Silk-based oral hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drug delivery, in particular to silk-based oral hydrogel as well as a preparation method and application thereof. The invention discloses and provides a preparation method of a hydrogel material which is based on fibroin, can be subjected to rapid gelation reaction and can be swallowed, subjected to gastric retention and enteric-coated. The material can be used for entrapment and multi-drug joint co-loading of water-phase soluble drugs or insoluble drugs. The hydrogel has the characteristics of excellent mechanical properties and shear thinning, is easy to swallow, and can effectively penetrate through the esophagus to enter the gastrointestinal tract. The hydrogel can be retained in the stomach for more than 24 hours, and responds to pancreatin in the enteric cavity to be degraded and release the entrapped medicine. In conclusion, the fibroin-based hydrogel capable of being swallowed, retained in the stomach and enteric, which can be subjected to rapid gelation reaction, has relatively high application value in the fields of oral drug carriers, oral health-care products, wound gel dressings, antibacterial materials and the like.

Description

Technical Field

[0001] This invention relates to the field of drug delivery technology, and in particular to a silk-based oral hydrogel, its preparation method, and its application. Background Technology

[0002] Currently, oral drug delivery, as the most commonly used and best-compliant non-invasive route of administration in clinical practice, still faces many challenges. First, many drug molecules with significant therapeutic potential have poor water solubility, resulting in low solubility and slow dissolution rates in gastrointestinal fluids. This leads to extremely low bioavailability after oral administration, limiting efficacy and requiring increased dosages to compensate for insufficient absorption, potentially causing toxic side effects. Second, the transport and retention time of traditional oral solid dosage forms (such as tablets and capsules) in the gastrointestinal tract is difficult to control, often being excreted within a few hours. For drugs requiring continuous absorption in the stomach or upper small intestine, this short retention time severely weakens their therapeutic effect. Existing oral drug delivery systems generally suffer from short gastrointestinal retention times, uneven release profiles, and susceptibility to destruction by gastric acid and digestive enzymes, leading to low drug utilization and unstable efficacy. Traditional gastric retention or enteric-coated formulations often rely on complex chemical cross-linking or special processing techniques, resulting in cumbersome preparation methods, complex formulations, poor swallowability, and poor biocompatibility. Especially for drugs that need to remain in the stomach and be specifically released in the intestines, there is currently a lack of a high-performance oral hydrogel carrier material that simultaneously possesses good swallowability, significant gastric retention, and intestinal environment-responsive degradation characteristics.

[0003] However, traditional hydrogels still have the following bottlenecks in oral applications: (1) Most hydrogels are highly hydrophilic networks with limited capacity to encapsulate hydrophobic (poorly soluble) drugs, often resulting in low drug loading and obvious drug burst release; (2) Ordinary hydrogels have insufficient mechanical properties and are prone to disintegration under strong peristalsis and shearing forces in the gastrointestinal tract, making it difficult to maintain structural integrity and achieve long-term gastrointestinal retention; (3) Existing gastric retention hydrogels are mostly prepared by chemical cross-linking, which is complicated and carries the risk of cross-linking agent residue. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a novel silk-based hydrogel material and its preparation and application method, which addresses the multiple limitations of existing oral drug delivery systems, so as to achieve efficient drug encapsulation, gastric retention and enteric release.

[0005] To address the aforementioned technical problems, this invention provides a method for preparing a silk-based oral hydrogel, comprising the following steps: S11: Mix the fibroin aqueous solution and the sericin aqueous solution to obtain a silk protein aqueous solution; the mass ratio of fibroin to sericin in the silk protein aqueous solution is 1-9:1-9; S12: Add alcohol to the aqueous solution of silk protein and perform a gelation reaction at 4-50℃ to obtain the silk-based oral hydrogel; keep the system at a preset temperature and allow it to stand for a period of time, and the system will undergo a sol-gel transition to finally form the silk-based hydrogel.

[0006] Specifically, the gelation reaction is carried out at a temperature of 37°C.

[0007] Preferably, the mass ratio of silk fibroin to sericin is 1-9:1.

[0008] Specifically, the mass ratio of silk fibroin to sericin is 3:1.

[0009] Preferably, the alcohol is selected from one or more of ethanol, isopropanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol and glycerol.

[0010] Specifically, the alcohol is selected from ethanol. The introduction of the mixed solvent system (water / alcohol) is not only as a solvent, but also as a structure inducer. Alcohol solvents (such as ethanol) change the solvation state of silk fibroin molecules, significantly improve the intermolecular interactions of silk fibroin molecules, and induce their molecular chains to accelerate the transformation from random coil structure to stable β-sheet domains.

[0011] Preferably, the volume ratio of the silk protein aqueous solution to the alcohol is 1-19:1-19.

[0012] Specifically, the volume ratio of the silk protein aqueous solution to the alcohol is 1:1.

[0013] Preferably, the concentration of the solute in the silk fibroin aqueous solution is 0.2-20 wt%.

[0014] Furthermore, the concentration of the solute in the silk protein aqueous solution is 4-10 wt%.

[0015] Specifically, the concentration of the solute in the silk protein aqueous solution is 4 wt%.

[0016] By controlling the concentration of silk fibroin, the SF / SS ratio, and the amount of alcohol solvent, this system can achieve rapid gelation within a temperature range from room temperature to near body temperature, with gelation reaction times as short as a few seconds. This rapid and controllable physical cross-linking process makes the formation of this gel network faster than traditional self-assembly and more suitable for industrial production.

[0017] Preferably, the method for preparing the sericin includes the following steps: S21: heating the raw silk yarn in an alkaline aqueous solution to separate and obtain crude sericin product; S22: dialyzing and concentrating the crude sericin product to obtain the sericin.

[0018] Preferably, the method for preparing the silk fibroin includes the following steps: S31: heating the raw silk yarn in an alkaline aqueous solution to separate and obtain crude silk fibroin fiber product; S32: adding the crude silk fibroin fiber product to an aqueous solution containing lithium bromide, dialyzing, and concentrating to obtain the silk fibroin.

[0019] Specifically, the preparation method of the sericin and silk fibroin includes the following steps: (a) Shredded raw silk yarn is added to an aqueous sodium carbonate solution and boiled to separate fibroin and sericin, resulting in crude sericin protein dissolved in the aqueous sodium carbonate solution and crude fibroin fiber with sericin removed; (b) The crude sericin protein dissolved in the aqueous sodium carbonate solution is dialyzed to remove inorganic salts and other impurities, resulting in a purified sericin protein solution, which is then concentrated at 4°C with a concentrated polyethylene glycol solution (20000-40000 Da, 30-50 wt%) to obtain a concentrated sericin protein solution of a specific mass concentration; (c) Dried degummed fibroin fiber is added to an aqueous lithium bromide solution to dissolve the fibroin fiber, and impurities in the system are removed by dialysis to obtain a purified fibroin protein aqueous solution, which is then concentrated at 4°C with a concentrated polyethylene glycol solution (20000-40000 Da, 30-50 wt%) to obtain a concentrated fibroin protein solution of a specific mass concentration.

[0020] Preferably, the silk-based oral hydrogel contains one or both of a water-soluble drug and a fat-soluble drug; the water-soluble drug is added by dissolving it in an aqueous solution of silk protein and then performing a gelation reaction, and the fat-soluble drug is added by dissolving it in an alcohol and then performing a gelation reaction.

[0021] Furthermore, the water-soluble drug is one or more of the following: water-soluble small molecule drugs (metformin, streptomycin, vitamin C, folic acid, tetracycline hydrochloride, nicotinamide adenine dinucleotide), water-soluble polypeptide drugs (ε-polylysine, GLP-1 analog liraglutide, exenatide, thymosin α1, salmon calcitonin), water-soluble protein drugs (insulin, interferon-α, erythropoietin, tissue plasminogen activator t-PA, catalase, uricase), and water-soluble nucleic acid drugs (siRNA drugs Patisiran and Inclisiran).

[0022] Furthermore, the final concentration of the water-soluble drug in the solution is 0.0001-10 mg / mL.

[0023] Specifically, based on the type of drug in the hydrogel, hydrogels are classified into hydrogels for loading water-soluble drugs, hydrogels for loading poorly soluble (lipid-soluble) drugs in the aqueous phase, and hydrogels for co-loading water-soluble drugs and poorly soluble (lipid-soluble) drugs in the aqueous phase.

