A colon-targeting silk fibroin-inulin loaded metal-organic framework composite hydrogel as well as a preparation method and application thereof

Abstract

This invention discloses a colon-targeted silk fibroin-inulin-loaded metal-organic framework (MOF) composite hydrogel, its preparation method, and its applications. The composite hydrogel is prepared by mixing an aqueous solution of silk fibroin, an aqueous solution of inulin, and an ethanol dispersion of MOF particles, followed by static gelation. This invention uses ethanol as a phase transition trigger, which can instantaneously induce a conformational change in silk fibroin to form a stable cross-linked backbone, while simultaneously promoting inulin precipitation, achieving rapid in-situ gelation of the two natural polymers. During the gel network formation process, dispersed MOF particles can be simultaneously encapsulated in situ, solving the technical problems of MOFs easily agglomerating and undergoing phase separation in traditional hydrogels. The composite hydrogel provided by this invention possesses excellent shear-thinning properties and good swallowability. The dual-network structure constructed by silk fibroin and inulin can withstand the highly acidic environment of the stomach, enabling a stepwise release of the drug in the digestive tract, achieving colon-targeted delivery.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a colon-targeting silk fibroin-inulin-loaded metal-organic framework composite hydrogel, its preparation method, and its application. Background Technology

[0002] Drug delivery systems (DDS) are a core research area in modern pharmaceutical science and biomedical engineering. Oral colon-targeted delivery systems have significant clinical value in treating lower gastrointestinal diseases such as inflammatory bowel disease and colorectal cancer. However, the human gastrointestinal tract has multiple physiological barriers, including gastric acid, various digestive enzymes, pH gradients, and intestinal peristalsis. Conventional oral formulations are prone to problems such as premature drug degradation and non-specific release throughout the body, making it difficult to accurately concentrate drugs at the colonic lesion site.

[0003] Hydrogels are often used as oral drug carriers due to their advantages such as high water content, excellent biocompatibility, and mechanical compatibility with human soft tissue. However, traditional single-component hydrogels have significant technical drawbacks: First, the highly hydrophilic three-dimensional network makes it difficult to efficiently encapsulate lipophilic drugs with extremely poor water solubility (such as various small-molecule targeted anti-tumor drugs and non-steroidal anti-inflammatory drugs), resulting in extremely low drug loading and easy drug aggregation. Second, the gel is mainly composed of macroporous structures with pore sizes of 50 nm or more, which has a weak binding effect on small-molecule drugs and is prone to severe burst release effects. Third, it is difficult to provide effective protection for biomacromolecules such as proteins and nucleic acids. Fourth, it only has a single pH response capability and lacks active colon-targeting function and microenvironment immune regulation capability.

[0004] To overcome these shortcomings, existing technologies have developed organic-inorganic hybrid hydrogels, incorporating metal-organic frameworks (MOFs) into polymer networks. MOFs possess ultra-high specific surface areas and well-ordered micro / mesoporous structures, enabling them to broadly load various drugs, including hydrophilic, lipophilic, peptides, and nucleic acids, through host-guest interactions. Their rigid crystalline channels also provide physical protection for biomolecules. However, MOF particles tend to aggregate in the gastrointestinal tract when taken orally alone, and some high-performance MOFs (such as the ZIF series) are prone to structural collapse due to the breakage of coordination bonds in the highly acidic environment of gastric juice. Furthermore, MOFs are mostly stably dispersed in organic solvents, exhibiting significant polarity differences compared to aqueous polymer precursor solutions. Simple blending can lead to phase separation and large-scale particle aggregation, making it impossible to prepare homogeneous gels.

[0005] In addition, existing technologies use natural functional polymers such as silk fibroin (SF) and inulin as oral carriers. Among them, silk fibroin hydrogel has good acid resistance and can be rapidly cross-linked and formed under ethanol induction, but its internal macroporous structure is prone to inducing drug burst release, and it is rapidly degraded by pancreatic enzymes after entering the small intestine, making it unable to complete distal colon delivery. Inulin, as a natural prebiotic polysaccharide, is not broken down by host digestive enzymes in the stomach and small intestine, and can only be fermented by colonic flora. It naturally has both colon-targeting and intestinal immune regulation functions. However, the gelation conditions of pure inulin are harsh, requiring a high concentration of raw materials of more than 30 wt%, and the curing time can reach 30-60 min. The resulting gel has poor mechanical properties and is difficult to withstand intestinal peristalsis shear. After incorporating inorganic particles, particle sedimentation and stratification will occur due to slow gelation.

[0006] Therefore, there is an urgent need for an organic-inorganic hybrid hydrogel with a simple preparation process, mild conditions, and the ability to overcome the compatibility barriers between components, in order to meet multiple clinical needs such as high drug loading capacity, broad-spectrum encapsulation, precise targeted release to the colon, and regulation of the intestinal microecology to achieve synergistic immune therapy. Summary of the Invention

[0007] This invention aims to overcome a series of defects in existing oral drug delivery carriers, solve the preparation problems such as the difficulty in mixing hydrophobic MOF organic dispersions with hydrophilic polymer aqueous solutions, and the uneven particle sedimentation caused by the long curing cycle of hybrid gels. It also aims to address how to design materials to enable carriers to possess multiple properties such as broad-spectrum and efficient encapsulation capacity for lipophilic / biomacromolecule drugs, protection against the extreme acidity of the stomach, a three-level intelligent cascade release characteristic of "stomach protection-small intestine transition-colon targeted burst", and regulation of intestinal flora and mediation of immune synergistic therapy.

[0008] This invention provides a colon-targeted silk fibroin-inulin-loaded metal-organic framework (MOF) composite hydrogel, its preparation method, and its application. MOF particles are uniformly and stably composited in a dual network matrix constructed from silk fibroin and inulin, achieving deep organic integration and complementary advantages among the three. This provides a high-performance carrier for the oral delivery of poorly soluble drugs and biomolecules, graded targeted drug release in the digestive tract, and combined intestinal immune therapy, and has outstanding industrialization value and clinical translation prospects.

[0009] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0010] The first aspect of this invention provides a silk fibroin-inulin-loaded metal-organic framework composite hydrogel, which is prepared by mixing an aqueous solution of silk fibroin, an aqueous solution of inulin and an ethanol dispersion of metal-organic framework particles and then allowing them to stand and gel.

[0011] The concentration of the silk fibroin aqueous solution is 0.5-20 wt%, the concentration of the inulin aqueous solution is 1-40 wt%, and the concentration of the metal-organic framework particle ethanol dispersion is 0.2-20 mg / g; the mass ratio of the silk fibroin aqueous solution to the inulin aqueous solution is 1:5-5:1; and the mass ratio of the total mass of the silk fibroin aqueous solution and the inulin aqueous solution to the mass of the metal-organic framework particle ethanol dispersion is 9.5:0.5-2:8.

[0012] This invention uses ethanol as a phase transition trigger, which can instantaneously induce a conformational change in silk fibroin and form a stable cross-linked backbone, while simultaneously promoting inulin precipitation, achieving rapid in-situ gelation of two natural polymers. During the gel network formation process, dispersed MOF particles can be simultaneously encapsulated in-situ, solving the technical problems of MOFs easily agglomerating and undergoing phase separation in traditional hydrogels. Compared to traditional macroporous hydrogels with pore sizes generally greater than 50 nm, the composite hydrogel of this invention introduces a rigid MOF structure, constructing a multi-level pore system that combines silk fibroin-inulin cross-linked macropores, MOF micropores, and mesopores. This system can efficiently and broadly encapsulate hydrophilic macromolecules and lipophilic drugs of different sizes, and significantly inhibit drug burst release behavior.

[0013] The silk fibroin aqueous solution was selected in the range of 0.5-20 wt% to provide sufficient sites for the formation of a β-sheet cross-linking network to maintain the support of the gel skeleton, while avoiding the problem of uneven mixing caused by excessively high system viscosity. The inulin aqueous solution was controlled at a mass concentration of 1-40 wt% to form a sufficiently dense anti-enzymatic protective matrix and maintain colon-targeting prebiotic activity. The metal-organic framework particle ethanol dispersion concentration was set at 0.2-20 mg / g to achieve monodisperse stability of submicron (0.05-1 μm) and micron (1-20 μm) crystals in the solvent, effectively inhibiting particle aggregation.

[0014] Preferably, the mass concentration of the silk fibroin aqueous solution is 2-10 wt%, the mass concentration of the inulin aqueous solution is 20-40 wt%, and the concentration of the metal-organic framework particle ethanol dispersion is 1-20 mg / g.

[0015] Adjusting the mass ratio of silk fibroin aqueous solution to inulin aqueous solution to 1:5-5:1 can balance the gel's resistance to gastric acid and its ability to detoxify in the colon. The mass ratio of the total mass of silk fibroin aqueous solution and inulin aqueous solution to the mass of the metal-organic framework particle ethanol dispersion is controlled at 9.5:0.5-2:8 to adapt the ethanol-induced phase transition kinetics to the aqueous system. This mass ratio is preferably 5:1-1:2, and more preferably 1:1.

[0016] Furthermore, the settling temperature is 4-60 ℃, which is a mild temperature range that allows for rapid gelation without high temperatures; preferably 4-37 ℃, which is beneficial for protecting heat-sensitive active drugs such as peptides and macromolecular proteins.

[0017] Furthermore, the settling time is 1-20 minutes, preferably 1-4 minutes.

[0018] Furthermore, the metal ions in the metal-organic framework particles are selected from zinc ions (Zn). 2+ ), iron ions (Fe 3 + ), zirconium ions (Zr) 4+ ), calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ) and copper ions (Cu 2+ One or more of the following.

[0019] Furthermore, the particle size of the metal-organic framework particles is 0.05-20 μm.

[0020] Furthermore, the metal-organic framework particles are selected from one or more of ZIF-8, ZIF-90, MOF-5, MIL-53, MIL-101, UiO-66, MIL-156, Mg-MOF and HKUST-1 particles.

[0021] Specifically, the metal-organic framework particles are porous crystalline materials assembled by coordination of metal ions and organic polydentate ligands, and can be selected from one or more of ZIF-8(Zn), ZIF-90(Zn), MOF-5(Zn), MIL-53(Fe), MIL-101(Fe), UiO-66(Zr), MIL-156(Ca), Mg-MOF(Mg), and HKUST-1(Cu).

[0022] Furthermore, the silk fibroin-inulin-loaded metal-organic framework composite hydrogel has a multi-level porous structure, including micropores and mesopores provided by the metal-organic framework particles, as well as macropores formed by the cross-linked network of silk fibroin and inulin.

[0023] Specifically, the aperture distribution range of this multi-level channel structure is as follows:

[0024] (1) Macropores: The pore size ranges from 50 nm to 10 μm. The macropore structure is mainly provided by a three-dimensional matrix network of polymers constructed by the conformational transformation of natural silk fibroin peptide chains, physical cross-linking and precipitation entanglement of long-chain inulin, which serves as the macroscopic mechanical framework of the gel and the diffusion channel of the macromolecular liquid.

[0025] (2) Mesopores: Their pore size ranges from 2 to 50 nm. These mesopore structures originate from two sources: firstly, some MOF particles with intrinsic mesoporous cage structures (such as the approximately 2.9 nm and 3.4 nm mesoporous cage structures present inside MIL-101(Fe); and secondly, from the inter-particle stacking gaps and lattice defects during the in-situ gelation process of MOF particles. This structure provides an extremely ample confined space for encapsulating hydrophilic / lipophilic biomacromolecules.

[0026] (3) Micropores: The pore size ranges from 0.3 to 2 nm. This micropore structure is entirely provided by the intrinsic metal-organic coordination topological framework of highly crystalline MOF particles (e.g., the window and cavity sizes of typical microporous MOFs such as ZIF-8, UiO-66, HKUST-1, and MOF-5 are mostly distributed between 0.3 nm and 1.5 nm). The extremely regular and dense micropore structure, with its strong van der Waals forces and host-guest interactions, is the core physical binding site for inhibiting the early burst release of small molecule drugs.

[0027] Furthermore, the silk fibroin-inulin-loaded metal-organic framework composite hydrogel is a colon-targeted silk fibroin-inulin-loaded metal-organic framework composite hydrogel.

[0028] A second aspect of this invention provides a method for preparing the silk fibroin-inulin-supported metal-organic framework composite hydrogel described in the first aspect, comprising the following steps:

[0029] (1) Mix the silk fibroin aqueous solution with the inulin aqueous solution to obtain a mixed aqueous solution;

[0030] (2) Disperse the metal-organic framework particles in ethanol to obtain a metal-organic framework particle ethanol dispersion;

[0031] (3) The mixed aqueous solution is mixed with the ethanol dispersion of metal-organic framework particles and then allowed to stand. Ethanol is used to induce the conformational change of silk fibroin and promote the precipitation of inulin. The silk fibroin-inulin-loaded metal-organic framework composite hydrogel is obtained by gelation.

[0032] Further, in step (1), the preparation method of the silk fibroin aqueous solution includes the following steps: degumming raw silk and dissolving it in lithium bromide aqueous solution, removing salt impurities by dialysis, and then concentrating it to obtain the silk fibroin aqueous solution.

[0033] Furthermore, the concentration is achieved by reverse osmosis concentration using a polyethylene glycol solution; the polyethylene glycol has a weight-average molecular weight of 20,000 Da, the concentration of the polyethylene glycol solution is 20-50 wt%, and the concentration temperature is 4-25 ℃.

[0034] Specifically, the preparation method of the silk fibroin aqueous solution includes the following steps:

[0035] S1. Add the shredded raw silk yarn to a sodium carbonate aqueous solution, boil to degumme, wash and dry to obtain silk fibroin fiber;

[0036] S2. Dissolve the silk fibroin in an aqueous lithium bromide solution, and remove salt impurities by dialysis with deionized water to obtain a purified silk fibroin aqueous solution.

[0037] S3. Using a polyethylene glycol solution with a concentration of 20-50 wt% and a weight-average molecular weight of 20000 Da, the purified silk fibroin aqueous solution is concentrated by reverse osmosis at 4-25 °C to obtain the silk fibroin aqueous solution.

[0038] The third aspect of this invention provides an application of the silk fibroin-inulin-loaded metal-organic framework composite hydrogel described in the first aspect in the preparation of drug delivery systems, particularly suitable for colon-targeted intelligent cascade delivery of lipophilic poorly soluble drugs and sensitive biomolecular drugs, as well as synergistic therapy combined with intestinal microenvironment immune regulation.

[0039] The composite hydrogel provided by this invention possesses excellent shear-thinning properties and good swallowability. The dual-network structure constructed from silk fibroin and inulin can withstand the highly acidic environment of the stomach, enabling a stepwise release of the drug in the digestive tract and achieving colon-targeted delivery. Simultaneously, inulin, as a prebiotic, can be fermented and decomposed by intestinal flora into short-chain fatty acids after entering the colon, actively regulating the intestinal microecology and producing a synergistic immune therapeutic effect. Therefore, the composite hydrogel of this invention exhibits significant advantages among various oral drug delivery systems and has broad prospects for research and clinical application.

[0040] Furthermore, the silk fibroin-inulin-loaded metal-organic framework composite hydrogel serves as an oral or injectable drug carrier; the oral drug carrier encapsulates a target drug, which is selected from one or more of hydrophilic drugs, lipophilic drugs, peptide drugs, nucleic acid drugs, and protein drugs. The target drug is adsorbed and encapsulated in the porous structure of the metal-organic framework particles through host-guest interactions.

