried for 48 hours, and the freeze-dried SFMA powder is stored at -80℃ for subsequent use.
[0017] In step (S20), the photocrosslinking agent used includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 2-hydroxy-2-methylphenylacetone, or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.
[0018] In step (S20), an aqueous solution of SFMA and LAP is irradiated under ultraviolet light to prepare an SFMA hydrogel.
[0019] In step (S20), an aqueous solution containing 10% SFMA and 0.2% LAP is irradiated under 405nm ultraviolet light for 5 minutes to prepare an SFMA hydrogel.
[0020] In step (S30), the prepared SFMA hydrogel is soaked in a tea polyphenol aqueous solution for a predetermined time to obtain silk fibroin tea polyphenol hydrogel. In application, the silk fibroin and the tea polyphenols gel in situ at the lesion site to form the silk fibroin tea polyphenol hydrogel.
[0021] In step (S30), the prepared SFMA hydrogel is soaked in a 10% TP aqueous solution for 3 hours to obtain SFMA / TP hydrogel.
[0022] The proportion of LAP ranges from 0.1% to 0.5%, and the concentration of tea polyphenols ranges from 5% to 20%.
[0023] According to another aspect of the present invention, the present invention also provides a silk fibroin tea polyphenol hydrogel, which is obtained by mixing and reacting silk fibroin hydrogel with tea polyphenols under predetermined conditions.
[0024] The silk fibroin tea polyphenol hydrogel mentioned above was prepared using the above-described preparation method.
[0025] The silk fibroin tea polyphenol hydrogel is suitable for adhering to the bladder wall.
[0026] The silk fibroin tea polyphenol hydrogel is suitable for the treatment of hemorrhagic cystitis.
[0027] The silk fibroin tea polyphenol hydrogel described herein is prepared using any one of the preparation methods described in claims 1 to 8.
[0028] The silk fibroin tea polyphenol hydrogel is suitable for adhesion to the mucosal layer of the bladder. Attached Figure Description
[0029] Figure 1 is a schematic diagram of the molecular structure and tissue interaction of silk fibroin and silk fibroin tea polyphenol hydrogel according to an embodiment of the present invention.
[0030] Figure 2 is a schematic diagram of the molecular structure and NMR spectrum of the silk fibroin according to the above embodiments of the present invention.
[0031] Figure 3 is a schematic diagram of the rheological properties of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.
[0032] Figure 4 is a schematic diagram of the mechanical properties of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.
[0033] Figure 5 is a schematic diagram of the tensile test of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.
[0034] Figure 6 is an electron microscope schematic diagram of the microstructure of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.
[0035] Figure 7 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.
[0036] Figure 8 is a schematic diagram of the operation process of intravesical instillation of the silk fibroin tea polyphenol hydrogel according to the above embodiment of the present invention.
[0037] Figure 9 is a schematic diagram of the silk fibroin tea polyphenol hydrogel staining section according to the above embodiment of the present invention.
[0038] Figure 10 is a schematic diagram of quantitative analysis of the silk fibroin tea polyphenol hydrogel used to treat mucosal bleeding according to the above embodiments of the present invention.
[0039] Figure 11 is a schematic diagram of the in vitro degradation test results of silk fibroin tea polyphenol hydrogel (SFMA / TP).
[0040] Figure 12 is a schematic diagram of HE staining results after in vivo degradation of silk fibroin / tea polyphenol hydrogel (SFMA / TP). Detailed Implementation
[0041] The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.
[0042] Example 1
[0043] The preparation method of silk fibroin and silk fibroin tea polyphenol hydrogel according to a preferred embodiment of the present invention is as follows:
[0044] (S10) Synthesis of Silk Fibroin (SFMA)
[0045] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using 12-14kDa dialysis tubes. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use. The obtained SFMA was analyzed on a 500MHz Bruker NMR spectrometer using D₂O as a solvent. 1 1H NMR analysis. A schematic diagram of the molecular structure and its organizational function is shown in Figure 1. 1 The H NMR analysis results are shown in Figure 2. In Figure 2, (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the NMR spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.
[0046] (S20) Synthesis of silk fibroin hydrogel
[0047] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 10% SFMA and 0.2% LAP under 405 nm ultraviolet light for 5 minutes. The LAP is lithium phenyl-2,4,6-trimethylbenzoyl hypophosphite, which is a photocrosslinking agent for the hydrogel.
