A covalent organic framework material, a preparation method thereof, an oral insulin delivery system and application thereof

Abstract

The application discloses a kind of covalent organic framework material and preparation method thereof, oral insulin delivery system and application.It provides a kind of keratin modification covalent organic framework material and preparation method thereof, oral insulin delivery system and application, belong to biological medicine technical field.The method comprises: condensation reaction is carried out to aldehyde group monomer and amino monomer in solvent, and covalent organic framework is obtained;Covalent organic framework is dispersed in buffer solution, and drug loading is carried out by adding insulin solution, and drug-loaded complex is obtained;Add keratin aqueous solution to drug-loaded complex, and be coated by electrostatic self-assembly, after stirring, freeze-drying.The application also provides the keratin modification covalent organic framework material with core-shell structure prepared by the above method, and the oral insulin delivery system comprising the material and insulin.The oral insulin delivery system has excellent gastrointestinal stability and glucose concentration-dependent release characteristics, and has good application prospect in the preparation of drugs for treating diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a covalent organic framework material and its preparation method, an oral insulin delivery system and its applications. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia, and the number of patients worldwide continues to rise. Insulin, as one of the most effective drugs for controlling blood sugar, has long relied on subcutaneous injection. However, injection not only causes pain and inconvenience for patients but also fails to mimic the physiological secretion pathway of insulin from the portal vein into the liver, easily leading to complications such as peripheral hyperinsulinemia. Oral insulin is considered an ideal alternative due to its convenient administration, high patient compliance, and better alignment with physiological metabolic patterns.

[0003] Covalent organic frameworks are a class of crystalline porous polymer materials formed by organic structural units linked by covalent bonds. These materials possess characteristics such as high specific surface area, regular pore structure, good chemical stability, and strong structural designability, showing broad application prospects in fields such as gas adsorption, catalysis, sensing, and drug delivery.

[0004] In the field of oral protein drug delivery, existing delivery carriers mainly face the following technical bottlenecks: First, protein drugs have large molecular weights and specific spatial conformations, and the pore size of existing carriers is not well matched with the size of drug molecules, resulting in low drug loading efficiency; Second, the strong acidic conditions and the presence of digestive enzymes in the gastrointestinal environment can easily lead to denaturation and inactivation of protein drugs, and delivery carriers need to maintain structural integrity and effectively protect drug activity in a complex physiological environment; Third, after oral administration, drugs are difficult to efficiently cross the intestinal mucosal barrier and enter the bloodstream, resulting in generally low bioavailability, which is difficult to meet clinical treatment needs.

[0005] Therefore, developing a novel protein drug delivery carrier that combines high drug loading efficiency, gastrointestinal stability, and good oral absorption performance has significant research value and clinical significance. Summary of the Invention

[0006] The purpose of this invention is to provide a covalent organic framework material that addresses the problems of insufficient stability of existing oral insulin delivery carriers in the gastrointestinal environment, low protein drug loading efficiency, and difficulty in achieving long-term hypoglycemic effects.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a keratin-modified covalent organic framework material, comprising the following steps: The condensation reaction of aldehyde monomers and amino monomers in a solvent is followed by washing, separation, and drying to obtain a covalent organic framework. The covalent organic framework was dispersed in a buffer solution, and then an insulin solution was added for drug loading to obtain a drug-loaded complex. A keratin aqueous solution was added to the drug-loaded complex, and the complex was coated by electrostatic self-assembly. After stirring, the complex was freeze-dried to obtain a keratin-modified covalent organic framework material.

[0008] Preferably, the solvent is selected from ionic liquids or organic mixed solvents; the ionic liquid is 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt, and the organic mixed solvent is a mixture of 1,4-dioxane and mesitylene; deionized water or acetic acid is added as a regulator in the condensation reaction, wherein the volume of deionized water added accounts for 0% to 50% of the total volume of the reaction system, and the concentration of acetic acid is 0.1 to 2.0 M.

[0009] Preferably, the aldehyde monomer is selected from pyromellitic pyrogallol or trialdehyde pyrogallol, and the amino monomer is selected from tris(4-aminophenyl)benzene or diaminoterphenyl; the mass ratio of the aldehyde monomer to the amino monomer in the condensation reaction is 1:1.5 to 1:2.5, and the condensation reaction time is 12 to 96 hours.

[0010] Preferably, the concentration of the insulin solution is 2-6 mg / mL; the mass concentration of the keratin aqueous solution is 4%-6%; and the stirring time is 1-4 hours.

[0011] The present invention also provides a keratin-modified covalent organic framework material, which is prepared by the above preparation method. The material has a core-shell structure, wherein the covalent organic framework is the core and the keratin is the shell.