[0041] Specifically, the target drugs have broad applicability, including but not limited to hydrophilic anticancer drugs, lipophilic / hydrophobic anti-inflammatory and analgesic targeted drugs, peptide hypoglycemic drugs, nucleic acid (siRNA / mRNA) gene therapy drugs, and macromolecular protein drugs (such as monoclonal antibodies).

[0042] Furthermore, when the target drug is a lipophilic drug, it is encapsulated in the silk fibroin-inulin-loaded metal-organic framework composite hydrogel using the following steps:

[0043] P1. Disperse metal-organic framework particles in an organic solvent to adsorb and encapsulate the lipophilic drug;

[0044] P2. After removing the organic solvent, the metal-organic framework particles loaded with lipophilic drugs are dispersed in ethanol to obtain a drug-loaded metal-organic framework particle ethanol dispersion; the silk fibroin aqueous solution is mixed with the inulin aqueous solution to obtain a mixed aqueous solution.

[0045] P3. The ethanol dispersion of the drug-loaded metal-organic framework particles is mixed with the mixed aqueous solution and allowed to stand. After gelation, a silk fibroin-inulin-loaded metal-organic framework composite hydrogel loaded with lipophilic drugs is obtained.

[0046] Furthermore, in step P1, the organic solvent is immiscible or poorly immiscible with water.

[0047] Further, in step P1, the organic solvent is selected from one or more of dichloromethane, chloroform, ethyl acetate, and n-hexane. This organic solvent is a good solvent for lipophilic drugs.

[0048] Furthermore, the oral drug carrier is a responsive colon-targeted drug delivery carrier, which exhibits a three-stage release characteristic during oral delivery:

[0049] Level 1: In the highly acidic environment of the stomach, the dual network physical barrier of silk fibroin and inulin protects MOF particles from acid degradation and inhibits drug burst release.

[0050] Second stage: In the neutral pH and pancreatic enzyme environment of the small intestine, the silk fibroin backbone undergoes slow local enzymatic hydrolysis, which loosens the gel network and achieves a smooth transitional release.

[0051] Level 3: After reaching the colon, the inulin is thoroughly fermented and degraded by specific intestinal symbiotic flora, achieving the disintegration of the gel skeleton and the targeted and accelerated release of the drug.

[0052] The above-described technical solution of the present invention has the following beneficial effects:

[0053] 1. This invention uses an ethanol dispersion of MOF particles as a crosslinking and phase transition triggering medium. Ethanol can maintain the monodispersity of MOFs and simultaneously induce the random coiling of silk fibroin to undergo a conformational transition to β-sheet, reduce the polarity of the system and promote the rapid precipitation of inulin. At room temperature, rapid gelation can be completed in one step in 1-4 minutes, which greatly shortens the unstable standing period of the system and avoids defects such as particle sedimentation and phase separation.

[0054] 2. After introducing MOFs, this invention constructs a full-scale hierarchical porous structure containing MOF micropores (pore size < 2 nm, derived from the intrinsic lattice of MOFs), mesopores (pore size 2-50 nm, derived from MOF defects or gaps) and silk fibroin-inulin cross-linked macropores. The specific surface area of ​​the material is significantly increased, and various drugs are firmly anchored by host-guest interactions, effectively inhibiting the burst release of drugs in the early stage of body fluid contact and steadily prolonging the sustained release period.

[0055] 3. The dense interpenetrating silk fibroin-inulin dual network of the present invention, with the help of spatial steric hindrance and shielding effect, prevents gastric acid protons and pepsin from penetrating into the internal pores, thus completely protecting the MOF backbone and active sensitive drugs such as peptides and nucleic acids in the upper digestive tract.

[0056] 4. This invention relies on the complementary degradation characteristics of silk fibroin and inulin to form a three-stage controllable release: the highly crystalline β-sheet network of silk fibroin in the stomach and the hydrogen bond network of inulin shrink densely, greatly inhibiting drug release; in the small intestine, silk fibroin is slowly enzymatically hydrolyzed, and inulin maintains the integrity of the matrix, and the gel gradually loosens to form a drug diffusion channel, achieving a smooth transition and sustained release; after reaching the colon, the flora specifically degrades inulin, the outer protective skeleton completely disintegrates, and the MOF particles, which serve as drug reservoirs, are fully exposed, allowing for concentrated and rapid drug release at the lesion site.

[0057] 5. The composite hydrogel provided by this invention is not only an inert delivery matrix, but also produces short-chain fatty acids from inulin through fermentation by colonic flora. This regulates the abundance of beneficial bacteria in the intestine and activates immune-related receptors, promoting the differentiation of anti-tumor memory T cells. When used in combination with chemotherapy drugs and immune checkpoint antibodies, it can form a synergistic treatment of flora and drugs, with anti-inflammatory and anti-tumor effects that are significantly better than those of single administration.

[0058] 6. The composite hydrogel provided by this invention has shear-thinning thixotropic properties. It is solid gel when standing, and its viscosity drops sharply and its fluidity increases under high shear conditions such as chewing and pushing, making it suitable for swallowing and injection processing. The degradation products are amino acids and monosaccharides, which are not toxic to the liver and kidneys, and are easy to make into oral preparations such as jelly and capsules, which are suitable for the elderly, children and critically ill patients, and have good prospects for industrialization. Attached Figure Description

[0059] Figure 1 A schematic diagram of the preparation process of the silk fibroin-inulin-loaded metal-organic framework composite hydrogel provided by the present invention and a schematic diagram of its application in an oral drug delivery system.

[0060] Figure 2The images show the isothermal adsorption-desorption curves and BET specific surface area histograms of the MOF particles of the present invention; wherein, (A) is the isothermal adsorption-desorption curve of ZIF-8 particles, (B) is the isothermal adsorption-desorption curve of MIL-101(Fe) particles, (C) is the isothermal adsorption-desorption curve of UiO-66(Zr) particles, (D) is the isothermal adsorption-desorption curve of Mg-MOF particles, (E) is the isothermal adsorption-desorption curve of HKUST-1(Cu) particles, and (F) is the BET specific surface area histogram of MOF particles.

[0061] Figure 3 Gel permeation chromatography (GPC) chromatograms and molecular weight distribution curves of short-chain inulin and long-chain inulin are shown below; where (A) is the GPC chromatogram of short-chain inulin, (B) is the molecular weight distribution curve of short-chain inulin, (C) is the GPC chromatogram of long-chain inulin, and (D) is the molecular weight distribution curve of long-chain inulin.

[0062] Figure 4 The images show the XRD patterns of the composite hydrogels prepared in Examples 30, 45, 60, 65, 70, and 75, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 after freeze-drying; wherein, (A) is Comparative Example 13 and Example 30, (B) is Comparative Example 13 and Example 45, (C) is Comparative Example 13 and Example 60, (D) is Comparative Example 13 and Example 65, (E) is Comparative Example 13 and Example 70, and (F) is Comparative Example 13 and Example 75.

[0063] Figure 5 The images show the morphology of the inulin-based hydrogels prepared in comparative examples 1-5 in an inverted bottle.

[0064] Figure 6 The images show the morphology of the inulin-based hydrogels prepared in Comparative Examples 7-8 and the silk fibroin-based hydrogels prepared in Comparative Examples 9-10 in inverted bottles.

[0065] Figure 7 The images show the morphology of the silk fibroin-inulin-loaded MIL-156(Ca) composite hydrogel prepared in Example 60 and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Examples 12-13 in an inverted bottle.

[0066] Figure 8The following are rheological test results for the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel prepared in Example 65, the inulin-based hydrogels prepared in Comparative Examples 3-4, the inulin-based hydrogel prepared in Comparative Example 7, the silk fibroin-based hydrogel prepared in Comparative Example 10, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13. Specifically, (A) is the temperature scan curve of the inulin-based hydrogel prepared in Comparative Example 3; (B) is the modulus-angular frequency scan curve of the inulin-based hydrogel prepared in Comparative Example 7, the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13, and the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel prepared in Example 65 at a temperature of 25 °C and a strain of 1% in an oscillating mode; and (C) is the temperature scan curve of the inulin-based hydrogel prepared in Comparative Example 4 and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 at a temperature of 37 °C. Viscosity-shear rate curves in rotation mode at ℃, (D) is the viscosity-shear rate curve of silk fibroin-based hydrogel prepared in Comparative Example 10 in rotation mode at 37 ℃.

[0067] Figure 9 The rheological test results of the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 and Comparative Example 17 are shown in the figure. Among them, (A) is the time-scan gelation kinetic curve of the mixed solution of silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 in the oscillation mode of temperature 25 ℃, oscillation frequency 1 Hz and strain 1%, and (B) is the comparison of the storage modulus of silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 and Comparative Example 17 in the oscillation mode of temperature 25 ℃, angular frequency 10 rad / s and strain 1%.

[0068] Figure 10 Syringe extrusion deformation morphology images of inulin-based hydrogels prepared in Comparative Examples 3-6, inulin-based hydrogels prepared in Comparative Example 8, silk fibroin-based hydrogels prepared in Comparative Example 9, and silk fibroin-inulin-based composite hydrogels prepared in Comparative Examples 12-13.

[0069] Figure 11 The syringe extrusion deformation morphology images show the composite hydrogels prepared in Examples 30, 60, and 65, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13.

[0070] Figure 12The figures show the drug loading and efficiency data of drug-loaded MOF particles for CUR, RFX, VCN, and Cy5-URC in Examples 86-97; wherein, (A) is the drug loading and efficiency data of drug-loaded MOF particles for CUR, (B) is the drug loading and efficiency data of drug-loaded MOF particles for RFX, (C) is the drug loading and efficiency data of drug-loaded MOF particles for VCN, and (D) is the drug loading and efficiency data of drug-loaded MOF particles for Cy5-URC.

[0071] Figure 13 The image shows the drug loading data of the silk fibroin-inulin-loaded metal-organic framework composite hydrogels containing drugs (CUR, RFX, VCN, Cy5-URC) prepared in Examples 86-97.

[0072] Figure 14 The image shows a fluorescence image of the mouse digestive tract after oral administration of the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel containing Cy5-URC prepared in Example 96 and a pure Cy5-URC solution.

[0073] Figure 15 The degradation kinetic curves of the composite hydrogels prepared in Examples 30, 60, and 65, the inulin-based hydrogels prepared in Comparative Examples 3-5, the inulin-based hydrogels prepared in Comparative Example 7, the silk fibroin-based hydrogels prepared in Comparative Example 9, and the silk fibroin-inulin-based composite hydrogels prepared in Comparative Example 13 in artificial gastric fluid, artificial small intestinal fluid, and artificial colonic fluid are shown in Figure 13. Among them, (A) is the degradation kinetic curve of Examples 30, 60, 65, Comparative Examples 7, Comparative Examples 9, and Comparative Example 13 in artificial gastric fluid, (B) is the degradation kinetic curve of Comparative Examples 3-5 and Comparative Example 7 in artificial gastric fluid, (C) is the degradation kinetic curve of Examples 30, 60, 65, Comparative Examples 9, and Comparative Example 13 in artificial small intestinal fluid, and (D) is the degradation kinetic curve of Examples 30, 60, 65, Comparative Examples 9, and Comparative Example 13 in artificial colonic fluid.

[0074] Figure 16 The drug release curves in simulated gastric fluid are as follows: the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC prepared in Example 96; the silk fibroin-inulin-based composite hydrogel loaded with Cy5-URC prepared in Comparative Example 14; the silk fibroin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 15; and the inulin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 16.

[0075] Figure 17The drug release curves in artificial intestinal fluid are shown for the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC prepared in Example 96, the silk fibroin-inulin-based composite hydrogel loaded with Cy5-URC prepared in Comparative Example 14, and the silk fibroin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 15.

[0076] Figure 18 The drug release curves in artificial colon fluid are shown for the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC prepared in Example 96, the silk fibroin-inulin-based composite hydrogel loaded with Cy5-URC prepared in Comparative Example 14, the silk fibroin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 15, and the inulin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 16. Detailed Implementation

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0078] This invention provides a silk fibroin-inulin-supported metal-organic framework composite hydrogel, which is prepared by mixing silk fibroin aqueous solution, inulin aqueous solution and metal-organic framework particle ethanol dispersion and then allowing it to stand for gelation.

[0079] The concentration of the silk fibroin aqueous solution is 0.5-20 wt%, the concentration of the inulin aqueous solution is 1-40 wt%, and the concentration of the metal-organic framework particle ethanol dispersion is 0.2-20 mg / g; the mass ratio of the silk fibroin aqueous solution to the inulin aqueous solution is 1:5-5:1; and the mass ratio of the total mass of the silk fibroin aqueous solution and the inulin aqueous solution to the mass of the metal-organic framework particle ethanol dispersion is 9.5:0.5-2:8.

[0080] To address the technical bottleneck of slow gelation rate of pure inulin in existing systems, this invention utilizes MOF particle ethanol dispersion as both a polarity regulator and a phase change catalyst in a ternary water-alcohol blend system, achieving a high degree of integration of the three components. Anhydrous ethanol has a dielectric constant of approximately 24.6, far lower than the 80.0 of pure water. When the MOF particle ethanol dispersion is rapidly added to the silk fibroin-inulin mixed aqueous solution, the system's polarity decreases sharply, the hydration layer on the inulin molecule surface is disrupted, solubility rapidly decreases, and rapid entanglement occurs through hydrogen bonds, accelerating the formation of a particle cross-linked network. Simultaneously, ethanol, as a potent conformational inversion inducer, rapidly removes water-bound components from the silk fibroin peptide chains, prompting thermodynamic rearrangement of the peptide chains through random coiling, forming a highly dense, physically cross-linked β-sheet crystalline network structure. Under this deeply interdependent and synergistic physical cross-linking mechanism—where the silk fibroin-dominated network undergoes rapid rigid cross-linking induced by ethanol, and the inulin-assisted network accelerates precipitation and formation driven by a sudden drop in polarity—the macroscopic apparent viscosity of the entire system undergoes a nonlinear abrupt change within seconds, and the entire gelation process of liquid-solid phase transition is completely completed within an extremely short time window of 1-4 minutes. This rapid in-situ gelation mechanism can instantly lock uniformly dispersed MOF particles in the fluid, confining them uniformly within a dense three-dimensional hydrogel network constructed by the interpenetration of silk fibroin and inulin. This solves the technical problems of macroscopic particle aggregation and phase separation, ensuring the uniformity of the overall structure of the resulting composite hydrogel.

[0081] The inulin described in this invention is a fructose polymer linked by β-(2→1) glycosidic bonds, typically containing a glucose residue at the end, and its molecular weight is mainly determined by its degree of polymerization (DP). Based on the degree of polymerization, the inulin can be divided into short-chain inulin and long-chain inulin. Specifically, short-chain inulin refers to inulin with a DP of 2-10, and the absolute molecular weight of each component is approximately 342-1639 Da; long-chain inulin refers to inulin with a DP of not less than 10. Preferably, the average degree of polymerization of the long-chain inulin is not less than 23; more preferably, the degree of polymerization of the long-chain inulin is 23-60, corresponding to an absolute molecular weight of each component of approximately 3746-9750 Da. Both the long-chain and short-chain inulin described in this invention include naturally sourced, processed, or commercially available inulin products whose degree of polymerization, average degree of polymerization, and absolute molecular weight fall within the above ranges.

[0082] In one embodiment of the present invention, the long-chain inulin or short-chain inulin may be inulin containing only a single degree of polymerization component, or it may be an inulin mixture composed of multiple inulin with different degrees of polymerization.

[0083] In one embodiment of the present invention, the metal-organic framework particles are selected from one or more of ZIF-8, ZIF-90, MOF-5, MIL-53 (Fe), MIL-101 (Fe), UiO-66 (Zr), MIL-156 (Ca), Mg-MOF and HKUST-1 (Cu) particles.