[0024] The method for loading water-soluble drugs includes the following steps: (a) a specific mass of water-soluble drug molecules is completely dissolved in a specific mass concentration and a specific SF / SS ratio of silk fibroin aqueous solution prepared above to prepare a homogeneous drug-containing silk fibroin solution, defined as solution C; (b) an alcohol solvent is used as solution B; (c) solution B and solution C are rapidly mixed according to a specific mass ratio, kept at a constant temperature, and subjected to a gelation reaction to obtain a uniformly loaded silk-based hydrogel containing water-soluble drug molecules.

[0025] The method for loading water-phase poorly soluble drugs comprises the following steps: (a) completely dissolving a specific mass of water-phase poorly soluble drug molecules in a specific mass of alcohol solvent to prepare a homogeneous drug-containing alcohol solution, defined as solution D; (b) using a specific mass concentration of silk fibroin aqueous solution with a specific SF / SS ratio as solution A; (c) rapidly mixing solution A and solution D according to a specific mass ratio, allowing them to stand at a constant temperature, and after undergoing a gelation reaction, a silk-based hydrogel uniformly loaded with water-phase poorly soluble drug molecules is obtained.

[0026] The method for co-loading water-soluble drugs and aqueous poorly soluble drugs comprises the following steps: (a) Prepare an aqueous solution C containing water-soluble drug molecules and an alcoholic solution D containing aqueous poorly soluble drug molecules, respectively, according to the above method; (b) Mix solution C and solution D rapidly in a specific volume ratio, let stand at a constant temperature, and after undergoing a gelation reaction, a silk-based hydrogel loaded with two different soluble drug molecules is obtained.

[0027] Furthermore, the lipid-soluble drug is a drug that has good solubility in alcohol solvents, such as one or more of curcumin, rifampin, all-trans retinoic acid, dexamethasone, α-tocopherol, roxithromycin, metronidazole, tannic acid, epigallocatechin gallate, rutin, triptolide, artemisinin, paclitaxel, paeoniflorin, kaempferol, luteolin, and quercetin.

[0028] Furthermore, the final concentration of the poorly soluble drug in alcohol is 0.0001-10 mg / mL.

[0029] Preferably, in step S12, the gelation reaction time is 5-180,000 s. The silk-based hydrogel prepared by this invention possesses a series of excellent physicochemical and biological properties, and the gelation reaction time can be achieved from 5 s to 1.8 × 10⁻⁶ s. 5 Precise control within the range of s.

[0030] This invention also provides a silk-based oral hydrogel prepared by the above-described method. The hydrogel uses fibroin and sericin together as a gel crosslinking network, and the gel matrix contains a mixed solvent composed of water and alcohol.

[0031] This invention also provides a gastric retention delivery formulation, comprising the aforementioned silk-based oral hydrogel. The gel material possesses excellent mechanical properties and shear-thinning characteristics, making it easy to swallow and effectively penetrating the esophagus to enter the gastrointestinal tract. The gastric retention delivery formulation maintains structural stability in simulated gastric fluid (pH 1.2, pepsin concentration 3.2 mg / mL), without significant swelling or degradation. Upon entering a simulated intestinal fluid environment (pH 6.8-7.4, pancreatic enzyme concentration 10 mg / mL), its silk protein network specifically degrades in response to pancreatic enzymes, thereby achieving targeted release of the encapsulated drug within the intestine.

[0032] Compared with the prior art, the above-described technical solution of the present invention has the following advantages: 1. The preparation method of this invention is simple, environmentally friendly, and suitable for industrialization: the entire preparation process is based entirely on physical cross-linking, without relying on any toxic chemical cross-linking agents or harsh high-temperature treatments. High-performance hydrogels can be obtained simply by adjusting the proportions of each component. The alcohol solvent used is one of the most widely used and longest-used excipients in global pharmaceutical formulations, and its safety has been extensively verified by major global regulatory agencies (such as the FDA), covering various dosage forms including oral, topical, and injectable formulations. Therefore, this solution is a mature, safe, and easily scalable technical solution recognized in the industry.

[0033] 2. The gelation reaction process of this invention is rapid and highly controllable: by inducing structural transformation of two silk proteins through alcohol solvents, the gelation reaction temperature and time can be precisely controlled. The gelation rate is fast, significantly superior to traditional physical gels of silk fibroin or sericin, greatly improving production efficiency.

[0034] 3. Excellent oral swallowability and patient compliance: Thanks to the excellent shear-thinning rheological properties imparted by the introduction of sericin, this hydrogel solves the problem of traditional gels being difficult to swallow, making it especially suitable for children, the elderly, or patients with swallowing difficulties.

[0035] 4. A perfect "gastric retention-enteric coagulation" intelligent response mechanism: This invention cleverly solves the seemingly contradictory needs of "gastric stability" and "intestinal release." In the gastric environment, the gel forms a stable cross-linked network matrix to achieve long-term retention (greater than 24 hours); after entering the intestine, it can be specifically degraded by pancreatic enzymes. This intelligent spatiotemporal controllability ensures both sufficient drug retention in the stomach and precise drug release in the intestine.

[0036] 5. Broad-spectrum drug loading capacity: Traditional hydrogels, due to the high hydrophilicity of their polymer networks, inherently face challenges in loading hydrophobic (poorly soluble in water) drugs. However, the "water-alcohol" binary solvent system employed in this invention successfully overcomes this limitation. Water-soluble drugs are soluble in the aqueous phase, while poorly soluble drugs are soluble in the alcohol phase; the mixture of the two can be uniformly embedded in the gel network. This superior broad-spectrum drug loading capacity enables the loading of both water-soluble and poorly soluble drugs, as well as multi-drug co-loading, providing a novel solution for combination therapy and personalized drug delivery.

[0037] In summary, the rapid gelation reaction silk-based hydrogel material provided by this invention has significant theoretical implications and broad application prospects in the field of oral drug delivery systems. This system not only successfully overcomes the limitations of traditional oral gels, such as short retention time in the digestive tract, difficulty in swallowing, and off-target drug release, but more importantly, it opens up a completely new material platform for significantly improving the bioavailability of poorly soluble drugs through a simple, green, and efficient material design. The design concept of this material provides a solid and feasible technical foundation for the development of novel intelligent oral formulations. Attached Figure Description