[0048] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0049] The prepared SFMA hydrogel was soaked in a 10% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ at the lesion site. That is, during application, TP was first injected into the bladder, followed by the injection of SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0050] Example 2
[0051] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:
[0052] (S10) Synthesis of Silk Fibroin (SFMA)
[0053] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using 12-14kDa dialysis tubes. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use. The obtained SFMA was analyzed on a 500MHz Bruker NMR spectrometer using D₂O as a solvent. 1 1H NMR analysis. A schematic diagram of the molecular structure and its organizational function is shown in Figure 1. 1 The H NMR analysis results are shown in Figure 2. In Figure 2, (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the NMR spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.
[0054] (S20) Synthesis of silk fibroin hydrogel
[0055] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 20% SFMA and 0.5% LAP under 405 nm ultraviolet light for 5 minutes. The LAP is lithium phenyl-2,4,6-trimethylbenzoyl hypophosphite, which is a photocrosslinking agent for the hydrogel.
[0056] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0057] The prepared SFMA hydrogel was soaked in a 15% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ 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) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0058] Example 3
[0059] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:
[0060] (S10) Synthesis of Silk Fibroin (SFMA)
[0061] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using 12-14kDa dialysis tubes. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use. The obtained SFMA was analyzed on a 500MHz Bruker NMR spectrometer using D₂O as a solvent. 1 1H NMR analysis. A schematic diagram of the molecular structure and its organizational function is shown in Figure 1. 1 The H NMR analysis results are shown in Figure 2. In Figure 2, (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the NMR spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.
[0062] (S20) Synthesis of silk fibroin hydrogel
[0063] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 10% SFMA and 0.35% LAP under 405 nm ultraviolet light for 4 minutes. The LAP is lithium phenyl-2,4,6-trimethylbenzoylphosphite, which is a photocrosslinking agent for the hydrogel.
[0064] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0065] The prepared SFMA hydrogel was soaked in a 20% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ 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) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0066] Example 4
[0067] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:
[0068] (S10) Synthesis of Silk Fibroin (SFMA)
[0069] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin. The silk was then washed repeatedly with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using 12-14kDa dialysis tubes. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use. The obtained SFMA was analyzed on a 500MHz Bruker NMR spectrometer using D₂O as a solvent. 1 1H NMR analysis. A schematic diagram of the molecular structure and its organizational function is shown in Figure 1. 1 The H NMR analysis results are shown in Figure 2. In Figure 2, (a) is a schematic diagram of the molecular structure of silk fibroin, and (b) is the NMR spectrum of silk fibroin, which proves that silk fibroin has been successfully synthesized.
[0070] (S20) Synthesis of silk fibroin hydrogel
[0071] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 10% SFMA and 0.1% LAP under 405 nm ultraviolet light for 6 minutes. The LAP is lithium phenyl-2,4,6-trimethylbenzoylphosphite, which is a photocrosslinking agent for the hydrogel.
[0072] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0073] The prepared SFMA hydrogel was soaked in a 5% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ 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) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0074] Example 5
[0075] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:
[0076] (S10) Synthesis of Silk Fibroin (SFMA)
[0077] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using a 12-14kDa dialysis tube. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use.
[0078] (S20) Synthesis of silk fibroin hydrogel
[0079] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 10% SFMA and 0.2% 2-hydroxy-2-methylphenylacetone under 405 nm ultraviolet light for 5 minutes. 2-hydroxy-2-methylphenylacetone served as the photocrosslinking agent for the hydrogel.
[0080] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0081] The prepared SFMA hydrogel was soaked in a 10% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ at the lesion site. That is, during application, TP was first injected into the bladder, followed by the injection of SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0082] Example 6
[0083] The preparation methods of silk fibroin and silk fibroin tea polyphenol hydrogel are as follows:
[0084] (S10) Synthesis of Silk Fibroin (SFMA)
[0085] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using a 12-14kDa dialysis tube. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use.
[0086] (S20) Synthesis of silk fibroin hydrogel
[0087] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 20% SFMA and 0.5% 2-hydroxy-2-methylphenylacetone under 405 nm ultraviolet light for 5 minutes. 2-hydroxy-2-methylphenylacetone served as the photocrosslinking agent for the hydrogel.
[0088] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0089] The prepared SFMA hydrogel was soaked in a 20% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ 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) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0090] Example 7
[0091] (S10) Synthesis of Silk Fibroin (SFMA)
[0092] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using a 12-14kDa dialysis tube. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use.
[0093] (S20) Synthesis of silk fibroin hydrogel
[0094] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 15% SFMA and 0.4% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone under 405 nm ultraviolet light for 5 minutes. 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone served as the photocrosslinking agent for the hydrogel.