[0084] In one embodiment of the present invention, ZIF-8 particles are available from Alfa Chemistry, catalog number ACM59061539-1; ZIF-90 is available from Alfa Chemistry, catalog number ACM1062147378-2; MIL-101 (Fe) is available from Guangdong Carbon Language New Materials Co., Ltd. (KARGEN), catalog number KAR-F49; UiO-66 (Zr) is available from Guangdong Carbon Language New Materials Co., Ltd., catalog number KAR-F30; HKUST-1 (Cu) is available from Guangdong Carbon Language New Materials Co., Ltd., catalog number KAR-F43.

[0085] In one embodiment of the present invention, the preparation method of ZIF-8 particles includes the following steps: 0.733 g of zinc nitrate hexahydrate is dissolved in 50 mL of methanol; 1.622 g of 2-methylimidazole is dissolved in 50 mL of methanol; the methanol solution of zinc nitrate hexahydrate is slowly added dropwise to the methanol solution of 2-methylimidazole while stirring (500 rpm), and stirring is continued for 1 h after the addition is complete. Subsequently, the mixed reaction solution is allowed to stand at room temperature for 2 h. Finally, the particle product is separated by centrifugation at 4 °C (10000 rpm, centrifugation time 10 min), washed 5 times with methanol to remove unreacted metal ions and organic ligands, and vacuum dried at 40 °C for 48 h to obtain ZIF-8 particle powder.

[0086] In one embodiment of the present invention, the preparation method of ZIF-90 particles includes the following steps: 220 mg of zinc acetate dihydrate is weighed and dissolved in 10 mL of N,N-dimethylformamide (DMF); 192 mg of imidazole-2-formaldehyde is weighed and dissolved in 10 mL of DMF; the above two solutions are mixed and stirred (500 rpm) for 5 min, and then, while stirring, an additional 50 mL of DMF is added to the reaction system, and stirring is continued for 1 h. After stirring is completed, the particle product is separated by centrifugation at 4 °C (10000 rpm, centrifugation time 10 min), washed 5 times with DMF to remove unreacted metal ions and organic ligands, and then washed 3 times with ethanol to remove residual DMF solvent. Finally, ZIF-90 particle powder is obtained by vacuum drying at 40 °C for 48 h.

[0087] In one embodiment of the present invention, the preparation method of MOF-5 particles includes the following steps: 5.1 g of terephthalic acid and 8.5 mL of triethylamine are dissolved in 400 mL of DMF to prepare an organic ligand solution; 17.0 g of zinc acetate dihydrate is dissolved in 500 mL of DMF to prepare a zinc salt solution. Under stirring, the zinc salt solution is added to the organic ligand solution, ensuring the addition is completed within 15 min, followed by continued stirring for 2.5 h. The product precipitate is obtained by filtration and soaked overnight in 250 mL of DMF. After filtration again, the product is transferred to 350 mL of chloroform (CHCl3) and soaked for 7 days, with the soaking solution CHCl3 being changed multiple times during the 7 days. After removing unreacted metal ions, organic ligands, and residual DMF solvent, MOF-5 particles are obtained by centrifugation at 4 °C (10000 rpm, centrifugation time 10 min). Finally, MOF-5 particle powder was obtained by vacuum drying at 40 °C for 48 h and then vacuum activation at 120 °C for 6 h.

[0088] In one embodiment of the present invention, the preparation method of MIL-53(Fe) particles includes the following steps: 0.27 g of ferric chloride hexahydrate (FeCl3·H2O) and 0.166 g of the organic ligand terephthalic acid (1,4-BDC) are added to 5 mL of deionized water, and the mixture is vigorously stirred at room temperature for about 20 min until a uniform suspension is formed; then the suspension is transferred to a stainless steel hydrothermal reactor with a polytetrafluoroethylene liner, sealed, and placed in an oven for constant temperature reaction at 150 °C for 24 h; after the reaction is completed and the reactor is naturally cooled, the crude product of MIL-53(Fe) particles is obtained by centrifugation at 4 °C (10000 rpm, centrifugation time 10 min). Subsequently, the crude product is washed five times each with deionized water, DMF, and anhydrous ethanol to thoroughly remove residual Fe. 3+ Unreacted ligands and DMF solvent were removed; finally, the product was placed in a vacuum oven and dried and activated at 120 °C for 12 h to obtain highly crystalline MIL-53(Fe) particle powder.

[0089] In one embodiment of the present invention, the preparation method of MIL-101(Fe) particles includes the following steps: 0.675 g FeCl3·6H2O and 0.206 g terephthalic acid are weighed into a beaker, and 30 mL DMF is added. The mixture is stirred at room temperature until it is uniformly dissolved. Then, it is transferred to a stainless steel hydrothermal reactor with a polytetrafluoroethylene liner and reacted at 110 °C for 20 h. After the reaction is complete, it is allowed to cool naturally to room temperature, and the crude product of MIL-101(Fe) particles is obtained by centrifugation at 4 °C (10000 rpm, centrifugation time 10 min). Subsequently, the sample is washed 5 times with DMF to remove unreacted metal ions and organic ligands, and then washed 3 times with ethanol to remove residual DMF solvent. Finally, the sample is vacuum dried at 40 °C for 48 h to obtain MIL-101(Fe) particle powder.

[0090] In one embodiment of the present invention, the preparation method of UiO-66(Zr) particles includes the following steps: 0.289 g of anhydrous zirconium tetrachloride (ZrCl4) and 0.206 g of terephthalic acid are weighed into a beaker, and 135.7 g of DMF is added. The mixture is stirred at room temperature until it is uniformly dissolved. Then, it is transferred to a stainless steel hydrothermal reactor with a polytetrafluoroethylene liner and reacted at 120 °C for 24 h. After the reaction is complete, it is allowed to cool naturally to room temperature, and the crude product of UiO-66(Zr) particles is obtained by centrifugation at 4 °C (10000 rpm, centrifugation time 10 min). Subsequently, the sample is washed 5 times with DMF to remove unreacted metal ions and organic ligands, and then washed 3 times with ethanol to remove residual DMF solvent. Finally, the sample is vacuum dried at 40 °C for 48 h to obtain UiO-66(Zr) particle powder.

[0091] In one embodiment of the present invention, the preparation method of MIL-156(Ca) particles includes the following steps: 0.376 g of gallic acid monohydrate is dispersed in 10 mL of distilled water at room temperature. Then, 0.236 g of calcium nitrate tetrahydrate is added, and 5 mol / L potassium hydroxide (KOH) aqueous solution is added dropwise to adjust the pH of the mixed solution to 8, followed by volume adjustment to 20 mL. The resulting reaction system is transferred to a polytetrafluoroethylene-lined high-pressure reactor and sealed. The reaction is carried out at 120 °C for 12 h, and the final product system has a pH of approximately 8. After the reaction is completed, the mixture is cooled to room temperature, filtered to obtain crude crystalline product of MIL-156(Ca) particles, washed repeatedly with distilled water three times, and vacuum dried at room temperature to obtain MIL-156(Ca) particle powder.

[0092] In one embodiment of the present invention, the preparation method of Mg-MOF particles includes the following steps: 0.2 g of anhydrous magnesium chloride and 0.79 g of gallic acid monohydrate are weighed and added to a round-bottom flask containing 10 mL of water, and the mixture is stirred under boiling and reflux conditions. Subsequently, the pH of the mixed reaction solution is adjusted to 8 using 10 mol / L KOH solution, and the resulting mixture is brought to a volume of 20 mL. The resulting reaction system is transferred to a polytetrafluoroethylene-lined high-pressure reactor and sealed, and heated at 140 °C for 24 h. After the reaction is completed, a light gray solid crude product is obtained by centrifugation (12000 rpm, 15 min, 4 °C), washed repeatedly with distilled water three times, and vacuum dried at room temperature to obtain Mg-MOF particle powder.

[0093] In one embodiment of the present invention, the preparation method of HKUST-1(Cu) particles includes the following steps: 0.435 g of copper nitrate trihydrate, 0.62 mL of acetic acid, 0.50 mL of triethylamine, and 12 mL of ethanol are mixed and stirred at room temperature. After 1 h, 0.210 g of trimesic acid is added to the above dark blue solution, and stirring is continued for 2 h to form a homogeneous solution. Subsequently, the reaction mixture is transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 85 °C for 24 h. After the reaction is completed, the crude product of HKUST-1(Cu) particles is collected by centrifugation (12000 rpm, 15 min, 4 °C) and washed three times with ethanol. The crude product is soaked in ethanol at 65 °C for 12 h and finally dried in a vacuum drying oven at 65 °C for 12 h to obtain HKUST-1(Cu) particle powder.

[0094] The pore structure characteristics of ZIF-8, MIL-101(Fe), UiO-66(Zr), Mg-MOF, and HKUST-1(Cu) particles prepared by the above method were characterized using nitrogen adsorption-desorption. Before testing, the MOF particles were placed in a vacuum degassing device and activated at 110 °C for 24 h to fully remove adsorbed solvent molecules and impurities from the pores. The activated MOF particles were then subjected to nitrogen adsorption-desorption tests at 77 K liquid nitrogen, and adsorption-desorption isotherms were obtained. The specific surface area was calculated using BET theory. The characterization results are as follows: Figure 2As shown, the MOF particles maintain a high specific surface area and typical micropore / mesopore distribution characteristics. This dense microstructure not only overcomes the barrier that traditional macroporous hydrogels cannot encapsulate lipophilic, poorly soluble drugs, but also endows the composite system with the ability to fundamentally inhibit the initial burst release of drugs through strong non-covalent interactions. The structural characterization in summary confirms that this invention overcomes the compatibility barrier and successfully constructs a multi-level porous system interwoven with macropores of a polymer cross-linked network and micropores / mesopores of MOF particles. This provides a solid material basis for subsequent broad-spectrum, high-load, and intelligent stepwise delivery in the digestive tract at both the microscopic morphology and thermodynamic levels.

[0095] This invention provides a method for preparing a silk fibroin-inulin-supported metal-organic framework composite hydrogel, comprising the following steps:

[0096] (1) Mix the silk fibroin aqueous solution with the inulin aqueous solution to obtain a mixed aqueous solution;

[0097] (2) Disperse the metal-organic framework particles in ethanol to obtain a metal-organic framework particle ethanol dispersion;

[0098] (3) The mixed aqueous solution is mixed with the ethanol dispersion of metal-organic framework particles and then allowed to stand. Ethanol is used to induce the conformational change of silk fibroin and promote the precipitation of inulin. The silk fibroin-inulin-loaded metal-organic framework composite hydrogel is obtained by gelation.

[0099] In one embodiment of the present invention, the preparation method of silk fibroin-inulin supported metal-organic framework composite hydrogel includes the following steps:

[0100] (a) Raw silk polymer peeling and degumming: Natural raw silk yarn is chopped and added to sodium carbonate aqueous solution (0.02mol / L) for high-temperature boiling degumming treatment for 20-80 min to peel off the outer sericin protein, and then washed and dried to obtain pure silk fibroin fiber.

[0101] (b) Hydrogen bond disruption and silk fibroin dissolution: The degummed silk fibroin fibers were added to a high-concentration lithium bromide aqueous solution (9.3 mol / L), with 1 g of silk fibroin fibers corresponding to 10 mL of lithium bromide solution. Under heating conditions of 60-80 °C, the strong disruptive effect of lithium bromide on intermolecular hydrogen bonds was utilized to carry out deep dissolution treatment for 4-8 h.

[0102] (c) Dialysis purification and salt removal: The solution is transferred into a dialysis bag (molecular weight cutoff of 3500-20000 Da), and non-high molecular weight impurities such as lithium bromide salts in the system are completely removed by gradient dialysis desalination in a large amount of deionized water for several days to obtain a purified and highly water-soluble SF aqueous solution.

[0103] (d) Reverse osmosis concentration: The SF aqueous solution in the dialysis bag is concentrated by external osmotic pressure suction at a low temperature of 4°C using a concentrated polyethylene glycol solution (Mw = 20000 Da, concentration of 20-50 wt%) to obtain a concentrated silk fibroin aqueous solution with a specific target mass concentration that does not undergo self-crosslinking.

[0104] (e) Preparation of binary aqueous precursor: The concentrated silk fibroin aqueous solution is thoroughly mixed with the inulin aqueous solution weighed according to the formula and heated to dissolve by mechanical stirring to obtain a highly homogeneous silk fibroin-inulin mixed aqueous solution. The temperature for heating and dissolving the inulin aqueous solution is 60-85 ℃, and the inulin dissolution time is 30-120 min.

[0105] (f) Phase transition induced gelation: The mixed aqueous solution and the ultrasonically homogenized MOF particle ethanol solution were rapidly vortex-mixed according to the specified ratio. After a short period of standing at a temperature of 4-60 °C, the mixture underwent a rapid gelation solid-phase transition triggered by the dual mechanisms of a sudden drop in ethanol polarity and conformational excitation, resulting in a silk fibroin-inulin-supported metal-organic framework composite hydrogel.

[0106] This invention provides an application of silk fibroin-inulin-loaded metal-organic framework composite hydrogel in the preparation of drug delivery systems.

[0107] To address the technical problem that traditional hydrogels are difficult to load hydrophobic drugs, this invention provides a segmented drug loading process of "pre-adsorption and encapsulation - post-mediated gelation", which is particularly suitable for lipophilic drugs.

[0108] In one embodiment of the present invention, the preparation method of the silk fibroin-inulin-loaded metal-organic framework composite hydrogel loaded with a lipophilic drug includes the following steps:

[0109] (i) Organic phase porous enrichment: Aqueous phase drug loading process is abandoned. High specific surface area and hydrophobic pores of MOF particles are used to disperse them in an organic solvent that is extremely immiscible or poorly immiscible with water and stirred and incubated. Lipophilic drugs are efficiently adsorbed, deeply enriched and tightly encapsulated into the micropores and mesopores of MOFs by relying on physicochemical mechanisms such as capillary force, van der Waals force and π-π conjugation.

[0110] (ii) Solvent replacement and alcohol phase dispersion: After removing excess free drug and thoroughly evaporating and drying the highly toxic organic solvent by centrifugation, the drug-loaded MOF particles are ultrasonically dispersed in low-toxicity anhydrous ethanol to obtain a uniform and stable drug-loaded MOF particle ethanol dispersion.

[0111] (iii) Macromolecular encapsulation and macroscopic shaping: Using the ethanol dispersion of drug-loaded MOF particles as a crosslinking trigger, it is rapidly mixed with a silk fibroin-inulin mixed aqueous solution. The system gels rapidly, and the microscopic drug-loaded MOF particles are completely encapsulated in the silk fibroin-inulin dual network framework to obtain the silk fibroin-inulin loaded metal-organic framework composite hydrogel loaded with the lipophilic drug.

[0112] The preparation method provided by this invention achieves "functional partitioning and isolation" of the drug carrier system, overcoming the inherent limitation that hydrophilic gels cannot accommodate hydrophobic drugs: it relies on the porous framework of MOFs to achieve efficient loading of hydrophobic drugs, and relies on the silk fibroin-inulin dual network to construct a stable protective outer layer framework. Even poorly soluble hydrophobic drugs that are difficult to dissolve with conventional alcohol co-solvents (PEG400, propylene glycol) can be prepared into hydrogel formulations with uniform drug loading and suitable for oral administration through this process.