[0038] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0039] Figure 1 This invention relates to the effect of different ethanol ratios on the gelation time of silk fibroin hydrogels (37℃, n=3); wherein, (A) the SS / SF mass ratio of silk fibroin is 1 / 9, (B) the SS / SF mass ratio of silk fibroin is 1 / 4, (C) the SS / SF mass ratio of silk fibroin is 2 / 3, (D) the SS / SF mass ratio of silk fibroin is 1 / 1, (E) the SS / SF mass ratio of silk fibroin is 3 / 2, and (F) the SS / SF mass ratio of silk fibroin is 4 / 1. Figure 2This invention relates to the effect of different SS / SF mass ratios (w / w) on the gelation time of silk fibroin hydrogels (37℃, n=3); where (A) the final ethanol concentration is 20wt%, (B) the final ethanol concentration is 33wt%, (C) the final ethanol concentration is 50wt%, and (D) the final ethanol concentration is 67wt%. Figure 3 The gelation time of silk fibroin hydrogels induced by different alcohols in this invention is affected by the alcohol content (37℃, n=3); wherein, (A) the gelation reaction time of silk fibroin aqueous solution mixed with different proportions of methanol at 37℃; (B) the gelation reaction time of silk fibroin aqueous solution mixed with different proportions of ethanol at 37℃; (C) the gelation reaction time of silk fibroin aqueous solution mixed with different proportions of isopropanol at 37℃; (D) the gelation reaction time of silk fibroin aqueous solution mixed with different proportions of n-butanol at 37℃; Figure 4 This invention relates to the effect of alcohol type on the gelation time of silk fibroin hydrogels (37°C, n=3); wherein, (A) the final concentration of alcohol solvent in the gel is 20 wt%, (B) the final concentration of alcohol solvent in the gel is 33 wt%, and (C) the final concentration of alcohol solvent in the gel is 50 wt%. Figure 5 This invention relates to the effect of temperature on the gelation time of silk protein hydrogel (n=3). Figure 6 This invention relates to the effect of drug encapsulation on the gelation time of silk protein hydrogel (37℃, n=3). Figure 7 The following are rheological tests (time-scan mode, temperature 37°C, frequency 1 Hz, strain 1%) of the silk protein hydrogels of this invention: (A) Hydrogel prepared in Example 32; (B) Hydrogel prepared in Example 33; (C) Hydrogel prepared in Example 36; (D) Hydrogel prepared in Example 37; (E) Hydrogel prepared in Example 44; (F) Hydrogel prepared in Example 45; (G) Hydrogel prepared in Example 52; (H) Hydrogel prepared in Example 53. Figure 8 Rheological tests (time scan mode, temperature 37°C, frequency 1 Hz, strain 1%) were performed on pure silk fibroin hydrogels of Comparative Examples 2 (A) and 3 (B) and Comparative Examples 6 (C) and 7 (D). Figure 9These are photographs of silk protein hydrogels containing different drugs according to the present invention; wherein, (A) is a photograph of a silk protein hydrogel containing curcumin (CUR), (B) is a photograph of a silk protein hydrogel containing rifampin (RIF), (C) is a photograph of a silk protein hydrogel containing all-trans retinoic acid (ATRA), (D) is a photograph of a silk protein hydrogel containing bovine serum albumin (BSA), and (E) is a photograph of a silk protein hydrogel containing ε-polylysine (EPL). Figure 10 This is the viscosity-shear rate curve (37°C) of the silk protein hydrogel of the present invention. Figure 11 These are photos showing the injectability of the silk protein hydrogel before and after drug loading according to the present invention. Figure 12 This invention relates to an MTT (thiazolyl blue colorimetric assay) cytotoxicity test of silk fibroin hydrogel extract on Caco-2 cells. The experiment was conducted in a 96-well plate, with 6 independent parallel sample wells (n=6) for each treatment condition to ensure the accuracy and repeatability of the results. The cell viability after 48 hours of co-incubation was over 90%, reflecting the excellent biocompatibility of the hydrogel. Figure 13 This is a degradation behavior diagram of the silk fibroin hydrogel, pure sericin hydrogel, and pure silk fibroin hydrogel of the present invention in artificial gastric fluid (AGF) and artificial intestinal fluid (AIF) (37℃, n=4); wherein, (A) degradation curve of silk fibroin hydrogel of Example 37 in artificial gastric fluid; (B) degradation curve of silk fibroin hydrogel of Example 37 in artificial intestinal fluid; (C) degradation curve of sericin hydrogel of Comparative Example 3 in artificial gastric fluid; (D) degradation curve of sericin hydrogel of Comparative Example 3 in artificial intestinal fluid; (E) degradation curve of silk fibroin hydrogel of Comparative Example 7 in artificial gastric fluid; (F) degradation curve of silk fibroin hydrogel of Comparative Example 7 in artificial intestinal fluid; Figure 14 This is a graph showing the sustained-release behavior of the silk protein hydrogel loaded with CUR and FITC-BSA in simulated gastric fluid (AGF) and simulated intestinal fluid (AIF) of the present invention (37°C, n=4); wherein, (A) release curve of silk protein hydrogel loaded with CUR in Example 69 in simulated gastric fluid; (B) release curve of silk protein hydrogel loaded with CUR in Example 69 in simulated intestinal fluid; (C) release curve of silk protein hydrogel loaded with FITC-BSA in Example 99 in simulated gastric fluid; (D) release curve of silk protein hydrogel loaded with FITC-BSA in Example 99 in simulated intestinal fluid; Figure 15This is a graph showing the sustained-release behavior of pure sericin and pure silk fibroin hydrogels coated with FITC-BSA in simulated gastric fluid (AGF) and simulated intestinal fluid (AIF) of the present invention (37°C, n=4); including the release curves of the sericin hydrogel coated with FITC-BSA (A) in Comparative Example 9 and the sericin hydrogel coated with FITC-EPL (B) in Comparative Example 10 in simulated gastric fluid; also including the release curves of the sericin hydrogel coated with FITC-BSA (C) in Comparative Example 11 and the sericin hydrogel coated with FITC-EPL (D) in simulated intestinal fluid; Figure 16 These are anatomical and fluorescence imaging images of the Cy5 fluorescently labeled silk protein hydrogel of the present invention in the stomach of mice after oral administration; Figure 17 This is a dynamic analysis of the fluorescence signal of Cy5-labeled silk fibroin hydrogel in the mouse gastrointestinal tract; wherein, A is a statistical graph of the total fluorescence intensity of the whole gastrointestinal tract in vitro imaging at different time points after oral administration, and B is a quantitative analysis graph of the fluorescence signal intensity of the stomach in vitro imaging at different time points after oral administration (n=3). Figure 18 This is a photograph of a silk protein hydrogel prepared in Example 105 of the present invention, which co-loads water-soluble drugs (uricase, URI) and lipid-soluble drugs (luteolin, LUT). Figure 19 The diagram shows the preparation method (A, B) of the hydrogel of this invention and the application process (C, D, E) of the corresponding formulation. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] Examples 1 to 30 The silk fibroin aqueous solution (solution A) used in this invention is prepared by blending equal masses of a silk fibroin (SF) solution and a sericin (SS) solution of a specific concentration. The total protein concentration in the solution is 0.2-20 wt%, and the mass ratio of SF to SS in solution A is 9:1 to 1:9. Table 1 shows the formulations of the component proportions of the mixed solution obtained by blending equal masses of silk fibroin (SF) solution and sericin (SS) solution of a specific concentration, namely Examples 1 to 20.

[0042] Table 1. Formulation of mixed solutions of silk fibroin (SF) solution and sericin (SS) solution in equal mass.

[0043] The SS solution and SF solution were prepared according to the following methods: (a) The shredded raw silk yarn is added to a sodium carbonate aqueous solution with a concentration of 0.02 mol / L, wherein the mass-volume ratio of raw silk yarn to sodium carbonate aqueous solution is 1 g: 10 mL. The fibroin and sericin are separated by heating and boiling to obtain crude sericin protein dissolved in sodium carbonate aqueous solution and crude fibroin fiber product after sericin removal. This step is called "degumming treatment" and the degumming time is 40 minutes. (b) The crude sericin product dissolved in sodium carbonate aqueous solution was dialyzed to remove inorganic salts and other impurities (dialysis bag molecular weight cutoff MWCO = 7000 Da, dialysis at room temperature for 72 hours) to obtain a purified sericin solution. The purified sericin solution was then concentrated at 4°C using a concentrated polyethylene glycol solution (polyethylene glycol concentration 40 wt%, molecular weight 40000 Da, linear polyethylene glycol). The mass ratio of the dilute sericin solution to the concentrated polyethylene glycol solution was 1:10 (w / w) to obtain a concentrated sericin solution of a specific mass concentration. Table 2 shows the mass concentration of SS solutions obtained at different concentration times, namely Examples 21-25. By adding deionized water to the concentrated SS mother liquor, SS solutions with a concentration range of 0.2-20 wt% can be prepared. (c) The dried, degummed silk fibroin fibers were added to a 9.3 mol / L lithium bromide aqueous solution and heated and stirred in a 60°C water bath for 4 hours to dissolve the silk fibroin fibers. The mass-to-volume ratio of silk fibroin fibers to lithium bromide aqueous solution was 1 g:10 mL. This step was called "fiber dissolution treatment". Finally, after the silk fibroin fibers were completely dissolved, impurities in the system were removed by dialysis (dialysis bag molecular weight cutoff MWCO = 7000 Da, dialysis at room temperature for 72 hours) to obtain a purified silk fibroin protein aqueous solution.

[0044] Table 2. Mass concentration of sericin (SS) aqueous solutions obtained at different concentration times.

[0045] Note: The concentration of sericin is obtained by heating a certain mass of the solution in an oven at 105℃ to evaporate the water until the mass remains constant, and then calculating the ratio of the mass of the remaining solute to the mass of the initial solution.