[0095] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0096] The prepared SFMA hydrogel was soaked in a 10% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ at the lesion site. That is, during application, TP was first injected into the bladder, followed by the injection of SFMA hydrogel. After photocuring, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0097] Example 8
[0098] (S10) Synthesis of Silk Fibroin (SFMA)
[0099] 40g of sliced silkworm cocoons were placed in 1L of 0.05M Na₂CO₃ solution and boiled at 100℃ for 30 minutes to remove sericin, followed by repeated washing with distilled water. The degummed silk was dried at room temperature. Subsequently, 20g of dried silk was dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour. Then, 6mL of glyceryl methacrylate (GMA, 424mM) was added to the mixture, and the mixture was stirred at 300rpm for 3 hours at 60℃. The resulting solution was filtered through gauze and dialyzed against distilled water for 4 days using a 12-14kDa dialysis tube. Finally, the glyceryl methacrylate solution was freeze-dried for 48 hours. The freeze-dried SFMA powder was stored at -80℃ for later use.
[0100] (S20) Synthesis of silk fibroin hydrogel
[0101] An SFMA hydrogel was prepared by irradiating an aqueous solution containing 5% SFMA and 0.2% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone under 405 nm ultraviolet light for 5 minutes. 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone served as the photocrosslinking agent for the hydrogel.
[0102] Synthesis of (S30) Silk Fibroin Tea Polyphenol Hydrogel (SFMA / TP)
[0103] The prepared SFMA hydrogel was soaked in a 15% TP aqueous solution for 3 hours and then photocured to obtain an SFMA / TP hydrogel for in vitro testing. Notably, during application, the silk fibroin tea polyphenol hydrogel (SFMA / TP) was formed in situ 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) was formed on the bladder wall surface, adhering to the bladder mucosa.
[0104] The silk fibroin tea polyphenol hydrogel (SFMA / TP) prepared by the above method is suitable for the treatment of hemorrhagic cystitis. As shown in Figure 1, the SFMA / TP hydrogel system can interact with urea in urine, thereby changing the molecular interactions in the system.
[0105] First, an animal model was established to observe the effect of silk fibroin / tea polyphenol hydrogel (SFMA / TP) in the treatment of hemorrhagic cystitis. Establishment of the animal model of hemorrhagic cystitis:
[0106] All animal-related procedures were performed in accordance with relevant national and international regulations for animal experiments. This invention used SD rats to establish a chemically induced hemorrhagic cystitis model. Cyclophosphamide (CYP) was administered intraperitoneally at a dose of 150 mg / kg, and the model was successfully established 24 hours later. That is, after intraperitoneal injection of 150 mg / kg into rats, we observed obvious hematuria and bladder bleeding visually, as well as submucosal hemorrhage observed by HE staining.
[0107] Intravesical injection of SMFA / TP hydrogel for the treatment of hemorrhagic cystitis: After anesthetizing rats, a midline incision of approximately 1 cm was made in the lower abdomen. A 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 complete contact between the TP and the bladder wall. Then, 0.2 ml of 10% SFMA was injected to inflate 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 polyphenol hydrogel (SFMA / TP) is shown in Figure 8(a).
[0108] The rheological properties of silk fibroin tea polyphenol hydrogel (SFMA / TP) are shown in Figure 3. As can be seen from Figure 3, after the SFMA hydrogel reacts with tea polyphenols (TP), the storage modulus and loss modulus also increase, and the silk fibroin tea polyphenol hydrogel (SFMA / TP) exhibits stronger mechanical properties and adhesion.
[0109] The mechanical properties of silk fibroin tea polyphenol hydrogel (SFMA / TP) are shown in Figure 4. As can be seen from Figure 4, the silk fibroin tea polyphenol hydrogel (SFMA / TP) formed after the reaction of SFMA hydrogel with tea polyphenol (TP) has significantly improved properties in terms of compression, cyclic compression and tensile strength. This allows the silk fibroin tea polyphenol hydrogel (SFMA / TP) to better adapt to the mechanical effects of bladder contraction and relaxation when applied to hemorrhagic cystitis. During bladder contraction and relaxation, the silk fibroin tea polyphenol hydrogel (SFMA / TP) can still maintain good adhesion to bladder tissue.
[0110] Figure 5 shows the tensile test of silk fibroin tea polyphenol hydrogel (SFMA / TP). As can be seen from the test results in Figure 5, silk fibroin tea polyphenol hydrogel (SFMA / TP) can be stretched several times and has good tensile properties.