[0113] A schematic diagram of the preparation process of the silk fibroin-inulin-supported metal-organic framework composite hydrogel provided by this invention and a schematic diagram of its application in an oral drug delivery system are shown below. Figure 1 As shown, this invention rapidly gels three raw materials—silk fibroin aqueous solution, inulin aqueous solution, and MOF particle ethanol dispersion—after mixing them using a vortex mixer. The accompanying enlarged image, using HKUST-1(Cu) as an example, illustrates the morphological differences between the solution state before mixing and the gel state after gel formation. The composite hydrogel possesses a macroporous structure formed by the cross-linked network of silk fibroin and inulin, a mesoporous structure formed by the interstices between MOF particles, and a microporous structure of the intrinsic crystal lattice of MOF particles. It can encapsulate drugs through "pre-adsorption," achieving the encapsulation of hydrophilic drugs, lipophilic drugs, protein drugs, nucleic acid drugs, and other drugs, exhibiting broad-spectrum drug loading capacity. After oral administration, the drug-loaded MOF composite hydrogel enters the human digestive tract. Upon reaching the stomach, its dense gel network structure blocks the penetration of hydrogen ions and pepsin, resisting gastric acid erosion and inhibiting drug burst release. In the small intestine, silk fibroin is slowly degraded by pancreatic enzymes, loosening the gel network and forming diffusion channels for sustained drug release. Upon reaching the colon, intestinal flora specifically degrades inulin, causing the gel network to disintegrate and fully exposing the drug-loaded MOF particles. The drug is then released rapidly and concentrated at the colonic lesion site. Inulin, as a prebiotic, can be fermented by intestinal flora in the colon to produce short-chain fatty acids, regulating intestinal flora structure and modulating the body's generation of memory T cells, reshaping the intestinal microenvironment immune system, and exerting a synergistic immunotherapeutic effect with anti-inflammatory and anti-tumor properties. Figure 1 This intuitively demonstrates the multiple innovative advantages of the present invention, including simple preparation of MOFs composite hydrogels, wide drug loading range, gastric protection, precise colonic delivery, and synergistic immune enhancement.

[0114] 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.

[0115] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0116] In the following examples, long-chain inulin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number: L709028-100g; short-chain inulin was purchased from Nanjing Dulai Biotechnology Co., Ltd., catalog number: C0154-10445; pepsin was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd. (TCI), catalog number: P0103, CAS number: 9001-75-6; and trypsin was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., catalog number: P0636, CAS No.: 8049-47-6; Inulinase purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number: S10241-100g, CAS No.: 9025-67-6; Artificial gastric fluid purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number: R29313-500ml; Artificial small intestinal fluid purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number: A1509631-500ml; Artificial colon fluid Purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number R29315-500ml; Curcumin purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number C400271-100g, CAS number: 458-37-7; Rifaximin purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S80963-25g, CAS number: 80621-81-4; Vancomycin purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number Y25829-5. g, CAS No.: 1404-90-6; uricase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S29799-2KU, CAS No.: 9002-12-4; sulfonyl Cy5 fluorescent dye (Sulfo-Cy5) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: S276215-5mg, CAS No.: 2230212-27-6; all MOFs used in the following examples were synthesized and prepared by the present invention.

[0117] Example 1

[0118] A method for preparing an aqueous solution of silk fibroin includes the following steps:

[0119] S1. Degumming treatment is performed by boiling the shredded raw silk yarn in a 0.02 mol / L sodium carbonate solution for 40 min, wherein the mass-volume ratio of raw silk yarn to sodium carbonate aqueous solution is 1 g : 10 mL. This degumming treatment is repeated 3 times. Then, the silk fibers are thoroughly washed with deionized water and dried at 50 °C.

[0120] S2. Dissolve the degummed silk fibroin in a 9.3 mol / L lithium bromide solution (60 °C, heated for 4 h), wherein the mass-to-volume ratio of silk fibroin to lithium bromide aqueous solution is 1 g : 10 mL. After cooling, dialyze in pure water for 3 days (dialysis bag molecular weight cutoff 7000 Da) to remove salt and other impurities, and obtain purified silk fibroin aqueous solution.

[0121] S3. The purified silk fibroin solution was concentrated with a concentrated polyethylene glycol solution at 4 °C (polyethylene glycol concentration 40 wt%, weight average molecular weight 20000 Da, linear polyethylene glycol). The mass ratio of the purified silk fibroin solution to the concentrated polyethylene glycol solution was 1:10 (w / w), and the concentration time was 8 h, resulting in a silk fibroin aqueous solution with a mass concentration of 11.3 wt%.

[0122] The concentration of the silk fibroin aqueous solution was determined using the drying-to-constant-weight method: an appropriate amount of silk fibroin aqueous solution was placed in a glass petri dish and dried to constant weight in a constant-temperature drying oven at 105 ℃. The dry weight of the silk fibroin was weighed, and the mass concentration of the solution was calculated by the ratio of the dry weight to the initial solution mass. In this invention, an 11.3 wt% silk fibroin aqueous solution was used as the stock solution. By adding deionized water, silk fibroin aqueous solutions with any mass concentration in the range of 1-10 wt% could be prepared. After preparation, the solutions were refrigerated at 4 ℃ for later use.

[0123] Example 2

[0124] A method for preparing an aqueous solution of long-chain inulin includes the following steps:

[0125] Weigh 40.0 g of long-chain inulin (GPC test results: weight average molecular weight Mw = 7733 Da, polydispersity index PDI = 2.07), and slowly pour it into 60 mL of continuously stirred deionized water in powder form to ensure complete dispersion. While stirring, heat the inulin solution to 85 °C in a water bath and maintain this temperature for 4 h to completely dissolve the inulin, resulting in a homogeneous, milky-white aqueous solution of long-chain inulin with a mass concentration of 40 wt%.

[0126] Example 3-22

[0127] The 40 wt% long-chain inulin (INU) aqueous solution of Example 2 was immediately mixed with the different saturated silk fibroin (SF) aqueous solutions of Example 1 in a specific ratio to obtain a mixed aqueous solution, which was then placed in a constant temperature incubator at 60 °C for later use.

[0128] The feeding table for preparing the mixed aqueous solution in Example 3-22 is shown in Table 1:

[0129] Table 1

[0130]

[0131] Examples 23-27

[0132] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0133] Weigh a certain amount of ZIF-8 particle powder and disperse it evenly in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution of a specific concentration. Quickly add the ZIF-8 particle ethanol solution to the mixed aqueous solution prepared in Example 5, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0134] Examples 28-32

[0135] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0136] Weigh a certain mass of ZIF-8 particle powder and disperse it evenly in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution of a specific concentration. Quickly add the ZIF-8 particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0137] Examples 33-37

[0138] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0139] Weigh a certain amount of ZIF-8 particle powder and disperse it evenly in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution of a specific concentration. Quickly add the ZIF-8 particle ethanol solution to the mixed aqueous solution prepared in Example 15, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0140] Examples 38-42

[0141] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0142] Weigh a certain amount of ZIF-8 particle powder and disperse it evenly in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution of a specific concentration. Quickly add the ZIF-8 particle ethanol solution to the mixed aqueous solution prepared in Example 20, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0143] The feed table for the preparation of silk fibroin-inulin-supported ZIF-8 composite hydrogels in Examples 23-42 is shown in Table 2:

[0144] Table 2

[0145]

[0146] Examples 43-47

[0147] A method for preparing a silk fibroin-inulin-loaded ZIF-90 composite hydrogel includes the following steps:

[0148] Weigh a certain amount of ZIF-90 particle powder and disperse it evenly in anhydrous ethanol to prepare a ZIF-90 particle ethanol solution of a specific concentration. Quickly add the ZIF-90 particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-90 composite hydrogel.

[0149] The feed table for the preparation of silk fibroin-inulin-loaded ZIF-90 composite hydrogels in Examples 43-47 is shown in Table 3:

[0150] Table 3

[0151]

[0152] Examples 48-52

[0153] A method for preparing a silk fibroin-inulin-loaded MOF-5 composite hydrogel includes the following steps:

[0154] Weigh a certain mass of MOF-5 particle powder and disperse it evenly in anhydrous ethanol to prepare a MOF-5 particle ethanol solution of a specific concentration. Quickly add the MOF-5 particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded MOF-5 composite hydrogel.

[0155] The feed table for the preparation of silk fibroin-inulin-supported MOF-5 composite hydrogels in Examples 48-52 is shown in Table 4:

[0156] Table 4

[0157]

[0158] Examples 53-57

[0159] A method for preparing a silk fibroin-inulin-loaded MIL-53(Fe) composite hydrogel includes the following steps:

[0160] A certain mass of MIL-53(Fe) particle powder was weighed and uniformly dispersed in anhydrous ethanol to prepare a MIL-53(Fe) particle ethanol solution of a specific concentration. The MIL-53(Fe) particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded MIL-53(Fe) composite hydrogel.

[0161] The feed table for the preparation of silk fibroin-inulin-supported MIL-53(Fe) composite hydrogels in Examples 53-57 is shown in Table 5:

[0162] Table 5

[0163]

[0164] Examples 58-62

[0165] A method for preparing a silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel includes the following steps:

[0166] A certain mass of MIL-101(Fe) particle powder was weighed and uniformly dispersed in anhydrous ethanol to prepare a MIL-101(Fe) particle ethanol solution of a specific concentration. The MIL-101(Fe) particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 25 °C for 4 min to obtain silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel.

[0167] The feed table for the preparation of silk fibroin-inulin-supported MIL-101(Fe) composite hydrogels in Examples 58-62 is shown in Table 6:

[0168] Table 6

[0169]

[0170] Examples 63-67

[0171] A method for preparing a silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel includes the following steps:

[0172] A certain mass of UiO-66(Zr) particle powder was weighed and uniformly dispersed in anhydrous ethanol to prepare a UiO-66(Zr) particle ethanol solution of a specific concentration. The UiO-66(Zr) particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel.

[0173] The feed table for the preparation of silk fibroin-inulin-supported UiO-66(Zr) composite hydrogels in Examples 63-67 is shown in Table 7:

[0174] Table 7

[0175]

[0176] Examples 68-72

[0177] A method for preparing a silk fibroin-inulin-loaded MIL-156(Ca) composite hydrogel includes the following steps:

[0178] A certain mass of MIL-156(Ca) particle powder was weighed and uniformly dispersed in anhydrous ethanol to prepare a MIL-156(Ca) particle ethanol solution of a specific concentration. The MIL-156(Ca) particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded MIL-156(Ca) composite hydrogel.

[0179] The feed table for the preparation of silk fibroin-inulin-loaded MIL-156(Ca) composite hydrogels in Examples 68-72 is shown in Table 8:

[0180] Table 8

[0181]

[0182] Examples 73-77

[0183] A method for preparing a silk fibroin-inulin-loaded Mg-MOF composite hydrogel includes the following steps:

[0184] Weigh a certain mass of Mg-MOF particle powder and disperse it uniformly in anhydrous ethanol to prepare a Mg-MOF particle ethanol solution of a specific concentration. Quickly add the Mg-MOF particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain a silk fibroin-inulin-loaded Mg-MOF composite hydrogel.

[0185] The feed table for the preparation of silk fibroin-inulin-supported Mg-MOF composite hydrogels in Examples 73-77 is shown in Table 9:

[0186] Table 9

[0187]

[0188] Examples 78-82

[0189] A method for preparing a silk fibroin-inulin-loaded HKUST-1(Cu) composite hydrogel includes the following steps:

[0190] Weigh a certain amount of HKUST-1(Cu) granules and disperse them evenly in anhydrous ethanol to prepare an HKUST-1(Cu) granule ethanol solution of a specific concentration. Quickly add the HKUST-1(Cu) granule ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let it stand at 25 °C for 4 min to obtain silk fibroin-inulin-loaded HKUST-1(Cu) composite hydrogel.

[0191] The feed table for the preparation of silk fibroin-inulin-supported HKUST-1(Cu) composite hydrogels in Examples 78-82 is shown in Table 10:

[0192] Table 10

[0193]

[0194] Example 83

[0195] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0196] ZIF-8 particles were uniformly dispersed in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution with a concentration of 10.0 mg / g. The ZIF-8 particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 4 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0197] Example 84

[0198] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0199] ZIF-8 particles were uniformly dispersed in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution with a concentration of 10.0 mg / g. The ZIF-8 particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 37 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0200] Example 85

[0201] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel includes the following steps:

[0202] ZIF-8 particles were uniformly dispersed in anhydrous ethanol to prepare a ZIF-8 particle ethanol solution with a concentration of 10.0 mg / g. The ZIF-8 particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the solution was allowed to stand at 50 °C for 4 min to obtain a silk fibroin-inulin-loaded ZIF-8 composite hydrogel.

[0203] Example 86

[0204] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel loaded with curcumin (CUR) includes the following steps:

[0205] P1. ZIF-8 granules were uniformly dispersed in chloroform to prepare a ZIF-8 granule chloroform solution with a concentration of 2.0 mg / g; CUR was dispersed and dissolved in chloroform to prepare a CUR chloroform solution with a concentration of 2.0 mg / g. The ZIF-8 granule chloroform solution and the CUR chloroform solution were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0206] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain CUR-loaded ZIF-8 particles (denoted as CUR@ZIF-8). The supernatant obtained from centrifugation was collected, and the mass of free CUR contained in it was tested to calculate the CUR loading capacity and loading efficiency of ZIF-8 particles. The collected CUR@ZIF-8 particles were dispersed in a small amount of water and freeze-dried to obtain CUR@ZIF-8 particle powder. The CUR@ZIF-8 particle powder was uniformly dispersed in ethanol to obtain a CUR@ZIF-8 particle ethanol solution with a concentration of 10 mg / g.

[0207] P3. Quickly add the CUR@ZIF-8 particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded ZIF-8 composite hydrogel with CUR encapsulated.

[0208] Example 87

[0209] A method for preparing a silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel with CUR encapsulated includes the following steps:

[0210] P1. Disperse MIL-101(Fe) granules uniformly in chloroform to prepare a chloroform solution with a concentration of 2.0 mg / g. Disperse and dissolve CUR in chloroform to prepare a chloroform solution with a concentration of 2.0 mg / g. Mix the MIL-101(Fe) granule chloroform solution and the CUR chloroform solution at a mass ratio of 1:1 and incubate in a constant temperature shaking oven at 80 rpm and 25 °C for 12 h in the dark.

[0211] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain CUR-loaded MIL-101(Fe) particles (denoted as CUR@MIL-101(Fe)). The supernatant obtained from centrifugation was collected, and the mass of free CUR contained in it was tested to calculate the drug loading capacity and drug loading efficiency of MIL-101(Fe) particles for CUR. The collected CUR@MIL-101(Fe) particles were dispersed in a small amount of water and freeze-dried to obtain CUR@MIL-101(Fe) particle powder; the CUR@MIL-101(Fe) particle powder was uniformly dispersed in ethanol to obtain a CUR@MIL-101(Fe) particle ethanol solution with a concentration of 10 mg / g.

[0212] P3. Quickly add the CUR@MIL-101(Fe) particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel with CUR encapsulated.

[0213] Example 88

[0214] A method for preparing a silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with CUR includes the following steps:

[0215] P1. UiO-66(Zr) granules were uniformly dispersed in chloroform to prepare a chloroform solution with a concentration of 2.0 mg / g. CUR was dispersed and dissolved in chloroform to prepare a chloroform solution with a concentration of 2.0 mg / g. The chloroform solutions of UiO-66(Zr) granules and CUR were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0216] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain CUR-loaded UiO-66(Zr) particles (denoted as CUR@UiO-66(Zr)). The supernatant obtained from centrifugation was collected, and the mass of free CUR contained in it was tested to calculate the CUR loading capacity and loading efficiency of UiO-66(Zr) particles. The collected CUR@UiO-66(Zr) particles were dispersed in a small amount of water and freeze-dried to obtain CUR@UiO-66(Zr) particle powder. The CUR@UiO-66(Zr) particle powder was uniformly dispersed in ethanol to obtain a CUR@UiO-66(Zr) particle ethanol solution with a concentration of 10 mg / g.