[0046] A concentrated SF solution of silk fibroin was obtained by concentrating a concentrated polyethylene glycol solution (40 wt%, molecular weight 40,000 Da, linear polyethylene glycol) at 4°C, with a mass ratio of 1:10 (w / w) between the dilute silk fibroin solution and the concentrated polyethylene glycol solution. Table 3 shows the mass concentrations of SF solutions obtained at different concentration times, i.e., Examples 26 to 30. By adding deionized water to the concentrated SF mother liquor, SF solutions of any concentration range of 0.2-20 wt% can be prepared. Extending the "degumming treatment" and "silk dissolving treatment" times can also yield corresponding SS and SF aqueous solutions, but it will reduce the molecular weight of the obtained sericin and silk fibroin, resulting in a decrease in the mechanical strength of the final silk fibroin aqueous solution after gelation. Therefore, selecting appropriate degumming and dissolving times is very important for preparing silk fibroin aqueous solutions. In the subsequent examples, the SS and SF aqueous solutions used had a degumming time of 40 minutes and a dissolving time of 4 hours, which are all particularly optimized results.

[0047] Table 3. Mass concentration of silk fibroin (SF) aqueous solutions obtained at different concentration times.

[0048] Examples 31 to 54 The silk fibroin aqueous solution (solution A) was prepared by blending equal masses of silk fibroin (SF) solution and sericin (SS) solution of a specific concentration. Then, using a silk fibroin aqueous solution of a specific concentration and a specific SS / SF mass ratio as solution A, and ethanol as solution B, solutions A and B were rapidly mixed according to a specific mass ratio (detailed ingredient addition is shown in Table 4). The mixture was then kept at a constant temperature of 37°C, allowing the system to undergo a gelation reaction, ultimately forming the silk-based hydrogel.

[0049] Examples 55 to 63 The silk fibroin aqueous solution (solution A) was prepared by blending equal masses of a 2 wt% sericin (SS) solution and an 8 wt% fibroin (SF) solution, resulting in a total protein content of 5 wt% and an SS to SF mass ratio of 1:4 (w / w). Subsequently, using this silk fibroin aqueous solution as solution A, and methanol, isopropanol, and n-butanol as solutions B, solutions A and B were rapidly mixed according to specific mass ratios (detailed feeding is shown in Table 5). The mixture was then kept at a constant temperature of 37°C, allowing the system to undergo a gelation reaction, ultimately forming the silk-based hydrogel.

[0050] Table 4. Feeding table for preparing silk fibroin hydrogels at 37 °C using ethanol as an inducing agent.

[0051] Examples 64 to 67 The scheme is the same as in Example 55, except that ethanol is used as solution B. The detailed feeding process is shown in Table 6. The system is then kept at a constant temperature of 25°C and undergoes a gelation reaction to finally form the silk-based hydrogel.

[0052] Table 5. Feeding table for preparing silk fibroin hydrogels at 37℃ using different alcohol solvents as inducing agents.

[0053] Table 6. Feeding table for preparing silk fibroin hydrogels at 25 °C using ethanol as an inducing agent.

[0054] Examples 68 to 85 This invention provides a method for using the silk-based hydrogel material as a drug delivery carrier to encapsulate poorly soluble drugs in the aqueous phase. The method for loading poorly soluble drugs in the aqueous phase includes the following steps (detailed feeding is shown in Table 7): (a) a specific mass of poorly soluble drug molecules in the aqueous phase (drug model molecules: curcumin CUR, rifampin RIF, all-trans retinoic acid ATRA) is completely dissolved in a specific mass of ethanol solvent to prepare a homogeneous drug-containing alcohol solution, defined as solution D; (b) a silk fibroin aqueous solution (solution A) is prepared by the following method: it is obtained by mixing equal masses of a 2 wt% sericin (SS) solution and an 8 wt% silk fibroin (SF) solution, the total protein content in the resulting solution A is 5 wt%, and the mass ratio of SS to SF is 1:4 (w / w); (c) 5 g of solution A and 5 g of solution D are taken respectively, rapidly mixed at a mass ratio of 1:1, and kept at a constant temperature of 37°C. After undergoing a gelation reaction, a silk-based hydrogel uniformly loaded with poorly soluble drug molecules in the aqueous phase is obtained.

[0055] Examples 86 to 97 The method is the same as in Example 68, except that the water-soluble drug molecules (drug model molecules: bovine serum albumin BSA, ε-polylysine EPL) are completely dissolved in the silk fibroin aqueous solution (solution A) to prepare a homogeneous drug-containing silk fibroin aqueous solution, defined as solution C; 5 g of ethanol as solution B and 5 g of solution C are rapidly mixed at a mass ratio of 1:1, and kept at a constant temperature of 37°C. After undergoing a gelation reaction, a silk-based hydrogel uniformly loaded with water-soluble drug molecules is obtained (detailed feeding is shown in Table 8).

[0056] Table 7. Feeding table for silk fibroin hydrogel loaded with poorly soluble aqueous drugs.

[0057] Table 8. Feeding table for water-soluble drugs loaded on silk fibroin hydrogel

[0058] Examples 98 to 103 This invention employs the following method to obtain FITC-labeled bovine serum albumin (BSA), named FITC-BSA: FITC fluorescent dye is added to an aqueous BSA solution to a concentration of 0.1% (by mass) of BSA. This mixture is then stirred at room temperature for 4 hours in the dark. After the reaction is complete, the resulting FITC-labeled BSA solution is purified by dialysis. After freeze-drying, FITC-labeled BSA powder is obtained. This powder is then directly dissolved in an aqueous silk fibroin solution to obtain an aqueous silk fibroin solution containing FITC-BSA at a specific drug concentration.

[0059] Similarly, FITC-labeled ε-polylysine (EPL), named FITC-EPL, was obtained using the following method: FITC fluorescent dye was added to an aqueous EPL solution to a concentration of 0.1% of the EPL mass. This mixture was protected from light and stirred at room temperature for 4 hours. After the reaction, the resulting FITC-labeled EPL solution was purified by dialysis. After freeze-drying, FITC-labeled EPL powder was obtained. This powder was then directly dissolved in an aqueous silk fibroin solution to obtain an aqueous silk fibroin solution containing FITC-EPL at a specific drug concentration.

[0060] This invention provides a method for using the aforementioned silk-based hydrogel material as a drug delivery carrier to encapsulate FITC-labeled water-soluble drugs. The method for loading water-soluble drugs includes the following steps (detailed material loading is shown in Table 9): (a) A silk fibroin aqueous solution (solution A) is prepared by mixing equal masses of a 2 wt% sericin (SS) solution and an 8 wt% fibroin (SF) solution, resulting in a total protein content of 5 wt% in solution A and a SS to SF mass ratio of 1:4 (w / w); (b) A specific mass of FITC-BSA and FITC-EPL lyophilized powder is completely dissolved in the silk fibroin aqueous solution (solution A) to prepare a homogeneous drug-containing silk fibroin aqueous solution, defined as solution C; (c) Ethanol is used as solution B; (d) 5 g of solution B and 5 g of solution C are rapidly mixed at a mass ratio of 1:1, and the mixture is kept at a constant temperature of 37 °C to undergo a gelation reaction, thereby obtaining a silk-based hydrogel uniformly loaded with water-soluble drug molecules.

[0061] Example 104 Fluorescent labeling of silk fibroin aqueous solution was performed using sulfonyl Cy5 fluorescent dye (Sulfo-Cy5). The method was as follows: Sulfonyl Cy5-NHS dye was added to the silk fibroin aqueous solution to a concentration of 0.1% of the silk fibroin mass. The mixture was stirred in the dark for 4 hours. After the reaction, the resulting Cy5-labeled silk fibroin solution was purified by dialysis to remove unlabeled free fluorescent molecules. Subsequently, after concentration, a Cy5-labeled silk fibroin solution with a mass concentration of 8 wt% was obtained. A 2 wt% sericin (SS) aqueous solution was mixed with an 8 wt% Cy5-labeled silk fibroin (Cy5-SF) solution to obtain a fluorescently labeled silk fibroin aqueous solution with a total protein concentration of 5 wt%, wherein the mass ratio of SS / SF was 1:4. Subsequently, 5 g of the Cy5 fluorescently labeled silk protein aqueous solution was rapidly mixed with an equal mass of 5 g of ethanol solvent and transferred to a syringe to obtain a fully gelled fluorescently labeled silk protein hydrogel. The final total protein concentration in the hydrogel was 2.5 wt%, and the ethanol content was 50 wt%. BALB / c mice were orally administered the gel via gavage at a dose of 1 g of hydrogel per kilogram of mouse body weight. After 30 minutes, the digestive tract of the mice was dissected to observe the formation and retention of the gel in the stomach.