[0012] Preferably, the covalent organic framework has a pore size of 0.5~5nm.

[0013] Preferably, the particle size of the material is 100 nm to 500 nm; the zeta potential of the material at pH 7.4 is -6.7 mV to -7.27 mV, and the zero charge point of the material is at pH... pzc It is 6.5.

[0014] The present invention also provides an oral insulin delivery system comprising the above-described keratin-modified covalent organic framework material and insulin loaded therein, the system having glucose concentration-dependent release characteristics.

[0015] Preferably, after the system is incubated in simulated gastric juice at pH 2.0 for 2 hours, the insulin release is less than 20%.

[0016] The present invention also provides the application of the above-described oral insulin delivery system in the preparation of drugs for treating diabetes.

[0017] The beneficial effects of this invention are: The covalent organic framework material provided by this invention possesses a regular pore structure and tunable pore size. The 3.06 nm pore size of the covalent organic framework is highly matched to the kinetic dimensions of the insulin molecule, achieving a drug loading capacity of 110 mg / g and an encapsulation efficiency of 89.88%. After incubation for 2 hours in simulated gastric juice at pH 2.0, the keratin-modified delivery system showed insulin release of less than 20%, while after 48 hours in PBS buffer containing 5 mg / mL glucose, insulin release exceeded 90%. In a type 1 diabetic rat model, oral administration of the RT~3~K-ins group resulted in sustained blood glucose reduction for up to 12 hours, with a relative bioavailability of 15.90%, and liver glycogen content recovered to 96.28% of the normal group. This invention represents a strategic evolution of covalent organic frameworks from surface modification to structural regulation, providing a new technical pathway for the design of oral protein drug delivery systems and showing broad application prospects in the field of diabetes treatment. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the design, synthesis, and oral insulin delivery of keratin-coated RT-COFs. Figure 2 A: PXRD patterns of RT-COF1 (ab), RT-COF2 (c), and RT-COF3 under different reaction conditions; B: PXRD and Pawley refinement comparison of RT-COF1 (a), RT-COF2 (b), and RT-COF3 (c); Figure 3 A: SEM images of RT-COF1, RT-COF2, RT-COF3 and RT1K-ins, RT2K-ins, and RT3K-ins; B: FE-TEM images of RT-COF1, RT-COF2, and RT-COF3; Figure 4 (a) RT-COF1; (b) RT-COF2; (c) RT-COF3: A: FT-IR spectrum; B: solid-state 13C CP-MAS NMR spectrum; Figure 5 (a) RT-COF1; (b) RT-COF2; (c) RT-COF3: (A) N2 adsorption-desorption isotherm; (B) BET plot; (C) TAG plot; Figure 6 Pore ​​size distribution maps of (A) RT-COF1; (B) RT-COF2; (C) RT-COF3 calculated by non-density functional theory (NLDFT); Figure 7(A) In vitro insulin release curves of RT1K-ins, RT2K-ins, and RT3K-ins under different pH sequence conditions (2 h at pH 2.0; 22 h at pH 6.8); (B) Cumulative in vitro insulin release of RT1K-ins, RT2K-ins, and RT3K-ins at 37°C and in PBS (10 mM, pH 7.4) at different glucose concentrations (0, 1, 3, and 5 mg / mL); (C) CD spectra of insulin release in each group; Figure 8 The toxicity of RT1K-ins, RT2K-ins and RT3K-ins to (A) Caco-2 cells; (B) LO2 cells; (C) Huh7 cells; Figure 9 (A) TEER and (B) ALP activity assays of Caco-2 cell monolayers at different growth times; (C) TEER of Caco-2 cells treated with different formulations; (D) P transported by RTK-ins across Caco-2 cells. app value; Figure 10 Changes in (A) body weight and (B) blood glucose levels over time in type 1 diabetes patients treated with different formulations of RTK-ins; Figure 11 Effects of different formulations administered continuously for 7 or 14 days on (A) liver and (B) kidney of diabetic rats. Detailed Implementation

[0019] This invention provides a method for preparing a keratin-modified covalent organic framework material, comprising the following steps: condensing an aldehyde monomer and an amino monomer in a solvent, followed by washing, separation, and drying to obtain a covalent organic framework; dispersing the covalent organic framework in a buffer solution, adding an insulin solution for drug loading to obtain a drug-loaded complex; adding an aqueous keratin solution to the drug-loaded complex, encapsulating it through electrostatic self-assembly, stirring, and then freeze-drying to obtain the keratin-modified covalent organic framework material.