[0111] Figure 6 shows the electron micrograph of silk fibroin tea polyphenol hydrogel (SFMA / TP). As can be seen from Figure 6, compared with silk fibroin hydrogel (SFMA), silk fibroin tea polyphenol hydrogel (SFMA / TP) exhibits a more regular and dense microstructure, indicating that silk fibroin tea polyphenol hydrogel (SFMA / TP) has good mechanical properties such as relaxation and contraction.
[0112] The adhesion performance test of silk fibroin tea polyphenol hydrogel (SFMA / TP) to bladder tissue is shown in Figure 7. As can be seen from Figure 7, after the silk fibroin tea polyphenol hydrogel (SFMA / TP) reacted with bladder tissue, the 180-degree glass experiment of the slide is shown in Figure 7 (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 with tea polyphenols was also improved.
[0113] In addition, the present invention also tested the adhesion of silk fibroin tea polyphenol hydrogel (SFMA / TP) to plastics, metals, rubbers and glass slides and its underwater adhesion ability, showing that silk fibroin tea polyphenol hydrogel (SFMA / TP) has good adhesion ability.
[0114] The present invention further tested the adhesion ability of silk fibroin tea polyphenol hydrogel (SFMA / TP) in artificial urine, as shown in Figure 7(d). It can be seen that the silk fibroin tea polyphenol hydrogel (SFMA / TP) can form a tight fit with the bladder wall and can adhere tightly even when the bladder is deformed.
[0115] Electron microscopy was performed on the lyophilized tissue of the hydrogel adhered to the bladder. The electron microscopy image of the adhesion between the hydrogel and the bladder tissue is shown in Figure 7(e). The results show that the hydrogel tissue and the bladder tissue are tightly adhered together, which confirms that the hydrogel has good adhesion properties.
[0116] The procedure and results of hydrogel intravesical instillation are shown in Figures 8(b) and (c):
[0117] Unlike humans, rats have a narrower urethra, making non-invasive intravesical drug administration via the urethra and bladder more difficult. To address this issue, based on simulating clinical bladder instillation therapy and minimizing bladder damage, we used a 3F (diameter = 1 mm) catheter for catheterization and intravesical drug administration. Female rats with shorter and straighter urethras were selected as animal models, and a series of procedures, including catheterization and intravesical injection of hydrogel, were successfully completed.
[0118] After anesthetizing the rats, a midline incision of approximately 1 cm was made in the lower abdomen. A urinary catheter was inserted to drain excess urine from the bladder. Subsequently, 0.2 ml of 10% TP was injected, and the bladder was massaged to ensure adequate contact between the TP and the bladder wall. Then, 0.2 ml of 10% SFMA was injected to expand and thin the bladder. The bladder was massaged again before exposure to UV light. Then, light curing was performed to form an SFMA / TP hydrogel on the bladder wall surface. Finally, the catheter was removed, and the abdominal cavity and skin were sutured. The bladder filled after instillation can be observed in Figure 8(c). Ultrasound images showed the real-time process of hydrogel instillation into the bladder.
[0119] Figure 9 shows the HE staining results of rat sections in the silk fibroin tea polyphenol hydrogel (SFMA / TP) group and the control group at specific time points. As can be seen from Figure 9, 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 hemorrhage can be observed.
[0120] Figure 10 shows the quantitative analysis results of submucosal hemorrhage in the experimental group and control group of the treatment of cystitis using silk fibroin tea polyphenol hydrogel (SFMA / TP). The results show that the silk fibroin tea polyphenol hydrogel (SFMA / TP) group has a significant hemostatic effect on the 1st and 3rd days after treatment.
[0121] Referring to Figure 11, which shows the in vitro degradation results of silk fibroin tea polyphenol hydrogel (SFMA / TP), it can be seen from Figure 11 that silk fibroin tea polyphenol hydrogel (SFMA / TP) has good degradation ability in vivo and will not produce toxic side effects in the human body.
[0122] Referring to Figure 12, which is a schematic diagram of HE staining results of silk fibroin tea polyphenol hydrogel (SFMA / TP) lysis and excretion in the bladder, the arrows in Figure 12 indicate the hydrogel, which fully demonstrates the biodegradability and safety of silk fibroin tea polyphenol hydrogel (SFMA / TP) in the bladder, and is an important property for ensuring urinary tract patency.
[0123] The above results indicate that SFMA / TP hydrogel can interact with urea in urine, altering the molecular dynamics within the system. SFMA / TP possesses good adhesion, tensile, and compressive properties, allowing it to adhere tightly to the bladder even during deformation. Therefore, hemostasis can be achieved by adhering SFMA / TP to bladder tissue. Furthermore, the experimental and control groups demonstrate that SFMA / TP exhibits significant hemostatic effects in the treatment of hemorrhagic cystitis.