[0062] Example 105 This invention also provides a method for co-loading water-soluble and lipid-soluble drugs in a silk fibroin hydrogel to achieve the co-delivery of two drugs with diametrically opposed solubilities. The specific method is the same as in Example 68, except that the water-soluble drug molecule (drug model molecule: uricase, abbreviated as URI) is completely dissolved in the silk fibroin aqueous solution (solution A) to prepare a homogeneous drug-containing silk fibroin aqueous solution, defined as solution C; the poorly soluble aqueous drug molecule (drug model molecule: luteolin, abbreviated as LUT) is completely dissolved in ethanol solvent to prepare a homogeneous drug-containing alcohol solution, defined as solution D; 5 g of solution C and 5 g of solution D are rapidly mixed at a mass ratio of 1:1, and allowed to stand at a constant temperature of 37°C to undergo a gelation reaction, thereby obtaining a uniform silk-based hydrogel co-loaded with water-soluble and lipid-soluble drug molecules. In this embodiment, the initial total protein concentration of the silk fibroin aqueous solution (solution A) was 5 wt%, the SS / SF mass ratio was 1 / 4, the URI drug concentration was 8 mg per g of solution C, and the LUT drug concentration was 4 mg per g of solution D. Accordingly, the final silk-based hydrogel prepared contained 4 mg / g of URI and 2 mg / g of LUT.

[0063] Comparative Examples 1 to 4 A 5 wt% pure sericin (SS) aqueous solution was used as solution A, and ethanol was used as solution B. Solutions A and B were rapidly mixed in a specific mass ratio (detailed feeding is shown in Table 10). The mixture was then kept at a preset temperature and allowed to stand. The system underwent a gelation reaction and finally formed the silk-based hydrogel.

[0064] Table 9. Feeding table for water-soluble drugs loaded on silk fibroin hydrogel

[0065] Table 10. Feeding table for preparing pure silk protein hydrogels at 37°C using ethanol as an inducing agent.

[0066] Table 11. Feeding table for preparing pure silk fibroin hydrogels at 37°C using ethanol as an inducing agent.

[0067] Comparative Examples 5 to 8 A 5 wt% pure silk fibroin (SF) aqueous solution was used as solution A, and ethanol was used as solution B. Solutions A and B were rapidly mixed in a specific mass ratio (detailed feeding is shown in Table 11). The mixture was then kept at a preset temperature and allowed to stand. The system underwent a gelation reaction and finally formed the silk-based hydrogel.

[0068] Comparative Examples 9 to 10 As a comparative example, pure sericin hydrogel material was used as a drug delivery carrier to encapsulate water-soluble drugs. The method for loading water-soluble drugs includes the following steps: (a) completely dissolving a specific mass of water-soluble drug molecules (drug model molecules: FITC-labeled bovine serum albumin FITC-BSA and FITC-labeled ε-polylysine FITC-EPL) in a 5 wt% aqueous solution of sericin (SS) to prepare a homogeneous drug-containing sericin solution, defined as solution C, with a drug concentration of 8 mg / g; (b) using ethanol as solvent as solution B; (c) rapidly mixing solution B and solution C at a mass ratio of 1:1, allowing to stand at a constant temperature, and undergoing a gelation reaction to obtain a pure sericin-based hydrogel uniformly loaded with water-soluble drug molecules. In this hydrogel, the final concentration of sericin is 2.5 wt%, the ethanol content is 50 wt%, and the final concentration of drug molecules is 4 mg / g. Comparative Example 9 is the one that carries FITC-BSA, while Comparative Example 10 is the one that carries FITC-EPL.

[0069] Comparative Examples 11 to 12 The method was the same as that of Comparative Examples 9 or 10, except that pure silk fibroin hydrogel material was used as a drug delivery carrier to encapsulate water-soluble drugs; among them, Comparative Example 11 encapsulated FITC-BSA, while Comparative Example 12 encapsulated FITC-EPL.

[0070] Test Example 1 The gelation transition time of silk fibroin hydrogels can be recorded using the inverted observation method. The detailed procedure for the inverted observation method is as follows: Mix an aqueous solution of silk fibroin with an alcohol solvent and incubate at a set temperature. Once the flow of the mixed solution is no longer observed when the sample tube is inverted, the gelation transition is considered complete, and this time should be recorded immediately.

[0071] Test Example 2 The mechanical properties of the fully gelled silk protein hydrogel were tested using a rotational rheometer. The instrument used was a Thermo Fisher HAAKE MARS-40. Test parameters included a circular flat fixture (20 mm in diameter), modulus-time scan in oscillation mode, a fixture spacing of 500 μm, a test temperature of 37 °C, an oscillation frequency of 1 Hz, and a strain of 1%. Furthermore, rheological tests were performed on the fully formed silk protein hydrogel. Shear thinning behavior was detected by measuring its viscosity-shear rate curve. The test mode was rotational viscosity-shear rate curve mode, with a flat fixture diameter of 20 mm, a fixture spacing of 500 μm, and a test temperature of 37 °C.

[0072] Test Example 3 Fluorescent labeling of silk fibroin aqueous solution was performed using sulfonyl Cy5 fluorescent dye (Sulfo-Cy5). The method was as follows: Sulfonyl Cy5-NHS dye was added to the silk fibroin aqueous solution to a concentration of 0.1% of the silk fibroin mass. The mixture was stirred in the dark for 4 hours. After the reaction, the resulting Cy5-labeled silk fibroin solution was purified by dialysis to remove unlabeled free fluorescent molecules. Subsequently, after concentration, a Cy5-labeled silk fibroin solution with a mass concentration of 8 wt% was obtained. A 2 wt% sericin (SS) aqueous solution was mixed with an 8 wt% Cy5-labeled silk fibroin (Cy5-SF) solution to obtain a fluorescently labeled silk fibroin aqueous solution with a total protein concentration of 5 wt%, wherein the mass ratio of SS / SF was 1:4. Subsequently, the Cy5-labeled silk fibroin aqueous solution was rapidly mixed with an equal mass of ethanol solvent and transferred to a syringe to obtain a fully gelled fluorescently labeled silk fibroin hydrogel. The final total protein concentration in the hydrogel was 2.5 wt%, and the ethanol content was 50 wt%. BALB / c mice were orally administered the hydrogel via gavage at a dose of 1 g per kilogram of mouse body weight. After 30 minutes, the mice's stomachs were dissected to observe the formation and retention of the gel in the stomach. Room temperature fluorescence imaging was performed using the "fluorescence imaging mode" of a small animal in vivo imaging system (IVIS-Lumina Series-III, PerkinElmer, USA), with the excitation filter wavelength set to 640 nm and the emission filter set to 680 nm.

[0073] A 2 wt% aqueous solution of sericin (SS) and an 8 wt% solution of Cy5-labeled silk fibroin (Cy5-SF) were mixed thoroughly to obtain a fluorescently labeled silk fibroin aqueous solution with a total protein concentration of 5 wt%, where the SS / SF mass ratio was 1:4. Subsequently, this Cy5-labeled silk fibroin aqueous solution was rapidly mixed with an equal mass of ethanol solvent and transferred to a syringe to obtain a fully gelled fluorescently labeled silk fibroin hydrogel with a final total protein concentration of 2.5 wt% and an ethanol content of 50 wt%. The hydrogel was administered orally to BALB / c mice via gavage at a dose of 1 g per kilogram of mouse body weight. The digestive tracts of the mice were dissected at different time points, and fluorescence imaging experiments were performed to evaluate the retention of the silk fibroin hydrogel in the mouse digestive tract.