[0217] P3. Quickly add the CUR@UiO-66(Zr) particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with CUR.

[0218] Example 89

[0219] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel loaded with rifaximin (RFX) includes the following steps:

[0220] P1. ZIF-8 granules were uniformly dispersed in chloroform to prepare a ZIF-8 granule chloroform solution with a concentration of 2.0 mg / g; RFX was dispersed and dissolved in chloroform to prepare an RFX chloroform solution with a concentration of 1.0 mg / g. The ZIF-8 granule chloroform solution and the RFX chloroform solution were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0221] P2. The incubated mixture was centrifuged at 4 °C (10000 rpm, 5 min) to obtain RFX-loaded ZIF-8 particles (denoted as RFX@ZIF-8). The supernatant obtained from centrifugation was collected, and the mass of free RFX contained in it was tested to calculate the drug loading capacity and efficiency of the ZIF-8 particles for RFX. The collected RFX@ZIF-8 particles were dispersed in a small amount of water and freeze-dried to obtain RFX@ZIF-8 particle powder. The RFX@ZIF-8 particle powder was uniformly dispersed in ethanol to obtain an ethanol solution of RFX@ZIF-8 particles with a concentration of 10 mg / g.

[0222] P3. The RFX@ZIF-8 particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded ZIF-8 composite hydrogel with RFX encapsulation.

[0223] Example 90

[0224] A method for preparing an RFX-encapsulated silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel includes the following steps:

[0225] P1. Disperse MIL-101(Fe) granules uniformly in chloroform to prepare a chloroform solution with a concentration of 2.0 mg / g. Disperse and dissolve RFX in chloroform to prepare a chloroform solution with a concentration of 1.0 mg / g. Mix the chloroform solution of MIL-101(Fe) granules and the chloroform solution of RFX at a mass ratio of 1:1 and incubate in a constant temperature shaking oven at 80 rpm and 25 °C for 12 h in the dark.

[0226] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain MIL-101(Fe) particles loaded with RFX (denoted as RFX@MIL-101(Fe)). The supernatant obtained from centrifugation was collected, and the mass of free RFX contained in it was tested to calculate the drug loading capacity and drug loading efficiency of MIL-101(Fe) particles for RFX. The collected RFX@MIL-101(Fe) particles were dispersed in a small amount of water and freeze-dried to obtain RFX@MIL-101(Fe) particle powder. The RFX@MIL-101(Fe) particle powder was uniformly dispersed in ethanol to obtain an ethanol solution of RFX@MIL-101(Fe) particles with a concentration of 10 mg / g.

[0227] P3. The ethanol solution of RFX@MIL-101(Fe) particles was rapidly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with RFX.

[0228] Example 91

[0229] A method for preparing an RFX-encapsulated silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel includes the following steps:

[0230] P1. UiO-66(Zr) granules were uniformly dispersed in chloroform to prepare a chloroform solution with a concentration of 2.0 mg / g. RFX was dispersed and dissolved in chloroform to prepare a chloroform solution with a concentration of 1.0 mg / g. The chloroform solutions of UiO-66(Zr) granules and RFX were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0231] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain RFX-loaded UiO-66(Zr) particles (denoted as RFX@UiO-66(Zr)). The supernatant obtained from centrifugation was collected, and the mass of free RFX contained in it was tested to calculate the drug loading capacity and drug loading efficiency of UiO-66(Zr) particles for RFX. The collected RFX@UiO-66(Zr) particles were dispersed in a small amount of water and freeze-dried to obtain RFX@UiO-66(Zr) particle powder. The RFX@UiO-66(Zr) particle powder was uniformly dispersed in ethanol to obtain an ethanol solution of RFX@UiO-66(Zr) particles with a concentration of 10 mg / g.

[0232] P3. The ethanol solution of RFX@UiO-66(Zr) particles was rapidly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with RFX.

[0233] Example 92

[0234] A method for preparing a vancomycin (VCN)-loaded silk fibroin-inulin-supported ZIF-8 composite hydrogel includes the following steps:

[0235] P1. Disperse ZIF-8 granules uniformly in water to prepare a ZIF-8 granule aqueous solution with a concentration of 2.0 mg / g; disperse and dissolve VCN in water to prepare a VCN aqueous solution with a concentration of 1.0 mg / g. Mix the ZIF-8 granule aqueous solution and the VCN aqueous solution at a mass ratio of 1:1 and incubate in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0236] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain VCN-loaded ZIF-8 particles (denoted as VCN@ZIF-8). The supernatant obtained from centrifugation was collected, and the mass of free VCN contained in it was tested to calculate the drug loading capacity and efficiency of the ZIF-8 particles for VCN. The collected VCN@ZIF-8 particles were washed with a small amount of ethanol and dried in a vacuum drying oven at 45 °C for 4 h to obtain VCN@ZIF-8 particle powder. The VCN@ZIF-8 particle powder was uniformly dispersed in ethanol to obtain a VCN@ZIF-8 particle ethanol solution with a concentration of 10 mg / g.

[0237] P3. The VCN@ZIF-8 particle ethanol solution was quickly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded ZIF-8 composite hydrogel with VCN.

[0238] Example 93

[0239] A method for preparing a silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with VCN includes the following steps:

[0240] P1. Disperse MIL-101(Fe) granules uniformly in water to prepare an aqueous solution with a concentration of 2.0 mg / g; disperse and dissolve VCN in water to prepare an aqueous solution with a concentration of 1.0 mg / g. Mix the MIL-101(Fe) granule aqueous solution and the VCN aqueous solution at a mass ratio of 1:1 and incubate in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0241] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain MIL-101(Fe) particles loaded with VCN (denoted as VCN@MIL-101(Fe)). The supernatant obtained from centrifugation was collected, and the mass of free VCN contained in it was tested to calculate the drug loading capacity and efficiency of MIL-101(Fe) particles for VCN. The collected VCN@MIL-101(Fe) particles were washed with a small amount of ethanol and dried in a vacuum drying oven at 45 °C for 4 h to obtain VCN@MIL-101(Fe) particle powder. The VCN@MIL-101(Fe) particle powder was uniformly dispersed in ethanol to obtain a VCN@MIL-101(Fe) particle ethanol solution with a concentration of 10 mg / g.

[0242] P3. The ethanol solution of VCN@MIL-101(Fe) particles was rapidly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel with VCN encapsulation.

[0243] Example 94

[0244] A method for preparing a silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with VCN includes the following steps:

[0245] P1. UiO-66(Zr) granules were uniformly dispersed in water to prepare an aqueous solution with a concentration of 2.0 mg / g. VCN was dispersed and dissolved in water to prepare an aqueous solution with a concentration of 1.0 mg / g. The aqueous solutions of UiO-66(Zr) granules and VCN were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0246] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain VCN-loaded UiO-66(Zr) particles (denoted as VCN@UiO-66(Zr)). The supernatant obtained from centrifugation was collected, and the mass of free VCN it contained was tested to calculate the VCN loading capacity and loading efficiency of UiO-66(Zr) particles. The collected VCN@UiO-66(Zr) particles were washed with a small amount of ethanol and dried in a vacuum drying oven at 45 °C for 4 h to obtain VCN@UiO-66(Zr) particle powder. The VCN@UiO-66(Zr) particle powder was uniformly dispersed in ethanol to obtain a VCN@UiO-66(Zr) particle ethanol solution with a concentration of 10 mg / g.

[0247] P3. The ethanol solution of VCN@UiO-66(Zr) particles was rapidly added to the mixed aqueous solution prepared in Example 10, and the mass ratio of the two solutions was 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with VCN.

[0248] Example 95

[0249] A method for preparing a silk fibroin-inulin-loaded ZIF-8 composite hydrogel containing Cy5-labeled uricase (Cy5-URC) includes the following steps:

[0250] P1. URC was fluorescently labeled with sulfonyl Cy5 fluorescent dye (Sulfo-Cy5). Sulfonyl Cy5-NHS dye was added to the URC aqueous solution to a concentration of 0.1% of the URC concentration. The mixture was stirred in the dark for 4 h. After the reaction, the resulting Cy5-labeled uricase solution was purified by dialysis to remove unlabeled free fluorescent molecules. The solution was then freeze-dried to obtain a Cy5-labeled uricase powder sample, designated Cy5-URC. ZIF-8 granules were uniformly dispersed in water to prepare a 2.0 mg / g ZIF-8 granule aqueous solution. Cy5-URC was dispersed and dissolved in water to prepare a 2.0 mg / g Cy5-URC aqueous solution. The ZIF-8 granule aqueous solution and the Cy5-URC aqueous solution were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 °C for 12 h in the dark.

[0251] P2. The incubated mixture was centrifuged at 4 °C (10000 rpm, 5 min) to obtain ZIF-8 particles loaded with Cy5-URC (denoted as Cy5-URC@ZIF-8). The supernatant obtained from centrifugation was collected, and the mass of free Cy5-URC it contained was tested to calculate the drug loading capacity and efficiency of ZIF-8 particles for Cy5-URC. The collected Cy5-URC@ZIF-8 particles were dispersed in a small amount of water and freeze-dried to obtain Cy5-URC@ZIF-8 particle powder. The Cy5-URC@ZIF-8 particle powder was uniformly dispersed in ethanol to obtain an ethanol solution of Cy5-URC@ZIF-8 particles with a concentration of 10 mg / g.

[0252] P3. Quickly add the Cy5-URC@ZIF-8 particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded ZIF-8 composite hydrogel loaded with Cy5-URC.

[0253] Example 96

[0254] A method for preparing a silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC includes the following steps:

[0255] P1. URC was fluorescently labeled with sulfonyl Cy5 fluorescent dye (Sulfo-Cy5). Sulfonyl Cy5-NHS dye was added to an aqueous solution of URC, and its concentration was increased to 0.1% of the URC concentration. The mixture was stirred in the dark for 4 h. After the reaction was complete, the resulting Cy5-labeled uricase solution was purified by dialysis to remove unlabeled free fluorescent molecules. The solution was then freeze-dried to obtain a Cy5-labeled uricase powder sample, designated Cy5-URC. MIL-101(Fe) granules were uniformly dispersed in water to prepare an aqueous solution with a concentration of 2.0 mg / g. Cy5-URC was dispersed and dissolved in water to prepare an aqueous solution with a concentration of 2.0 mg / g. The aqueous solution of MIL-101(Fe) particles and the aqueous solution of Cy5-URC were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0256] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain MIL-101(Fe) particles loaded with Cy5-URC (denoted as Cy5-URC@MIL-101(Fe)). The supernatant obtained from centrifugation was collected, and the mass of free Cy5-URC contained in it was tested to calculate the drug loading capacity and drug loading efficiency of MIL-101(Fe) particles for Cy5-URC. The collected Cy5-URC@MIL-101(Fe) particles were dispersed in a small amount of water and freeze-dried to obtain Cy5-URC@MIL-101(Fe) particle powder. The Cy5-URC@MIL-101(Fe) particle powder was uniformly dispersed in ethanol to obtain an ethanol solution of Cy5-URC@MIL-101(Fe) particles with a concentration of 10 mg / g.

[0257] P3. Quickly add the Cy5-URC@MIL-101(Fe) particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC.

[0258] Example 97

[0259] A method for preparing a silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with Cy5-URC includes the following steps:

[0260] P1. URC was fluorescently labeled with sulfonyl Cy5 fluorescent dye (Sulfo-Cy5). Sulfonyl Cy5-NHS dye was added to an aqueous solution of URC, and its concentration was increased to 0.1% of the URC concentration. The mixture was stirred in the dark for 4 h. After the reaction was complete, the resulting Cy5-labeled uricase solution was purified by dialysis to remove unlabeled free fluorescent molecules. The solution was then freeze-dried to obtain a Cy5-labeled uricase powder sample, designated Cy5-URC. UiO-66(Zr) particles were uniformly dispersed in water to prepare an aqueous solution with a concentration of 2.0 mg / g. Cy5-URC was dispersed and dissolved in water to prepare an aqueous solution with a concentration of 2.0 mg / g. The aqueous solution of UiO-66(Zr) particles and the aqueous solution of Cy5-URC were mixed at a mass ratio of 1:1 and incubated in a constant temperature shaking oven at 80 rpm and 25 ℃ for 12 h in the dark.

[0261] P2. The incubated mixed solution was centrifuged at 4 °C (10000 rpm, 5 min) to obtain UiO-66(Zr) particles loaded with Cy5-URC (denoted as Cy5-URC@UiO-66(Zr)). The supernatant obtained from centrifugation was collected, and the mass of free Cy5-URC contained in it was tested to calculate the drug loading capacity and drug loading efficiency of UiO-66(Zr) particles for Cy5-URC. The collected Cy5-URC@UiO-66(Zr) particles were dispersed in a small amount of water and freeze-dried to obtain Cy5-URC@UiO-66(Zr) particle powder; the Cy5-URC@UiO-66(Zr) particle powder was uniformly dispersed in ethanol to obtain an ethanol solution of Cy5-URC@UiO-66(Zr) particles with a concentration of 10 mg / g.

[0262] P3. Quickly add the Cy5-URC@UiO-66(Zr) particle ethanol solution to the mixed aqueous solution prepared in Example 10, with the mass ratio of the two solutions being 1:1. After thorough mixing, let stand at 25 °C for 4 min to obtain the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel loaded with Cy5-URC.

[0263] Comparative Examples 1-5

[0264] A method for preparing an inulin-based hydrogel includes the following steps:

[0265] In Example 2, a 40 wt% INU aqueous solution was mixed with deionized water at 60 °C in a specific mass ratio to prepare INU aqueous solutions of different mass concentrations. Subsequently, the INU aqueous solutions were allowed to stand at 25 °C for 24 h and then allowed to cool naturally to slowly form inulin-based hydrogels through physical cross-linking.

[0266] The feed table for preparing inulin-based hydrogels in Comparative Examples 1-5 is shown in Table 11:

[0267] Table 11

[0268]

[0269] Comparative Examples 6-8

[0270] A method for preparing an inulin-based hydrogel includes the following steps:

[0271] The 40 wt% INU aqueous solution from Example 2 was mixed with deionized water at 60 °C in a specific mass ratio to prepare INU aqueous solutions of different mass concentrations. Then, a certain mass of anhydrous ethanol (25 °C) was added and mixed thoroughly. The mixture was allowed to stand at 25 °C for 10 min to obtain inulin-based hydrogels that were gelled by ethanol induction.

[0272] The feed table for preparing inulin-based hydrogels in Comparative Examples 6-8 is shown in Table 12:

[0273] Table 12

[0274]

[0275] Comparative Examples 9-11

[0276] A method for preparing a silk fibroin-based hydrogel includes the following steps:

[0277] The 6 wt% SF aqueous solution from Example 1 was mixed with deionized water in a specific mass ratio to prepare SF aqueous solutions of different mass concentrations. Then, a certain mass of anhydrous ethanol was added and mixed thoroughly. The mixture was allowed to stand at 25 °C for 4 min to obtain silk fibroin-based hydrogels that were gelled by ethanol induction.

[0278] The feed table for the preparation of silk fibroin-based hydrogels in Comparative Examples 9-11 is shown in Table 13:

[0279] Table 13

[0280]

[0281] Comparative Example 12

[0282] A method for preparing a silk fibroin-inulin-based composite hydrogel includes the following steps:

[0283] Deionized water was rapidly added to the mixed aqueous solution prepared in Example 10, with a mass ratio of 1:1 between the two solutions. After thorough mixing, the mixture was allowed to stand at 25 °C for 24 h and then allowed to cool naturally to slowly form a silk fibroin-inulin-based composite hydrogel through physical cross-linking.