[0020] In this invention, the condensation reaction refers to the process of forming an imine bond (C=N) between an aldehyde monomer and an amino monomer through a Schiff base reaction, which can be carried out at room temperature. The washing, separation, and drying are conventional post-processing operations in the art. Washing can be performed multiple times using tetrahydrofuran, anhydrous ethanol, or a combination of both. Separation can be achieved by centrifugation or filtration. Drying can be carried out under vacuum at 40-60°C. The buffer solution is used to maintain the pH environment of the solution to facilitate the preservation of insulin activity; conventional protein preservation buffers such as citrate buffer can be used. The electrostatic self-assembly refers to the process by which keratin molecules with opposite charges spontaneously form a coating layer on the surface of a covalent organic framework through electrostatic attraction. The freeze-drying process involves freezing an aqueous sample at low temperature and then directly sublimating the ice crystals in a vacuum environment to obtain a dry powder. This method can effectively maintain the activity of the protein drug and improve the storage stability of the product. In some embodiments of this invention, the condensation reaction time can be 12-96 hours, preferably 48-72 hours, and more preferably 72 hours.

[0021] In this invention, the solvent is selected from ionic liquids or organic mixed solvents. The ionic liquid refers to a salt compound composed entirely of anions and cations that is liquid at or near room temperature, possessing extremely low vapor pressure, good thermal stability, and controllable solubility. In this invention, the ionic liquid is 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt. The organic mixed solvent refers to a homogeneous solvent system formed by mixing two or more organic solvents in a certain proportion. In this invention, the organic mixed solvent is a mixture of 1,4-dioxane and mesitylene, which can be mixed in a 1:1 volume ratio. Deionized water or acetic acid is added as a regulator in the condensation reaction. The regulator controls the reaction rate and the crystallinity of the product. The volume of deionized water added accounts for 0% to 50% of the total volume of the reaction system, preferably 5% to 40%, more preferably 10% to 30%. The concentration of acetic acid is 0.1 to 2.0 M, preferably 0.5 to 1.5 M, more preferably 1.0 M.

[0022] In this invention, the aldehyde monomer refers to an organic compound whose molecular structure contains at least two aldehyde (-CHO) functional groups, which can undergo a condensation reaction with an amino monomer to form a covalent organic framework linked by imine bonds. The aldehyde monomer is selected from trimesonol or trialdehyde-resorcinol. Triesonol's chemical name is 1,3,5-benzenetriformol, and its molecular structure contains three symmetrically distributed aldehyde groups. Trialdehyde-resorcinol's chemical name is 2,4,6-trihydroxy-1,3,5-benzenetriformol, and its molecular structure contains both three aldehyde groups and three hydroxyl groups. The amino monomer refers to an organic compound whose molecular structure contains at least two amino (-NH2) functional groups. The amino monomer is selected from tris(4-aminophenyl)benzene or diaminoterphenyl. Tris(4-aminophenyl)benzene's chemical name is 1,3,5-tris(4-aminophenyl)benzene, and its molecular structure contains three para-substituted aminophenyl groups. The chemical name of diaminoterphenyl is [1,1':4',1''-terphenyl]-4,4''-diamine, and its molecular structure contains two amino groups. In the condensation reaction, the mass ratio of the aldehyde monomer to the amino monomer is 1:1.5 to 1:2.5, preferably 1:1.8 to 1:2.3, and more preferably 1:2. The condensation reaction time is 12 to 96 hours, preferably 24 to 72 hours, and more preferably 48 to 72 hours. In some embodiments of the present invention, when the aldehyde monomer is pyromellitic pyroxenaldehyde and the amino monomer is tris(4-aminophenyl)benzene, the pore size of the resulting covalent organic framework is approximately 1.05 nm; when the aldehyde monomer is trialdehyde phloroglucinol and the amino monomer is tris(4-aminophenyl)benzene, the pore size of the resulting covalent organic framework is approximately 1.38 nm; and when the aldehyde monomer is trialdehyde phloroglucinol and the amino monomer is diaminoterphenyl, the pore size of the resulting covalent organic framework is approximately 3.06 nm. In some embodiments of the present invention, the condensation reaction further includes a step of ultrasonically dispersing the aldehyde monomer and amino monomer in a solvent for 20-40 minutes. In some embodiments of the present invention, when the aldehyde monomer is trialdehyde phloroglucinol and the amino monomer is tris(4-aminophenyl)benzene, the reaction further includes a step of stirring and pre-reacting at room temperature for 1-6 hours after adding the amino monomer solution, followed by a static reaction.