[0074] Test Example 4 The fully formed silk protein hydrogel was subjected to in vitro degradation tests to evaluate its degradation behavior in a simulated digestive tract environment, such as... Figure 18As shown in Figures C, D, and E. The in vitro degradation assay method is as follows: Physiological saline (PBS, pH=7.4), artificial gastric fluid (AGF, pH=1.2), artificial intestinal fluid (AIF, pH=6.8), artificial gastric fluid containing pepsin (pH=1.2, enzyme concentration: 3.2 mg / mL), and artificial intestinal fluid containing pancreatic enzyme (pH=6.8, enzyme concentration: 10 mg / mL) were prepared according to the pharmacopoeia formulations. These buffer solutions were used as degradation solutions for the silk protein hydrogel; 2 mL of degradation solution was used for 1 g of hydrogel. At specific time points, all degradation solution was removed from the hydrogel sample vials, and the remaining weight was weighed. A degradation curve was plotted based on the percentage of the remaining solution relative to the initial hydrogel weight. The degradation experiment was conducted in a constant-temperature shaking incubator at 37°C and a shaking rate of 100 rpm.

[0075] Test Example 5 The release curves of drug-loaded silk fibroin hydrogels in different media were tested using the following method: Artificial gastric fluid containing pepsin (pH=1.2, enzyme concentration: 3.2 mg / mL) and artificial intestinal fluid containing pancreatin (pH=6.8, enzyme concentration: 10 mg / mL) were prepared according to the pharmacopoeia formulation. These enzyme-containing buffer solutions were used as release solutions for the drug-loaded silk fibroin hydrogels, with 2 mL of release solution used for 1 g of drug-loaded hydrogel. At specific time points, 200 μL of release solution was collected, and the same volume of fresh buffer was added. The collected FITC-BSA and FITC-EPL release solutions were subjected to fluorescence emission spectroscopy to calculate their cumulative drug release and plot release curves. The collected CUR, RIF, and ATRA release solutions were subjected to UV-Vis absorption spectroscopy to calculate their cumulative drug release and plot release curves. All release experiments were conducted in a constant-temperature shaking chamber at 37 ℃ and a shaking rate of 100 rpm.

[0076] Test Example 6 Two g of the silk fibroin hydrogel prepared in Example 37 was immersed in 4 mL of physiological saline (PBS). At different time points (0 h, 2 h, 24 h, and 48 h), 1 mL of the extract was collected, and 1 mL of fresh PBS was added. Finally, the cytotoxicity of the hydrogel extract at each time point was tested using the MTT assay. Human colorectal adenocarcinoma cells (Caco-2) were used as the model cells. The test method was as follows: Caco-2 cells were seeded at a density of 3000 cells / well in 96-well cell culture plates; the initial concentration of the MTT reagent was 5 mg / mL, and after being added to each test well according to the experimental design, the final concentration of the system was adjusted to 0.5 mg / mL; the test material (the extract obtained after co-incubating the hydrogel with PBS for different times) and Caco-2 cells were co-incubated in a standard cell culture environment of 37°C and 5% carbon dioxide for 48 hours. Toxicity assessment and biocompatibility verification were completed by detecting cell viability.

[0077] Effect evaluation like Figure 1 and Figure 2 As shown, the experimental results collectively demonstrate that, at 37℃, the gelation kinetics of silk fibroin hydrogels can be precisely and reversibly regulated by two key parameters: the final ethanol concentration and the sericin (SS) / sericin (SF) mass ratio. However, the direction and mechanism of their influence on the gelation process differ fundamentally. Figure 1 As shown, under different SS / SF mass ratios, the gelation time of silk fibroin hydrogels significantly decreased with increasing final ethanol concentration, and this trend remained consistent across all ratios, indicating that ethanol plays a dominant role as a gelation inducing agent in this system. The introduction of ethanol effectively weakens the hydration around silk fibroin molecules, promotes the establishment of hydrophobic interactions between fibroin molecules, and accelerates their transformation from random coils to β-sheet structures, thus significantly accelerating the sol-gel transition process. These results demonstrate that by adjusting the ethanol content, rapid hydrogel formation can be achieved in a shorter time, providing a basic condition for in-situ gelation under oral administration conditions. In contrast, as... Figure 2 As shown, under the condition of constant final ethanol concentration, the gelation time of silk fibroin hydrogels showed a significant increasing trend with the gradual increase of the proportion of sericin in the system. This result is inconsistent with the general understanding in the prior art that sericin can promote network formation through hydrogen bonding and molecular chain entanglement.

[0078] Further analysis revealed that in the water / alcohol mixed solvent system used in this invention, the gelation reaction process is not dominated by system viscosity, but rather by the formation and stacking of silk fibroin β-sheet structures. Increased sericin content reduces the effective participation ratio of silk fibroin in the system, and its highly hydrophilic and random structural characteristics interfere with the hydrophobic interactions and β-sheet stacking between silk fibroin molecules, thus kinetically delaying the gel network construction process.

[0079] therefore, Figure 1 and Figure 2 This invention reveals the essential characteristics of the silk fibroin hydrogel formation mechanism from two different dimensions: ethanol acts as a structure inducer, accelerating the gelation process, while sericin acts as a regulator of gelation kinetics, slowing down and finely regulating the gelation rate. By simultaneously controlling the final ethanol concentration and the SS / SF mass ratio, a controllable transition from rapid to slow gelation can be achieved over a wide range, while maintaining the structural stability and mechanical properties of the gel after formation.

[0080] Figure 3 and Figure 4 The experimental results collectively demonstrate that, under the same initial concentration of silk fibroin (5 wt%) and the same SS / SF mass ratio (1:4), the type of alcohol solvent and its final concentration in the system significantly affect the gelation kinetics of silk fibroin hydrogels. This effect is not a simple concentration effect but is highly dependent on the chemical structural characteristics of the alcohol molecules. Specifically, in different alcohol-induced systems (methanol, ethanol, isopropanol, and n-butanol), as the final alcohol concentration increased from 20 wt% to 33 wt% and 50 wt%, the gelation time of the hydrogel generally decreased, indicating that increasing the alcohol content can enhance the induction of conformational changes in silk fibroin molecules. However, the magnitude of the change in gelation rate differed significantly among different alcohol systems, indicating that even under the same concentration conditions, different alcohol molecules do not have the same ability to promote the gelation process. Further cross-sectional comparisons of different alcohol types under the same final alcohol concentration revealed significant differences in the gelation time of silk fibroin hydrogels induced by different alcohols. This indicates that structural factors such as the carbon chain length, polarity, number of hydroxyl groups, and steric hindrance of alcohol molecules significantly affect their ability to alter the solvation state and intermolecular interactions of silk fibroin. Comparatively, alcohols with shorter carbon chains and higher polarity are more effective in disrupting the hydration layer surrounding silk fibroin molecules in water / alcohol mixtures, inducing the transformation of silk fibroin molecules from a random coil structure to a stable β-sheet structure, thereby accelerating the construction of the gel network. Conversely, alcohols with longer carbon chains or greater steric hindrance exhibit relatively lower structure induction efficiency, resulting in a significantly prolonged gelation time. In conclusion, Figure 3 and Figure 4The results clearly demonstrate that in the silk fibroin hydrogel system of this invention, alcohol solvents do not merely exist as dilution or mixing media, but rather participate in the formation of the gel network as key structure inducers and gelation kinetic regulators. By rationally selecting the type of alcohol and precisely controlling its content in the system, the gelation rate of silk fibroin hydrogels can be controllably adjusted over a wide range, providing an important parameter control method for different oral drug delivery applications and industrial preparation processes.

[0081] Figure 5 The results show that the gelation time of the silk fibroin hydrogel is significantly shortened with increasing temperature, indicating that increased temperature can accelerate the molecular motion and conformational rearrangement of silk fibroin. This demonstrates that the hydrogel system of this invention can achieve gelation regulation within the range of room temperature to body temperature, making it particularly suitable for applications requiring rapid gelation reactions at body temperature under oral administration conditions.

[0082] Figure 6 The results showed that after introducing different types of drugs into the silk fibroin hydrogel system, the hydrogel could still form smoothly under alcohol-induced conditions. However, the gelation time was prolonged to varying degrees compared to the uncoated system, and the variation in gelation time was closely related to the molecular structure and physicochemical properties of the coated drugs. Specifically, different drugs exhibited significant differences in molecular size, hydrophobicity, charge characteristics, and functional group types. During hydrogel formation, these drugs participated in or interfered with the interaction network between silk fibroin molecules through hydrophobic interactions, hydrogen bonding, or electrostatic interactions, thus affecting the kinetics of the transition of silk fibroin from random coils to β-sheet structures to varying degrees. Some drugs, due to their strong interactions with silk fibroin molecules, delayed the rapid construction of the gel network to some extent, resulting in a prolonged gelation time. However, it should be noted that although the gelation time changed after drug coating, the systems under all tested drug conditions were able to complete the gelation reaction within an acceptable time range, and the formed hydrogels did not show significant deterioration in macroscopic morphology and structural stability. The results show that the silk protein hydrogel system of the present invention has good compatibility with drugs of different structures and properties. Although its gelation kinetics can be modulated by drug molecules, they are not disrupted, demonstrating the applicability and stability of the system in practical drug delivery applications.