[0284] Comparative Example 13

[0285] A method for preparing a silk fibroin-inulin-based composite hydrogel includes the following steps:

[0286] Anhydrous ethanol was rapidly added to the mixed aqueous solution prepared in Example 10, with a mass ratio of 1:1 between the two solutions. After thorough mixing, the resulting mixed solution was allowed to stand at 25 °C for 4 min to obtain a silk fibroin-inulin-based composite hydrogel induced by ethanol gelation.

[0287] Comparative Example 14

[0288] A method for preparing a silk fibroin-inulin-based composite hydrogel loaded with Cy5-URC includes the following steps:

[0289] Cy5-URC was dissolved in the mixed aqueous solution prepared in Example 10 to prepare a drug solution with a concentration of 0.08 mg / g. Anhydrous ethanol was rapidly added to the drug solution at a mass ratio of 1:1. After thorough mixing, the solution was allowed to stand at 25 °C for 4 min to finally obtain a silk fibroin-inulin-based composite hydrogel loaded with Cy5-URC that underwent ethanol-induced gelation.

[0290] Comparative Example 15

[0291] A method for preparing a silk fibroin-based hydrogel loaded with Cy5-URC includes the following steps:

[0292] Cy5-URC was dissolved in a 2 wt% SF aqueous solution from Example 1 to prepare a drug solution with a concentration of 0.08 mg / g. Anhydrous ethanol was rapidly added to the drug solution at a mass ratio of 1:1. After thorough mixing, the solution was allowed to stand at 25 °C for 4 min to obtain a silk fibroin-based hydrogel containing Cy5-URC that underwent ethanol-induced gelation.

[0293] Comparative Example 16

[0294] A method for preparing an inulin-based hydrogel loaded with Cy5-URC includes the following steps:

[0295] The 40 wt% INU aqueous solution from Example 2 was mixed with pure water at a 1:1 mass ratio to obtain a 20 wt% INU aqueous solution. Cy5-URC was then dissolved in this 20 wt% INU aqueous solution to prepare a drug solution with a concentration of 0.08 mg / g. Anhydrous ethanol was rapidly added to the drug solution at a 1:1 mass ratio, and after thorough mixing, the solution was allowed to stand at 25 °C for 10 min to finally obtain an inulin-based hydrogel containing Cy5-URC that underwent ethanol-induced gelation.

[0296] Comparative Example 17

[0297] A method for preparing a silk fibroin-inulin-based composite hydrogel includes the following steps:

[0298] Weigh 40.0 g of short-chain inulin (GPC test results: weight average molecular weight Mw = 1063 Da, polydispersity index PDI = 2.74), and slowly pour it into 60 mL of continuously stirred deionized water in powder form to ensure complete dispersion. While stirring, heat the inulin solution in a water bath to 85 °C and maintain this temperature for 4 h to completely dissolve the inulin, resulting in a homogeneous, milky-white aqueous solution of short-chain inulin with a mass concentration of 40 wt%.

[0299] A 40 wt% aqueous solution of short-chain inulin was immediately mixed with a 4 wt% aqueous solution of silk fibroin from Example 1 at a mass ratio of 1:1 to obtain a mixed aqueous solution, which was then placed in a constant temperature incubator at 60 °C for later use.

[0300] Anhydrous ethanol was rapidly added to the above mixed aqueous solution at a mass ratio of 1:1. After thorough mixing, the mixture was allowed to stand at 25 °C for 10 min to obtain a silk fibroin-inulin-based composite hydrogel induced by ethanol gelation.

[0301] GPC diagrams and molecular weight distribution curves of short-chain inulin and long-chain inulin are shown below. Figure 3 As shown, long-chain inulin exhibits a high weight-average molecular weight (Mw = 7733 Da) and a reasonable distribution coefficient, which lays a solid polymeric structural foundation for its rapid precipitation and formation of a particulate physical cross-linked network with sufficient mechanical support when polarity drops sharply. In contrast, short-chain inulin (Mw = 1063 Da) is insufficient in chain length and cannot provide corresponding macroscopic skeletal support. Its gelation rate induced by ethanol is slightly slower, and its gel strength is much lower than that of long-chain inulin.

[0302] Test Example 1

[0303] The composite hydrogels prepared in Examples 30, 45, 60, 65, 70 and 75, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 were freeze-dried and their crystal structures were characterized by X-ray diffraction (XRD). The test conditions were: Cu Kα target radiation source (wavelength λ=0.15406 nm), and diffraction angle 2θ scanning range of 5°-80°.

[0304] Characterization results as follows Figure 4 As shown, the XRD patterns directly confirm that the rapid phase transition preparation process of this invention is mild and has significant advantages. After the silk fibroin and inulin form a double-network cross-linking coating, characteristic diffraction peaks corresponding to the doped MOF particles can still be observed in the composite hydrogels of each embodiment. This indicates that the phase transition process using ethanol as a polarity modifier and conformation inducer does not destroy the sensitive metal coordination lattice of the MOF particles, achieving a complete and undamaged composite of the organic polymer matrix and the inorganic crystalline framework.

[0305] Test Example 2

[0306] The silk fibroin-inulin-loaded MIL-156(Ca) composite hydrogel prepared in Example 60, the inulin-based hydrogel prepared in Comparative Examples 1-5, the inulin-based hydrogel prepared in Comparative Examples 7-8, the silk fibroin-based hydrogel prepared in Comparative Examples 9-10, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Examples 12-13 were subjected to static molding tests in inverted bottles. The inverted bottle method was used to visually determine whether the system formed a gel with self-supporting ability. The temperature-controlled gelation concentration threshold of pure inulin, the phase transition induction effect of ethanol on a single component (silk fibroin / inulin), and the influence of the presence or absence of MOFs / ethanol on the molding behavior of the dual polymer blend were investigated.

[0307] Test results are as follows Figure 5-7 As shown, Figures 5-7 The top image was taken under natural light, while the bottom image was taken under flash. The flash image in the bottom image shows the transparency of the gel more clearly. Figure 5 The concentration threshold for pure inulin to gel solely through cooling can be visually determined: Comparative Example 1 (5 wt% INU concentration in the hydrogel) and Comparative Example 2 (10 wt%) flowable and exist in a solution state after inversion; Comparative Examples 3 (20 wt%), 4 (30 wt%), and 5 (40 wt%) stabilize into a gel state after cooling by forming an anti-gravity three-dimensional network with microcrystals through molecular hydrogen bonds. The results indicate that inulin gelation is highly concentration-dependent when induced solely by temperature, and low concentrations of inulin (10 wt% and below) are insufficient to form a complete self-supporting gel framework.

[0308] Ethanol can rapidly induce gelation in both types of systems, but from Figure 6 As can be seen, the two hydrogels exhibit significant differences in appearance: the inulin-based hydrogels (Comparative Examples 7 and 8) are milky white and opaque. This is because ethanol, as a non-solvent, drastically lowers the dielectric constant of the system, causing the long polysaccharide chains to instantly undergo large-scale aggregation and phase separation of microcrystalline structures. The silk fibroin-based hydrogels (Comparative Examples 9 and 10) are transparent and clear, indicating that when ethanol induces the transformation of silk fibroin macromolecules from random coils to a dense β-sheet structure, it constructs a long-range cross-linked network with an extremely uniform spatial grid, high density, and no macroscopic aggregation. In contrast, the inulin-based hydrogel (Comparative Example 2, with an INU mass concentration of 10 wt%) cannot be formed upon cooling alone, but it can rapidly gel and form a stable gel structure after being induced by ethanol. This confirms that ethanol can promote the interaction between INU molecules and the formation of the gel network, overcoming the concentration limitation of inulin for low-temperature gelation.

[0309] Based on the aforementioned rules governing single-component gel formation, Figure 7The inverted morphology of the composite hydrogels prepared in Example 60 and Comparative Examples 12 and 13 was compared. All three composite hydrogels were prepared using a mixed aqueous solution of 2 wt% silk fibroin and 20 wt% inulin. In Comparative Example 12, the mixed aqueous solution was diluted with an equal mass of deionized water, reducing the concentrations of silk fibroin and inulin to 1 wt% and 10 wt%, respectively. Since the system lacked ethanol as a phase transition inducer, it could not induce the conformational change of SF at room temperature. Furthermore, because the concentration of INU dropped below the critical gelation boundary, it could not crystallize under temperature control, resulting in the system exhibiting a completely milky white fluid solution state during the inverted test. In Comparative Example 13, an equal mass of anhydrous ethanol was mixed with the mixed aqueous solution. The introduction of ethanol instantly activated the dual induction effect, simultaneously triggering the β-sheet transition of SF and the microcrystalline precipitation of INU, thus successfully forming a milky white interpenetrating double network solid gel state without the aid of added inorganic particles. In Example 60, an ethanol solution containing 10 mg / g MIL-101(Fe) particles was mixed with an equal mass of a mixed aqueous solution to prepare a brownish-yellow homogeneous gel containing 1 wt% silk fibroin, 10 wt% inulin, and 5 mg / g MIL-101(Fe). The gel remained stable and showed no flow deformation after inversion. This result confirms that the process of pre-dispersing MOFs in ethanol and then mediating in-situ gelation can rapidly solidify the polymer network with ethanol while uniformly confining crystals within a dual-network framework. This overcomes the defects of inorganic particle aqueous systems, such as easy sedimentation and phase separation, and provides a reliable structural basis for targeted drug release and stable drug loading in the digestive tract.

[0310] Test Example 3

[0311] The silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel prepared in Example 65, the inulin-based hydrogel prepared in Comparative Examples 3-4, the inulin-based hydrogel prepared in Comparative Example 7, the silk fibroin-based hydrogel prepared in Comparative Example 10, the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 17 were comprehensively rheologically characterized using a HAAKE MARS-40 rotational rheometer to evaluate their mechanical properties, gelation stability, gelation process, and gelation kinetics. All hydrogel test samples were uniformly prepared as cylindrical specimens with a diameter of 20 mm and a thickness of 500 μm, and the tests were conducted using a circular parallel plate clamp with a diameter of 20 mm and a clamp spacing of 500 μm.

[0312] Test results are as follows Figure 8 and Figure 9 As shown, dynamic rheological testing first quantitatively confirmed the mechanical response characteristics of the hydrogel's internal network under temperature, frequency, and shear force stimuli. Figure 8(A) shows the temperature scan curve of the inulin-based hydrogel prepared in Comparative Example 3. The test temperature range was 20-90 °C, the heating rate was 1 °C / min, the oscillation frequency was 1 rad / s, and the strain was 1%. The test results show that the inulin-based hydrogel formed solely by temperature crystallization has poor thermal stability. As the temperature increases from 20 °C to the physiological temperature of 37 °C and above, the storage modulus G' and loss modulus G'' of the hydrogel both decrease significantly. This indicates that the inulin network constructed solely by temperature-controlled crystallization is highly sensitive to temperature and is prone to thermal dissociation and network collapse in the warm environment of the body. At the same time, the storage modulus of this 20 wt% inulin-based hydrogel is only 3 Pa, and its mechanical strength is extremely low.

[0313] Figure 8 (B) shows the modulus-angular frequency scanning curves of the inulin-based hydrogel prepared in Comparative Example 7, the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13, and the silk fibroin-inulin-loaded UiO-66(Zr) composite hydrogel prepared in Example 65 under oscillation mode at 25 °C and 1% strain. Throughout the entire tested angular frequency range, the storage modulus G' of all hydrogel samples was consistently significantly higher than the loss modulus G''. The modulus curves were stable and almost frequency-independent, exhibiting typical solid-state elastic characteristics, demonstrating that each gel system successfully constructed a structurally complete, rigid, and highly stable three-dimensional cross-linked network.

[0314] Figure 8 (C) shows the viscosity-shear rate curves of the inulin-based hydrogel prepared in Comparative Example 4 and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 in rotation mode at a temperature of 37 °C. Figure 8 Figure (D) shows the viscosity-shear rate curve of the silk fibroin-based hydrogel prepared in Comparative Example 10 under rotational mode at 37 °C, reflecting the injectability of the fully formed hydrogel. All hydrogel samples exhibited significant shear-thinning behavior, with viscosity decreasing significantly with increasing shear rate, demonstrating good rheological processing characteristics. Among them, the high-concentration inulin-based hydrogel (Comparative Example 4) had extremely high initial viscosity, a dense and rigid network, and poor shear flowability, making it difficult to achieve smooth injection extrusion. In contrast, the silk fibroin-inulin-based composite hydrogel induced by ethanol (Comparative Example 13) had moderate initial viscosity, and the network could quickly untangle and deconstruct under applied shear force, resulting in a significant decrease in viscosity and excellent injection smoothness. The silk fibroin-based hydrogel (Comparative Example 10) itself possesses low initial viscosity and sensitive shear-thinning response, confirming that silk fibroin can serve as an excellent rheology modifier, effectively improving the defects of high viscous resistance and poor processability in polysaccharide gel systems, enabling the composite gel to balance structural rigidity after molding with ease of injection processing.

[0315] Figure 9(A) shows the time-scan gelation kinetics curve of the mixed solution of silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13, under oscillation mode at 25 °C, 1 Hz, and 1% strain, characterizing the gelation process of the ethanol-induced system. After the addition of the ethanol phase change inducer, the system can trigger a modulus mutation within seconds, and within 1-4 min, the storage modulus G' rapidly surpasses the loss modulus G'' and quickly reaches a stable plateau, achieving extremely rapid in-situ solidification. This extremely rapid gelation mechanism, triggered in seconds and solidified in minutes, can complete network solidification before MOF particles settle, aggregate, and separate, achieving uniform confined dispersion of MOF particles within the gel framework.

[0316] Figure 9 Figure (B) shows a comparison of the storage modulus of the silk fibroin-inulin-based composite hydrogels prepared in Comparative Example 13 and Comparative Example 17 under oscillation mode at 25℃, 10 rad / s angular frequency, and 1% strain. Storage modulus reflects the hydrogel's ability to resist elastic deformation and can be used to characterize the rigidity and mechanical strength of the gel network structure, and to evaluate the density and cross-linking strength of its three-dimensional network structure. Generally, a higher storage modulus indicates that the hydrogel has stronger mechanical properties and a more stable network structure. The results show that the storage modulus of the dual-network gel constructed with long-chain inulin can reach about 3000 Pa, and its mechanical strength is nearly three times that of the short-chain inulin gel (about 1200 Pa), fully demonstrating that long-chain inulin plays an irreplaceable role in constructing high-strength, high-stability gel networks and providing macroscopic mechanical support.

[0317] In summary, the rheological test results show that the silk fibroin-inulin-loaded metal-organic framework composite hydrogel constructed by the ethanol rapid phase transition induction strategy of this invention effectively solves the problems of poor thermal stability, low mechanical strength, and insufficient processability of pure inulin-based hydrogels. It also has excellent thermodynamic stability, shear processing compliance, and rapid in-situ gelation and curing ability, and can effectively withstand gastrointestinal mechanical peristalsis and fluid erosion, providing a stable structural basis for achieving intestinal targeted stepwise drug release.

[0318] Test Example 4

[0319] Syringe extrusion flow deformation morphology tests were performed on the composite hydrogels prepared in Examples 30, 60, and 65, the inulin-based hydrogels prepared in Comparative Examples 3-6, the inulin-based hydrogels prepared in Comparative Example 8, the silk fibroin-based hydrogels prepared in Comparative Example 9, and the silk fibroin-inulin-based composite hydrogels prepared in Comparative Examples 12-13.