[0124] Therefore, the silk fibroin tea polyphenol hydrogel (SFMA / TP) provided by this invention can be applied to the treatment of hemorrhagic cystitis.
[0125] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are merely examples and do not limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments, and any variations or modifications may be made to the implementation of the present invention without departing from the stated principles.
Claims
1. A process for the preparation of a silk fibroin tea polyphenol hydrogel, characterized by, Includes the following steps: (S10) Preparation of silk fibroin; (S20) Preparation of silk fibroin hydrogel; and (S30) Preparation of silk fibroin tea polyphenol hydrogel.
2. The method of claim 1, wherein the step (S10) comprises the steps of: (S101) Boil the sliced silkworm cocoons in Na2CO3 solution to remove sericin, wash and dry; (S102) Dissolve the dried silk in LiBr solution, add glyceryl methacrylate, stir, filter the resulting solution and dialyze; (S103) Freeze-dry the silk fibroin glycidyl methacrylate solution and store the freeze-dried SFMA powder for later use. 3. The method for preparing silk fibroin tea polyphenol hydrogel according to claim 2, wherein in step (S10), 40g of sliced silkworm cocoons are placed in 1L of 0.05M Na2CO3 solution and boiled at 100℃ for 30 minutes to remove sericin; then washed repeatedly with distilled water, and the degummed silk is dried at room temperature. Subsequently, 20g of dried silk is dissolved in 100mL of 9.3M LiBr solution and dissolved at 60℃ for 1 hour; 6mL of glyceryl methacrylate is added to the mixture and stirred at 300rpm for 3 hours at 60℃. The resulting solution is filtered through gauze and dialyzed against distilled water for 4 days using a 12-14kDa dialysis tube; finally, the glyceryl methacrylate solution of silk fibroin is freeze-dried for 48 hours, and the freeze-dried SFMA powder is stored at -80℃ for subsequent use.
4. The method for preparing silk fibroin tea polyphenol hydrogel according to any one of claims 1 to 3, wherein the photocrosslinking agent used in step (S20) includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 2-hydroxy-2-methylphenylacetone or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.
5. The method for preparing silk fibroin tea polyphenol hydrogel according to claim 2, wherein in step (S20), an aqueous solution of SFMA and LAP is irradiated under ultraviolet light to prepare SFMA hydrogel.
6. The method for preparing silk fibroin tea polyphenol hydrogel according to claim 3, wherein in step (S20), an aqueous solution containing 10% SFMA and 0.2% LAP is irradiated under 405nm ultraviolet light for 5 minutes to prepare SFMA hydrogel.
7. The method for preparing silk fibroin tea polyphenol hydrogel according to claim 5, wherein in step (S30), the prepared SFMA hydrogel is soaked in a 10% TP aqueous solution for 3 hours to obtain SFMA / TP hydrogel.
8. The method for preparing silk fibroin and tea polyphenol hydrogel according to claim 6, wherein in step (S30), when applied, the silk fibroin and the tea polyphenols undergo in-situ gelation at the lesion site to form the silk fibroin and tea polyphenol hydrogel.
9. The method for preparing silk fibroin tea polyphenol hydrogel according to claim 7, wherein the proportion of LAP ranges from 0.1% to 0.5%, and the concentration of tea polyphenols ranges from 5% to 20%.
10. A silk fibroin tea polyphenol hydrogel, characterized by, The silk fibroin-tea polyphenol hydrogel is obtained by mixing and reacting silk fibroin hydrogel with tea polyphenols under predetermined conditions.
11. The silk fibroin tea polyphenol hydrogel according to claim 10, wherein the silk fibroin tea polyphenol hydrogel is prepared by any one of the preparation methods according to claims 1 to 8.
12. The silk fibroin tea polyphenol hydrogel according to claim 11, wherein the silk fibroin tea polyphenol hydrogel is adapted to adhere to the bladder wall.
13. Use of a silk fibroin tea polyphenol hydrogel, characterized in that, The silk fibroin tea polyphenol hydrogel is suitable for the treatment of hemorrhagic cystitis.
14. The application of the silk fibroin tea polyphenol hydrogel according to claim 13, wherein the silk fibroin tea polyphenol hydrogel is prepared by any one of the preparation methods according to claims 1 to 8.
15. The application of the silk fibroin tea polyphenol hydrogel according to claim 14, wherein the silk fibroin tea polyphenol hydrogel is suitable for adhesion to the mucosal layer of the bladder.