[0083] like Figure 7 and 8 As shown, rheological testing revealed that the storage modulus (G') of the gel material is as high as 0.1 × 10⁻⁶. 2 Up to 8.2×10 5 Pa, loss modulus (G'') is 2.8 to 7 × 10 3The Pa and G' values ​​are significantly greater than the G'' value, indicating the formation of a stable and robust silk fibroin gel network structure with excellent mechanical properties to resist physical compression by the gastrointestinal tract. The hydrogel material exhibits significant shear-thinning properties, meaning its viscosity decreases at high shear rates and recovers at low shear rates or upon rest. This property makes it easy to squeeze out with a syringe or swallow directly, effectively penetrating the esophagus and entering the gastrointestinal tract.

[0084] Regarding the stability of gel formation, except for the pure silk fibroin comparative group, the storage modulus (G') of all other examples and the pure silk fibroin comparative group was significantly higher than the loss modulus (G"), and remained stable during the 600-second test period, indicating that the physical cross-linked network constructed by alcohol induction has excellent structural stability at physiological temperatures. This was compared with pure component hydrogels (…). Figure 8 ) and SS / SF blended hydrogel ( Figure 7 Rheological data revealed that the ratio of silk fibroin (SF) to sericin (SS) is a key factor determining the mechanical strength of the gel. Comparative Examples 6 and 7 show that pure silk fibroin hydrogels exhibit extremely high mechanical strength, with G' values ​​ranging from 17.04 to 23.54 kPa, demonstrating strong rigidity. While strong, this excessive rigidity can lead to difficulty in oral swallowing and a lack of flexibility. Comparative Examples 2 and 3 show that pure sericin hydrogels have extremely low mechanical strength, with G' values ​​ranging from only 0.061 to 0.095 kPa, making it difficult to form a gel network with sufficient support. This weak gel network is unable to resist gastric peristalsis and cannot achieve gastric retention. However, this invention, by blending the two (Examples 32 to 53), successfully achieved precise control of the hydrogel's mechanical strength over a broad range from 0.35 kPa to 11.23 kPa. As the proportion of sericin in the system increases (i.e., the SS / SF mass ratio gradually increases from 1:9 to 4:1), the storage modulus of the hydrogel shows a significant decreasing trend, confirming that the flexible sericin effectively "softens" the rigid silk fibroin backbone. By introducing SS, the defects of excessive stiffness in pure SF gel are avoided, while also compensating for the deficiency of excessive softness in pure SS gel. In summary, this invention effectively overcomes the limitations of single-component hydrogels by controlling the ratio of SF to SS. The excessive stiffness of pure silk fibroin gel can easily lead to difficulty in swallowing, while the excessively low strength of pure sericin gel cannot meet the mechanical requirements of gastric retention; in contrast, the blended hydrogel of this invention (especially in the range of SS / SF ratio between 1:4 and 1:1) exhibits the best mechanical balance. This controllable characteristic of "combining rigidity and flexibility" not only endows the material with suitable flexibility to facilitate oral swallowing, but also ensures that it has sufficient mechanical strength to resist gastric peristalsis, thereby achieving long-term gastric retention.

[0085] Figure 9 The results clearly show that different types of drugs can be uniformly encapsulated in the silk fibroin hydrogel, with the gel remaining intact and showing no obvious phase separation. This demonstrates that the hydrogel system of this invention has broad-spectrum drug loading capacity and is suitable for the stable encapsulation of both water-soluble and poorly soluble drugs in the aqueous phase.

[0086] Figure 10 The results show that the viscosity of the silk protein hydrogel prepared in Example 37 decreases significantly with increasing shear rate, exhibiting typical shear-thinning behavior. This property makes the hydrogel behave as a low-viscosity fluid during swallowing or extrusion, while rapidly recovering its structure after settling, thereby significantly improving its oral swallowability.

[0087] like Figure 11 As shown, the silk protein hydrogel before and after drug loading was extruded onto the glass slide using a syringe and formed perfectly, demonstrating its excellent injectability. Figure 11 The results show that, regardless of whether the hydrogel is drug-loaded, it can be successfully extruded through a syringe and maintain its shape, indicating that drug loading does not affect the injectability and structural integrity of the hydrogel. This result further demonstrates the operability and stability of this hydrogel system in actual drug delivery processes.

[0088] Figure 12 The results showed that after co-incubating the silk fibroin hydrogel extract prepared in Example 37 with Caco-2 cells for 48 hours, the cell viability remained above 90%, indicating that the hydrogel material had no significant toxicity to the cells. This result demonstrates that the hydrogel of the present invention has excellent biocompatibility and meets the safety requirements for orally delivered materials.

[0089] based on Figure 13As shown in (A to F) of the in vitro enzymatic stability test results, the silk-based hydrogel prepared by this invention (Example 37) perfectly demonstrates the intelligent response characteristics of "gastric stability and intestinal degradation" necessary for oral drug delivery carriers, overcoming the significant defects of single-component hydrogels. First, in a simulated gastric juice environment (AGF, containing pepsin), the pure silk fibroin hydrogel (Comparative Example 3) exhibited extremely poor stability, rapidly undergoing enzymatic hydrolysis and erosion after an initial brief swelling, and completely degrading in just 8 hours, confirming that it could not maintain its structure to protect the drug in the gastric acid and pepsin environment. In contrast, both the pure silk fibroin hydrogel (Comparative Example 7) and the blended hydrogel of this invention (Example 37) showed excellent resistance to acid and protease hydrolysis. During the 72-hour test period, the gel mass remained above 90% of the initial mass, demonstrating that the β-sheet skeleton of silk fibroin provides robust structural protection for the blended hydrogel, ensuring that the carrier can resist the erosion of gastric digestive juices and providing a structural basis for achieving long-acting gastric retention. Secondly, in a simulated intestinal fluid environment (AIF, containing pancreatic enzymes), the three hydrogels exhibited distinctly different degradation kinetics, further highlighting the advantages of the blend system. The pure sericin hydrogel (Comparative Example 3) degraded too rapidly, disappearing completely within 12 hours, easily leading to drug burst release; while the pure silk fibroin hydrogel (Comparative Example 7), due to its high crystallinity, showed poor responsiveness to pancreatic enzymes, retaining approximately 65% ​​of its mass after 72 hours. This excessively slow degradation rate severely hindered effective drug release, resulting in reduced bioavailability. In stark contrast, the blend hydrogel of this invention (Example 37) exhibited ideal linear degradation characteristics, continuously and stably degrading under the action of pancreatic enzymes, with approximately 25% of its mass remaining after 72 hours. This moderate degradation rate is attributed to the introduction of sericin, which reduced the overall crystallinity of the gel network and increased the number of enzyme cleavage sites, enabling it to undergo moderate degradation and swelling in response to the intestinal environment. In summary, the hydrogel of Example 37, through the synergistic effect of SF and SS, successfully achieved intelligent spatiotemporal characteristics, maintaining high physical and chemical stability in the gastric fluid environment to protect the encapsulated drug, while releasing the drug through controlled degradation induced by pancreatic enzymes in the intestinal environment. This precisely meets the dual clinical requirements of oral enteric delivery systems for carrier stability and release kinetics.