[0320] Test results are as follows Figure 10 and 11As shown, in inulin-based hydrogels formed solely by cooling, high-concentration comparative examples 4 and 5 can still exhibit relatively complete macroscopic gel morphology after extrusion. Low-concentration comparative example 3 has insufficient three-dimensional physical mesh density, and its structure undergoes irreversible damage after shearing, macroscopically appearing as an amorphous paste-like fluid that has lost its self-supporting ability. In ethanol-induced inulin-based hydrogels, although low-concentration comparative example 8 can barely undergo physical phase separation to form a gel under static conditions, its physical mesh obviously cannot resist strong shear stress after extrusion with a syringe, resulting in severe collapse and a paste-like flow. Comparative example 6, with insufficient concentration, failed to reach the critical concentration for ethanol-induced crystallization and gel formation, making macroscopic gelation completely impossible at the process level. Comparative Example 9: The ethanol-induced 1 wt% silk fibroin-based hydrogel exhibited excellent morphological retention and high rigidity self-supporting gel state after extrusion via syringe, due to the efficient induction of macromolecular chains by ethanol to form a dense and highly uniform β-sheet cross-linked backbone. Comparative Example 12, which did not contain ethanol, lacked the powerful phase transition trigger ethanol, resulting in the inability of macromolecular chains to undergo conformational mutation and in-situ solidification, thus failing to form a gel. Consequently, it maintained a low-viscosity fluid solution state after extrusion via syringe. Comparative Example 13: A two-component interpenetrating network hydrogel was constructed by combining the advantages of SF and INU. Thanks to the introduction of a long-range SF protein network backbone and the spatial synergistic toughening effect, this system exhibited significantly improved macroscopic mechanical strength and shear failure resistance, and maintained a highly intact morphology and good support after extrusion via syringe.

[0321] The composite hydrogels prepared in Examples 30, 60, and 65, after being extruded using a syringe, exhibited shear thinning and network self-healing characteristics highly consistent with, or even superior to, Comparative Example 13. They could be smoothly extruded through the needle and instantly regained their macroscopic strength after the shear force was removed, maintaining a continuous, smooth, and highly intact complex coiled strip-shaped solid profile without any macroscopic fragmentation, local splitting, or flow phenomena. The test results indicate that the introduction of MOF particles does not compromise the gel's injectability. Furthermore, through the efficient synergy between inorganic microcrystals and organic macromolecular networks in the interfacial microregions, a more robust hybrid physical barrier is locked, ensuring the structural integrity of the drug-loaded formulation during oral administration, injection, and gastrointestinal peristalsis.

[0322] Test Example 5

[0323] The drug loading capacity (DLC) and drug loading efficiency (DLE) of the drug-loaded MOF particles in Examples 86-97, as well as the drug loading capacity of the prepared silk fibroin-inulin-loaded metal-organic framework composite hydrogels, were tested. The supernatant obtained from centrifugation in step P2 of each example was collected, and the testing method was as follows:

[0324] (1) The mass of free CUR contained in it was tested by ultraviolet-visible absorption spectroscopy (UV). Based on the mass of CUR fed, the drug loading capacity and drug loading efficiency of MOF particles to CUR were calculated.

[0325] (2) The mass of free RFX contained in the sample was determined by high performance liquid chromatography (HPLC). The supernatant was diluted with the mobile phase, brought to a final volume, and filtered through a 0.22 μm microporous membrane. The filtrate was used as the test solution. Chromatographic separation was performed using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) with acetonitrile-0.05 mol / L potassium dihydrogen phosphate buffer (volume ratio approximately 60:40) as the mobile phase for isocratic elution. The flow rate was set at 1.0 mL / min, the column temperature was maintained at 30 ℃, the UV detection wavelength was set at 276 nm, and the injection volume was 20 μL. After the test, the peak area was recorded, and the mass of RFX in the supernatant was quantitatively calculated based on the RFX standard curve. Based on the RFX feed mass, the drug loading capacity and drug loading efficiency of the MOF particles were calculated.

[0326] (3) The mass of free VCN contained in the sample was determined by HPLC. The supernatant was diluted with ultrapure water, brought to a final volume, and filtered through a 0.22 μm microporous membrane. The filtrate was used as the test solution. Chromatographic separation was performed using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of 0.05 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 3.2 with phosphoric acid) and acetonitrile (volume ratio approximately 85:15). Isocratic elution was performed to improve peak shape. The flow rate was set to 1.0 mL / min, the column temperature was maintained at 30℃, the UV detection wavelength was set to 280 nm, and the injection volume was 20 μL. After the test, the peak area of ​​vancomycin was recorded. The mass of VCN in the supernatant was quantitatively calculated based on the VCN standard curve. Based on the mass of VCN fed, the drug loading capacity and drug loading efficiency of MOF particles to VCN were calculated.

[0327] (4) The mass of free Cy5-URC contained in it was tested by fluorescence emission spectroscopy. The excitation wavelength was set to 649 nm and the emission wavelength was set to 670 nm. Based on the mass of Cy5-URC, the drug loading capacity and drug loading efficiency of MOF particles on Cy5-URC were calculated.

[0328] The formulas for calculating the drug loading capacity and drug loading efficiency of MOF particles are as follows:

[0329] DLE MOFs (%) = (W 包载 / W 初始 ) × 100%;

[0330] DLC MOFs(%) = (W 包载 / W 总质量 ) × 100%;

[0331] Among them, W 包载 = W 初始 - W 游离 W 初始 For initial drug administration quality; W 游离 W represents the mass of unadsorbed free drug in the supernatant. 总质量 The total mass of the separated and dried drug-loaded MOF particles.

[0332] The formula for calculating the drug loading capacity of the silk fibroin-inulin-loaded metal-organic framework composite hydrogel is as follows:

[0333] DLC 凝胶 (%) = (W 最终包载 / W 凝胶 ) × 100%;

[0334] W 最终包载 = W 载药MOFs × DLC MOFs ;

[0335] Among them, W 最终包载 To determine the actual drug quality ultimately retained within the gel after the gelation process, W 凝胶 W represents the total dry weight of the silk fibroin-inulin-loaded metal-organic framework composite hydrogel containing the drug. 载药MOFs The feed quality of drug-loaded MOF particles during hydrogel preparation.

[0336] The DLC and DLE test results of MOF particles (ZIF-8, MIL-101(Fe), UiO-66(Zr)) on CUR, RFX, VCN and Cy5-URC are as follows: Figure 12 As shown, the superior loading performance of three MOF particles as primary carriers is revealed. Thanks to their highly developed micro-mesoporous structure and large specific surface area, different MOFs exhibit specific and efficient enrichment capabilities for drugs with varying physicochemical properties. Among them, ZIF-8 particles demonstrate excellent loading capacity for CUR and macromolecular Cy5-URC, with DLCs reaching approximately 32% and 24%, respectively, and corresponding DLEs of approximately 55% and 32%. Meanwhile, MIL-101(Fe) particles exhibit the highest affinity for hydrophilic VCN, achieving a drug loading of approximately 16%, with a drug loading efficiency approaching 39%.

[0337] DLC test results of drug-loaded silk fibroin-inulin-supported metal-organic framework composite hydrogels are as follows: Figure 13As shown, the final macroscopic drug loading of the composite hydrogels remained at a high level with significant clinical application value. The absolute drug loading of the ZIF-8-based composite hydrogel for the hydrophobic drug CUR and the macromolecular Cy5-URC reached approximately 1.4% and 1.05%, respectively; the final drug loading of the MIL-101(Fe)-based composite hydrogel for VCN also remained stable at approximately 0.7%.

[0338] Test results confirm the advanced nature of the "functional partitioning isolation" and "two-level confined encapsulation" drug delivery strategy of this invention: the inner MOF particle core utilizes strong physical adsorption and pore confinement to fundamentally overcome the technical weakness of traditional hydrophilic hydrogels in being unable to stably carry high concentrations of hydrophobic drugs; while the rapidly cross-linked silk fibroin-inulin dual network of the outer layer forms a dense physical shielding layer at the moment of gelation, which greatly inhibits the secondary expansion and synergistic leakage of drugs during the processing and molding process, providing a reliable pharmaceutical basis for achieving precise targeted release in the complex environment of the digestive tract.

[0339] Test Example 6

[0340] The silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC prepared in Example 96 was subjected to in vivo targeted delivery test by fluorescence imaging of the in vitro digestive tract of mice. The test method was as follows: two parallel experiments were set up. One group was the silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC, and the other group was pure Cy5-URC solution with the same dosage. BALB / c mice were administered the drugs by gavage or oral administration at a dosage of 7.55 mg per kilogram of mouse body weight. Mice were sacrificed at five time points after drug administration: 0 h, 4 h, 8 h, 12 h, and 24 h. The digestive tract tissues (stomach, small intestine, and colon) were completely isolated. The IVIS-Lumina Series-III small animal in vivo imaging system was used to perform in vitro fluorescence imaging of the digestive tract in fluorescence mode with an excitation filter wavelength of 640 nm and an emission filter wavelength of 680 nm to track the drug transport pathway, distribution concentration, and residence time in the gastrointestinal tract.

[0341] Test results are as follows Figure 14 As shown, in the Cy5-URC pure solution control group, the macromolecular protein drug underwent rapid mechanical propulsion and physiological elimination in vivo: at 0 h after administration, fluorescence was concentrated only in the stomach; at 4 h, the fluorescence in the stomach decreased significantly, with only a weak signal visible in the small intestine; by 8 h, due to the lack of skeletal protection and mucosal anchoring function of free biomolecules, they were rapidly propulsed downwards and degraded under strong intestinal peristalsis, and the fluorescence signal throughout the entire intestine was completely extinguished. This indicates that ordinary protein drugs are easily lost or diluted and degraded in the upper digestive tract after oral administration, and cannot achieve effective concentration enrichment at the local lesion site in the lower digestive tract.

[0342] The drug-loaded hydrogel exhibited the dual technological advantages of highly precise spatiotemporally controlled delivery and ultra-long-lasting tissue adhesion and retention in the colon. During the initial 0-4 h of drug administration, the fluorescent signal in the mouse digestive tract descended slowly and safely along the gel framework in a highly encapsulated, dense mass morphology, gradually migrating and accumulating from the stomach across the small intestine to the colon. This strongly confirms that the dual-network framework formed by the instantaneously triggered conformational change of ethanol, consisting of tightly interwoven silk fibroin β-sheet chains and long-chain inulin microcrystals, possesses excellent physical barrier and structural drug-locking functions in the upper digestive tract environment (gastric acid and highly active enzymes in the small intestine), effectively preventing early burst release and synergistic leakage of the drug. When the formulation reached the lower digestive tract (4-12 h), the fluorescence intensity in the cecum and colon gradually increased, with a signal abundance several times higher than that of the pure solution group. This indicates that when the gel precisely reaches the lower digestive tract, the targeted degradation of the inulin matrix by colon-specific microbial enzymes successfully triggers the "in-situ dissolution and self-disintegration" of the gel network, thereby precisely releasing the uniformly confined drug-loaded MOF particles and spreading them on the colonic mucosal surface. Within a long period of 24 hours after administration, the fluorescence signal in the pure solution group had already completely extinguished, while the drug-loaded composite hydrogel group still firmly maintained a high-density, localized strong fluorescent patch signal in the colonic tissue segment. This confirms that the released MIL-101(Fe) particles, with their well-developed pore structure, large specific surface area, and multi-site physical / chemical anchoring effect with the colonic mucosa, endow the delivery system with excellent resistance to intestinal peristalsis. In summary, the composite hydrogel of this invention not only successfully achieves comprehensive inhibition of upper digestive tract leakage and burst release, but also achieves ultra-long-term tissue adhesion and retention in the colonic region for a period of time (≥ 24 h), providing a long-term, targeted drug delivery basis for colon-related diseases such as chronic inflammation and local tumors of the lower digestive tract.

[0343] Test Example 7

[0344] The composite hydrogels prepared in Examples 30, 60, and 65, the inulin-based hydrogels prepared in Comparative Examples 3-5, the inulin-based hydrogel prepared in Comparative Example 7, the silk fibroin-based hydrogel prepared in Comparative Example 9, and the silk fibroin-inulin-based composite hydrogel prepared in Comparative Example 13 were subjected to in vitro simulated digestive tract segmental degradation kinetic tests to verify the multi-level structural tolerance and colon-specific bioenzyme degradation response of the composite hydrogels of the present invention under complex digestive tract physiological environment. Quantitative data on the continuous mass loss rate of each group of hydrogels in the in vitro simulated digestive tract environment were also tested. The testing method was as follows: Three standardized simulated digestive solutions were prepared: artificial gastric juice (AGF, pH = 1.2, containing pepsin, concentration 3.2 mg / mL), artificial small intestinal juice (AIF, pH = 6.8, containing pancreatin, concentration 10 mg / mL), and artificial colonic juice (ACF, pH = 7.4, containing inulinase, concentration 25 U / mL). All three solutions were standardized commercial buffer solutions. 2g of each hydrogel sample was initially immersed in 4 mL of the corresponding degradation medium (AGF, AIF, or ACF) and incubated in a 37 ℃, 100 rpm constant temperature shaking incubator. Fresh digestive solution was replaced every 8 h to maintain medium activity and pH stability. At preset time points, the hydrogel samples were removed, surface moisture was aspirated, and the weight was calculated. The percentage of remaining hydrogel mass at each time point was calculated. Each group was divided into triplicate, and degradation kinetic curves were plotted.

[0345] Test results are as follows Figure 15 As shown, firstly, in Figure 15 In the simulated gastric fluid environment of Example (A), the composite hydrogels of each embodiment (Examples 30, 60, and 65), the silk fibroin-inulin-based composite hydrogel (Comparative Example 13), and the silk fibroin-based hydrogel (Comparative Example 9) all exhibited extremely excellent resistance to acid erosion and pepsin degradation during an acidic incubation period of up to 12 hours. Quantitative test data showed that the cumulative mass loss rate of the above systems was generally robustly controlled below 10%, and the macroscopic morphology always maintained a completely rigid, intact three-dimensional block structure, without any local edge dissolution or matrix fragmentation. This effectively confirms that the high-density silk fibroin β-sheet crystalline region and long-chain inulin microcrystalline network formed by the rapid conformational transformation triggered by anhydrous ethanol in this invention have an excellent physical barrier effect against strong acid and pepsin environments, effectively locking in the internal primary carrier MOFs and preventing the early disordered release of guest drugs. In contrast, the inulin-based hydrogel (Comparative Example 7) underwent rapid dissociation and collapse in gastric fluid. Figure 15The degradation curves of the inulin-based hydrogels (Comparative Examples 3, 4, 5, and 7) in (B) further reflect that the degradation behavior in the gastric environment significantly accelerates as the inulin concentration in the hydrogel decreases (from a high concentration of 40 wt% in Comparative Example 5 to a low concentration of 10 wt% in Comparative Example 7). This demonstrates that the fatal defects of inulin hydrogels, such as low mechanical strength and poor acid resistance, severely limit their structural stability in the stomach; while the introduction of silk fibroin to construct an interpenetrating double network in this invention can compensate for this defect, achieving complementary advantages and synergistic anti-enzyme effects between components.