[0090] based on Figure 14 and Figure 15 The in vitro drug release curves shown demonstrate that the silk-based composite hydrogel prepared in this invention exhibits significantly superior enteric-coated, precisely controlled-release properties compared to single-component (pure sericin or pure fibroin) carriers. Firstly, Figure 14(A, C) demonstrate the superior protective performance of the blended hydrogels of this invention in simulated gastric juice (AGF). Whether encapsulating the hydrophobic small molecule drug CUR (Example 69) or the large molecule protein drug FITC-BSA (Example 99), the cumulative release rate remained low (approximately 10%–20%) over 24 hours in the presence of pepsin, with a flat release curve and no burst release observed. In contrast, Figure 15 (A, B) show that pure sericin hydrogels performed extremely poorly in the gastric environment. The cumulative release rates of the encapsulated FITC-BSA (Comparative Example 9) and FITC-EPL (Comparative Example 10) surged to 41% and 55% respectively only one hour after administration, and were almost completely released within eight hours. This confirms that pure sericin carriers are highly susceptible to acid-induced disintegration in the stomach and cannot effectively protect the drug against the gastric environment. In contrast, the blended system, utilizing the crystalline framework of silk fibroin (SF), significantly improved the chemical and physical stability of the carrier in the stomach. Secondly, Figure 14 (B, D) reveal the intelligent responsive release advantages of the vector of the present invention in a simulated intestinal fluid (AIF) environment. Under pancreatic enzyme induction, Examples 69 and 99 both exhibited stable and efficient drug release kinetics. Especially the macromolecular delivery system encapsulating FITC-BSA ( Figure 14 It achieves a release rate of nearly 80% within 24 hours, realizing highly efficient delivery within the intestinal absorption window. In contrast... Figure 15 The performance of pure silk fibroin carriers in intestinal fluid in (C and D) showed that due to the excessive hydrophobicity and high crystallinity of the SF backbone, the carriers responded extremely slowly to pancreatic enzyme degradation. The cumulative release rates of comparative examples 11 and 12 after 24 hours were only 25% and 37%, respectively. This delayed release means the drug cannot be completely dissolved within the effective absorption time, severely limiting its bioavailability. In conclusion, Figure 14 and Figure 15 The comparative experimental results strongly demonstrate that this invention, through the blending of silk fibroin and sericin, successfully overcomes the existing technical defects of "gastric burst release" for pure sericin carriers and "intestinal release obstruction" for pure silk fibroin carriers. This blended system not only provides a reliable barrier for broad-spectrum drugs in strong acid and pepsin-rich environments, but also ensures efficient and precise drug release in the intestinal tract through the enzyme-responsive sensitivity introduced by sericin, making it an ideal new intelligent oral delivery platform.

[0091] Figure 16 The results showed that after oral administration, the Cy5-labeled silk protein hydrogel could pass smoothly through the esophagus of mice and form a clear shape in the stomach, indicating that the hydrogel has good shaping ability and retention properties in the stomach. These results validate the swallowability and gastric retention characteristics of the hydrogel of this invention from an in vivo experimental perspective.

[0092] Similarly, Figure 17The results showed that the fluorescence signal of the silk fibroin hydrogel in the mouse gastrointestinal tract initially concentrated in the stomach and then gradually shifted into the intestine, with the stomach signal remaining for a relatively long time. These results further quantitatively demonstrate that the hydrogel possesses significant gastric retention capacity and subsequent intestinal transport and release characteristics in vivo. Figure 18 This image shows a silk protein hydrogel prepared in Example 105 of this invention, which co-loads a water-soluble drug (uricase, URI) and a lipid-soluble drug (luteolin, LUT). The image visually illustrates the macroscopic process of the transformation from a "solution state" to a "gel state" after the drug-containing silk protein aqueous solution and the drug-containing ethanol solution are mixed, ultimately forming a cross-linked, stable, and uniformly loaded dual-drug co-loaded hydrogel system containing URI and LUT.

[0093] In summary, the SF / SS blend system proposed in this invention achieves synergistic effects of the advantages of both components, effectively solving a major bottleneck in existing technologies. Imparting "Easy-to-swallow" properties (solving the stiffness problem of SF): The introduction of sericin (SS) significantly improves the defects of excessive stiffness and poor shear-thinning properties of pure silk fibroin (SF) gels. As a flexible hydrophilic component incorporated into the SF backbone, SS effectively modulates the rheological behavior of the gel, endowing the blended hydrogel with unique shear-thinning characteristics. This means that under the shear force of swallowing, the gel viscosity decreases instantaneously, exhibiting a liquid-like state, thus ensuring that the carrier can easily pass through the narrow esophagus, avoiding swallowing obstacles associated with traditional solid dosage forms or high-stiffness gels.

[0094] Structure and function work synergistically to ensure "gastric retention" (solving the SS strength problem): In the gastric environment, shear forces disappear, and the gel rapidly recovers its structure. At this point, the β-folded framework of SF provides the high strength and acid resistance required to resist gastric mechanical peristalsis and chemical erosion, forming a solid physical basis for gastric retention; simultaneously, the excellent bioadhesive activity of SS helps the hydrogel adhere to the gastric mucosa surface, further enhancing the carrier's gastric retention capacity. Experiments show that this hydrogel can maintain structural stability for more than 24 hours under simulated gastric fluid conditions.

[0095] The introduction of SS (Supervisory Surface Antibody) effectively overcomes the limitations of pure SF gel, which is too hydrophobic and degrades slowly. The addition of SS increases the overall hydrophilicity of the carrier and introduces more enzyme cleavage sites. When the gel enters the intestinal environment, it specifically responds to digestive enzymes such as pancreatic enzymes, swelling and degrading to achieve "enteric-coated" properties. This ensures effective and rapid drug release within the intestinal absorption window, significantly improving drug bioavailability and avoiding the problem of drugs being excreted along with the poorly degradable gel carrier.

[0096] Therefore, the purpose of this invention is to provide an ingestible, gastric-retention, and enteric-coated silk-based hydrogel material. By constructing a controllable gelation reaction structure with silk fibroin and sericin as the main cross-linking network and an aqueous / alcoholic mixed solvent system, it achieves rapid gelation, gastrointestinal retention, and responsive degradation by intestinal proteases. The silk-based hydrogel of this invention can simultaneously and efficiently encapsulate both water-soluble and poorly soluble drugs in the aqueous phase, significantly prolonging the drug's retention time in the gastrointestinal tract and achieving controllable release under the specific protease environment of the intestine, thereby improving the oral delivery efficiency and long-term therapeutic stability of the drug.

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing silk based oral hydrogel, characterized in that, The method comprises the following steps: S11: mixing a silk fibroin aqueous solution and a sericin aqueous solution to obtain a silk protein aqueous solution; in the silk protein aqueous solution, the mass ratio of silk fibroin to sericin is 1-9:1-9; S12: adding an alcohol to the silk protein aqueous solution to obtain the silk-based oral hydrogel through a gelation reaction at 4-50℃.

2. The process for the preparation of silk based oral hydrogel according to claim 1, characterized in that: The alcohol is selected from one or more of ethanol, isopropanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol and glycerol.

3. The process for the preparation of silk based oral hydrogel as claimed in claim 1 wherein: The volume ratio of the silk protein aqueous solution to the alcohol is 1-19:1-19.

4. The process for the preparation of silk based oral hydrogel as claimed in claim 1 wherein: In the silk protein aqueous solution, the concentration of solutes is 0.2-20wt%.

5. The process for the preparation of silk based oral hydrogel as claimed in claim 1 wherein: The preparation method of the sericin comprises the following steps: S21: heating raw silk yarn in an alkaline aqueous solution to separate a sericin crude product; and S22: dialyzing and concentrating the sericin crude product to obtain the sericin.

6. The process for the preparation of silk based oral hydrogel as claimed in claim 1 wherein: The preparation method of the silk fibroin comprises the following steps: S31: heating raw silk yarn in an alkaline aqueous solution to separate a silk fibroin crude product; and S32: adding the silk fibroin crude product into an aqueous solution containing lithium bromide, dialyzing and concentrating to obtain the silk fibroin.

7. The process for the preparation of silk based oral hydrogel as claimed in claim 1 wherein: The silk-based oral hydrogel contains one or both of a water-soluble drug and a fat-soluble drug; the water-soluble drug is dissolved in the silk protein aqueous solution before the gelation reaction, and the fat-soluble drug is dissolved in the alcohol before the gelation reaction.

8. The process for the preparation of silk based oral hydrogel as claimed in claim 1 wherein: In the step S12, the gelation reaction is performed for 5-180000 s.

9. A silk-based oral hydrogel prepared by the preparation method in any one of claims 1-8.

10. A gastric residence delivery formulation characterized by: The silk-based oral hydrogel in claim 9 is included.

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