[0346] Subsequently, Figure 15 In the artificial small intestinal fluid environment of (C), the silk fibroin-based hydrogel (Comparative Example 9) underwent rapid hydrolytic collapse under the strong catalysis of pancreatic enzymes; conversely, the silk fibroin-inulin-based composite hydrogel (Comparative Example 13) and the composite hydrogels prepared in each example (Examples 30, 60, and 65) all exhibited an extremely gradual and controllable slow degradation trend. After 24 hours of continuous incubation and rinsing, the remaining mass ratio of the composite hydrogel was still as high as 80% or more, and the macroscopic morphology did not undergo a precipitous destruction. This result verifies the scientific nature of the dual-network system of the present invention: due to the absolute resistance and high stability of the inulin polysaccharide network in the formulation to intestinal pancreatic enzymes, even when the silk fibroin component undergoes localized weak dissolution, the entire framework can still maintain the mechanical interpenetrating stability of the overall topological structure. This loosening of the network due to local selective degradation transforms the gel from its initial dense state into a sponge-like porous structure. This not only endows the gel with good flexibility and compliance to adapt to the physiological peristalsis of the small intestine, but also provides a reasonable diffusion channel for the gradual release of drugs, ensuring that most of the drug-loaded contents can continue to move to the cecum and colon in a highly intact dosage form.

[0347] Finally, Figure 15 In the targeted response evaluation of the artificial colonic fluid environment in (D), the silk fibroin-based hydrogel (Comparative Example 9) exhibited the slowest degradation rate due to its lack of responsiveness to colon-specific bioenzymes. However, the silk fibroin-inulin-based composite hydrogel (Comparative Example 13) and the composite hydrogels prepared in each example (Examples 30, 60, and 65) all showed a highly significant rapid degradation trend under the action of specific inulinase in the artificial colonic fluid. The composite hydrogels incorporating drug-loaded MOF particles (Examples 30, 60, and 65) showed a finely regulated overall degradation rate that was appropriately slowed down compared to the silk fibroin-inulin-based composite hydrogel (Comparative Example 13) without MOF particles. This indicates that there is a microscopic trapping and physical confinement effect between the inorganic MOF core and the polymer cross-linked backbone at the microscale, which further optimizes the disintegration kinetics of the gel in the colonic segment and effectively avoids the instantaneous release of the guest drug.

[0348] In summary, the composite hydrogel provided by this invention possesses an extremely precise intelligent oral targeted drug release behavior characterized by "stable concealment in the stomach, moderate loosening in the small intestine, and rapid cascade disintegration in the colon." Specifically, the system maintains high stability in the highly acidic environment of the stomach to fully protect the contents; in the small intestinal environment, pancreatic enzymes mediate partial degradation of SF, driving moderate sponging of the network for a smooth transition; and once it precisely enters the colonic environment, inulinase specifically secreted by the intestinal flora efficiently cleaves the polysaccharide backbone, triggering a complete in-situ self-collapse of the gel backbone, thereby explosively releasing drug-loaded MOF particles locally in the colonic lesion, thus achieving precise targeted release of high-abundance drugs.

[0349] Test Example 8

[0350] The silk fibroin-inulin-loaded MIL-101(Fe) composite hydrogel loaded with Cy5-URC prepared in Example 96, the silk fibroin-inulin-based composite hydrogel loaded with Cy5-URC prepared in Comparative Example 14, the silk fibroin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 15, and the inulin-based hydrogel loaded with Cy5-URC prepared in Comparative Example 16 were subjected to in vitro segmented simulated digestive fluid drug cumulative release tests to verify the colon-targeted controlled release and long-acting sustained release effects of the composite hydrogel of the present invention in the complex physiological environment of the digestive tract. The test method was as follows: each group of drug-loaded hydrogels were placed in three media: artificial gastric fluid (AGF, pH = 1.2, containing pepsin, concentration 3.2 mg / mL), artificial small intestinal fluid (AIF, pH = 6.8, containing pancreatic enzyme, concentration 10 mg / mL), and artificial colonic fluid (ACF, pH = 7.4, containing inulinase, concentration 25 U / mL) to carry out in vitro release experiments and monitor the cumulative release rate of the drug. Two g of drug-loaded hydrogel was immersed in 4 mL of release medium (AGF, AIF, or ACF) and incubated in a constant temperature shaking incubator at 37 ℃ and 100 rpm. At the preset time point, 500 μL of release solution was taken out and the same volume of fresh medium was added to maintain the system volume. The release solution was diluted to 50 mL (100-fold dilution) with PB buffer (0.2 mol / L, pH = 7.4). The fluorescence intensity was detected by fluorescence spectrophotometer at an excitation wavelength of 649 nm and an emission wavelength of 670 nm. The cumulative drug release rate at each time point was calculated by combining the Cy5-URC standard curve. Each group was divided into three parallel samples, and the drug release curve was plotted.

[0351] Test results are as follows Figure 16-18 As shown, in an artificial gastric fluid environment, Figure 16The study demonstrated significant structure-dependent differences in the drug release kinetics of the various drug-loaded hydrogels. The drug-loaded composite hydrogel prepared in Example 96 exhibited a superior "gastric lock-in" effect, with its cumulative release rate strictly suppressed to an extremely low level of approximately 20% within 12 hours. This value is significantly lower than that of Comparative Example 16 (drug-loaded inulin-based hydrogel), fully confirming that the dual physical barrier constructed by the "MOF pore confinement" and the "SF-INU macroscopic dual network" can effectively inhibit the free diffusion of sensitive protein drugs in strong acid, fundamentally preventing early drug leakage and providing a solid pharmaceutical guarantee for improving the effective delivery rate of macromolecular drugs across the gastric barrier. Furthermore, in the cross-sectional comparison of the control group of hydrogels without MOFs, Comparative Example 14 (drug-loaded silk fibroin-inulin-based composite hydrogel) exhibited the best drug release inhibition ability, with its 12-hour cumulative release rate (approximately 40%) significantly lower than that of the single-component Comparative Examples 15 (drug-loaded silk fibroin-based hydrogel) and 16 (drug-loaded inulin-based hydrogel). This further confirms that the heterogeneous synergistic network formed between silk fibroin and inulin molecular chains possesses higher topological density and acid swelling resistance. In contrast, the pure inulin backbone of Comparative Example 16 mainly relies on a single non-covalent physical entanglement for maintenance, lacking sufficient structural support under continuous erosion in an extremely acidic environment, leading to accelerated network loosening and rapid drug diffusion.

[0352] When in an artificial small intestinal fluid environment Figure 17All drug-loaded hydrogels exhibited responsive accelerated drug release characteristics. Comparative Example 15 (drug-loaded silk fibroin-based hydrogel) showed the fastest release, with its cumulative release rate surging to over 60% after 12 hours. This was mainly attributed to the skeletal breakage of the single silk fibroin network under the specific hydrolysis of potent trypsin, leading to the rapid release of the internally loaded Cy5-URC. In contrast, the drug release process of Comparative Example 14 (drug-loaded silk fibroin-inulin-based composite hydrogel) was significantly delayed, with the 12-hour release rate controlled at approximately 50%. This confirms that the natural resistance of the inulin component to trypsin effectively compensates for the easy enzymatic degradation of silk fibroin, and the synergistic effect of the two components significantly reduces the overall structural damage of the gel under enzymatic conditions, achieving sustained physical protection of the protein drug. Even more impressively, the drug-loaded composite hydrogel prepared in Example 96 exhibited the strongest drug release inhibition ability, with its cumulative sustained release amount after 12 hours being less than 40%. This is because the inulin polysaccharide network in the formulation has a certain resistance to pancreatic enzymes in the small intestine. Even if the silk fibroin component undergoes localized slight dissolution under enzymatic action, the entire drug-carrying framework can still maintain the mechanical interpenetrating stability of the macroscopic topology. This "matrix loosening" induced by localized selective degradation allows the gel to smoothly transition from the extremely dense state of the stomach to a sponge-like porous structure. This structural evolution not only endows the material with good flexibility and compliance to the physiological peristalsis of the small intestine, but also constructs a suitable diffusion channel for the gradual release of the drug, fundamentally ensuring that the vast majority of the drug-carrying core can continue to advance to the cecum and colonic lesions in a highly intact dosage form.

[0353] In an artificial colonic fluid environment Figure 18The drug-loaded hydrogels exhibited differentiated enzyme-responsive drug release characteristics. Comparative Example 16 (drug-loaded inulin-based hydrogel), due to its network backbone being entirely composed of polysaccharides specifically degradable by inulinase, underwent rapid enzymatic depolymerization and structural disintegration, exhibiting the most aggressive release behavior, with a cumulative release rate of nearly 90% after 48 hours. Comparative Example 15 (drug-loaded silk fibroin-based hydrogel), lacking colon-targeted degradation sites, relied solely on passive diffusion for drug release, resulting in a release rate of less than 50% after 48 hours, failing to meet the demand for high-concentration drug release at the lesion site. Comparative Example 14 (drug-loaded silk fibroin-inulin-based composite hydrogel) showed a moderate transitional release state, confirming that the introduction of inulin successfully endowed the inert silk fibroin network with a colon-specific degradation "switch." The drug-loaded composite hydrogel prepared in Example 96 of this invention exhibits optimal synergistic effects of targeted triggering and long-term sustained release, with a cumulative drug release rate of approximately 65% ​​over 48 hours. Its excellent controlled-release performance stems from a multi-level confinement mechanism: at the macroscopic level, the ACF environment specifically degrades the inulin components in the network, triggering the gel's targeted drug release switch; while at the microscopic level, the large mesoporous structure of MIL-101(Fe) particles and their strong host-guest adsorption forces, combined with the steric hindrance provided by the residual silk fibroin backbone, exert a strong secondary physical constraint on the Cy5-URC macromolecule. This deep synergy at both the macro and micro scales not only ensures precise drug exposure to colonic lesions but also completely avoids the burst release risk easily triggered by single-component matrices, achieving long-term controlled release in the colon. In summary, the drug-loaded inulin-based hydrogel has a strong colonic-responsive release capability but limited gastrointestinal protection; the drug-loaded silk fibroin-based hydrogel has good structural stability but insufficient colon-specific response. The drug-loaded composite hydrogel formed by combining SF / INU and MOF particles has the advantages of gastrointestinal protection, enzyme-responsive degradation and sustained-release delivery. It can effectively reduce drug loss in the upper gastrointestinal tract and promote the sustained release of Cy5-URC in the colon, showing good application prospects in the field of oral protein drug delivery.

[0354] In summary, this invention successfully constructed a composite hydrogel system with efficient drug loading, stable protection, and precise colonic delivery through a multi-level structural design of "MOF-confined carrier + silk fibroin / inulin dual-network hydrogel". System characterization results confirmed that long-chain inulin provides a good crystalline support for the gel network, and the silk fibroin and inulin achieve rapid synergistic phase transition crosslinking through ethanol induction, further endowing the system with excellent mechanical stability, shear thinning properties, and injection compliance. Simultaneously, the introduction of MOF particles did not disrupt the gel network structure; instead, it significantly improved the loading capacity and storage stability of different types of drugs through microporous / mesoporous confinement. In vitro digestion simulation experiments showed that the composite hydrogel can maintain structural integrity in the strong acid and small intestinal enzyme environment of the stomach, effectively inhibiting premature drug leakage, and undergoing responsive degradation under the action of colon-specific inulinase, achieving sustained drug release at the target site. Furthermore, in vivo fluorescence tracing results further demonstrated that this system can effectively protect large molecular protein drugs across the upper gastrointestinal barrier and promote their long-term retention and local accumulation in the colon. It is evident that the composite hydrogel constructed in this invention overcomes the technical bottlenecks of traditional hydrogels, such as low drug loading capacity, insufficient structural stability, and limited colon targeting efficiency. It realizes an intelligent delivery process of "gastric stability protection - small intestinal sustained release transition - colonic response release," demonstrating significant application potential in the field of precise delivery of oral protein drugs and various types of active drugs.

[0355] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A silk fibroin-inulin-supported metal-organic framework composite hydrogel, characterized in that, It was prepared by mixing an aqueous solution of silk fibroin, an aqueous solution of inulin and an ethanol dispersion of metal-organic framework particles, followed by static gelation. The silk fibroin aqueous solution has a mass concentration of 0.5-20 wt%, the inulin aqueous solution has a mass concentration of 1-40 wt%, and the metal-organic framework particle ethanol dispersion has a concentration of 0.2-20 mg / g; the mass ratio of the silk fibroin aqueous solution to the inulin aqueous solution is 1:5-5:1; the mass ratio of the total mass of the silk fibroin aqueous solution and the inulin aqueous solution to the mass of the metal-organic framework particle ethanol dispersion is 9.5:0.5-2:8; the inulin is long-chain inulin with a degree of polymerization of not less than 10; the metal-organic framework particles are selected from one or more of ZIF-8, ZIF-90, MOF-5, MIL-53 (Fe), MIL-101 (Fe), UiO-66 (Zr), MIL-156 (Ca), Mg-MOF, and HKUST-1 (Cu) particles.

2. The silk fibroin-inulin-supported metal-organic framework composite hydrogel according to claim 1, characterized in that, The settling temperature is 4-60 ℃; the settling time is 1-20 min.

3. The silk fibroin-inulin-supported metal-organic framework composite hydrogel according to claim 1, characterized in that, The particle size of the metal-organic framework particles is 0.05-20 μm.

4. The silk fibroin-inulin-supported metal-organic framework composite hydrogel according to claim 1, characterized in that, The silk fibroin-inulin-loaded metal-organic framework composite hydrogel has a multi-level porous structure, including micropores and mesopores provided by the metal-organic framework particles, as well as macropores formed by the cross-linked network of silk fibroin and inulin.

5. A method for preparing the silk fibroin-inulin-supported metal-organic framework composite hydrogel according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Mix the silk fibroin aqueous solution with the inulin aqueous solution to obtain a mixed aqueous solution; (2) Disperse the metal-organic framework particles in ethanol to obtain a metal-organic framework particle ethanol dispersion; (3) The mixed aqueous solution is mixed with the ethanol dispersion of metal-organic framework particles and then allowed to stand. Ethanol is used to induce the conformational change of silk fibroin and promote the precipitation of inulin. The silk fibroin-inulin-loaded metal-organic framework composite hydrogel is obtained by gelation.

6. The preparation method according to claim 5, characterized in that, In step (1), the preparation method of the silk fibroin aqueous solution includes the following steps: degumming raw silk and dissolving it in lithium bromide aqueous solution, removing salt impurities by dialysis, and then concentrating it to obtain the silk fibroin aqueous solution.

7. The application of the silk fibroin-inulin-supported metal-organic framework composite hydrogel according to any one of claims 1-4 in the preparation of a drug delivery system.

8. The application according to claim 7, characterized in that, The silk fibroin-inulin-loaded metal-organic framework composite hydrogel can be used as an oral drug carrier or an injectable drug carrier.

9. The application according to claim 8, characterized in that, The oral drug carrier contains a target drug, which is selected from one or more of hydrophilic drugs, lipophilic drugs, polypeptide drugs, nucleic acid drugs, and protein drugs.

10. The application according to claim 9, characterized in that, When the target drug is a lipophilic drug, the following steps are used to encapsulate it in the silk fibroin-inulin-loaded metal-organic framework composite hydrogel: P1. Disperse metal-organic framework particles in an organic solvent to adsorb and encapsulate the lipophilic drug; P2. After removing the organic solvent, the metal-organic framework particles loaded with lipophilic drugs are dispersed in ethanol to obtain a drug-loaded metal-organic framework particle ethanol dispersion; the silk fibroin aqueous solution is mixed with the inulin aqueous solution to obtain a mixed aqueous solution. P3. The ethanol dispersion of the drug-loaded metal-organic framework particles is mixed with the mixed aqueous solution and allowed to stand. After gelation, a silk fibroin-inulin-loaded metal-organic framework composite hydrogel loaded with lipophilic drugs is obtained.

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