[0023] In this invention, the concentration of the insulin solution is 2-6 mg / mL, preferably 3-5 mg / mL, and more preferably 4 mg / mL. Insulin is a protein hormone secreted by pancreatic β-cells that has a hypoglycemic effect, and its molecular dynamics size is approximately 2.5-3.0 nm. The mass concentration of the keratin aqueous solution is 4%-6%, preferably 4.5%-5.5%, and more preferably 5%. Keratin is an insoluble fibrous structural protein derived from animal tissues such as hair, hooves, and feathers. Its molecular chain contains a large number of charged groups such as carboxyl and amino groups, and it can form a polyelectrolyte with good biocompatibility and degradability in aqueous solution. The stirring time is 1-4 hours, preferably 1.5-3 hours, and more preferably 2 hours.

[0024] The present invention also provides a keratin-modified covalent organic framework material, which is prepared by the above preparation method. The material has a core-shell structure, wherein the covalent organic framework is the core and the keratin is the shell.

[0025] In this invention, the core-shell structure refers to a composite structure consisting of an inner core and an outer shell, wherein the core is a covalent organic framework material and the shell is a keratin coating layer. The pore size of the covalent organic framework is 0.5–5 nm, preferably 1.0–4.0 nm, and more preferably 1.0–3.5 nm. In some embodiments of this invention, the pore size of the covalent organic framework is 1.05 nm, 1.38 nm, or 3.06 nm, and more preferably 3.06 nm. When the pore size of the covalent organic framework is 3.06 nm, it highly matches the kinetic size (2.5–3.0 nm) of the insulin molecule, enabling higher drug loading and encapsulation efficiency. The particle size of the material is 100 nm–500 nm, preferably 150 nm–450 nm, and more preferably 200 nm–400 nm. The material exhibits a Zeta potential ranging from -6.7 mV to -7.27 mV at pH 7.4, indicating that its surface carries a certain amount of negative charge. This negative charge is beneficial for reducing non-specific protein adsorption and prolonging in vivo circulation time. The zero-charge point (pHpzc) of the material is 6.5, meaning that the material surface carries a positive charge when the ambient pH is below 6.5 and a negative charge when the ambient pH is above 6.5.

[0026] The present invention also provides an oral insulin delivery system comprising the above-described keratin-modified covalent organic framework material and insulin loaded therein, the system having glucose concentration-dependent release characteristics.

[0027] In this invention, the oral insulin delivery system refers to a drug delivery system that uses a keratin-modified covalent organic framework material as a carrier and insulin as the therapeutic active ingredient, administered orally. The glucose concentration-dependent release characteristic refers to the intelligent response release behavior of insulin in the delivery system, where the release rate is relatively slow in low glucose concentration environments and significantly accelerated in high glucose concentration environments. This characteristic facilitates the automatic adjustment of the insulin release rate according to the body's blood glucose level, achieving stable blood glucose control.

[0028] In some embodiments of the invention, after incubation for 2 hours in simulated gastric juice at pH 2.0, the insulin release is less than 20%. Methods for preparing simulated gastric juice are well known in the art, typically involving adjusting the pH of deionized water to 1.0-3.0 with hydrochloric acid, and optionally adding pepsin to simulate the gastric digestive environment. In some embodiments of the invention, after incubation for 48 hours in PBS buffer (pH 7.4) containing 5 mg / mL glucose, the insulin release is greater than 90%.

[0029] The present invention also provides the application of the above-described oral insulin delivery system in the preparation of drugs for treating diabetes.

[0030] In this invention, the diabetes includes type 1 diabetes. Type 1 diabetes, also known as insulin-dependent diabetes mellitus, is caused by the destruction of pancreatic β cells by the autoimmune system, leading to an absolute deficiency of insulin. Patients require exogenous insulin supplementation to maintain normal blood glucose levels. The oral insulin delivery system of this invention can be administered orally, absorbed through the gastrointestinal tract, and enters the bloodstream to exert a hypoglycemic effect. In some embodiments of this invention, the dosage of the oral insulin delivery system is 75 IU / kg (in insulin). In some embodiments of this invention, the application includes the preparation of a drug for sustained hypoglycemic action lasting more than 12 hours. In some embodiments of this invention, the application includes the preparation of an oral insulin drug with a relative bioavailability of 15.90%. In some embodiments of this invention, the application includes the preparation of a drug for promoting liver glycogen synthesis, restoring liver glycogen content to 96.28% of the normal level. In some embodiments of this invention, the oral insulin delivery system, after oral administration, can form an insulin reservoir in the portal vein, promoting insulin accumulation in the liver and mimicking the physiological insulin secretion pathway.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The experimental materials used in the following examples are as follows: (1) Experimental reagents: Trimethylbenzene, trialdehyde-resorcinol, tris(4-aminophenyl)benzene, diaminoterphenyl, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIm][NTf2]), tetrahydrofuran, N,N-dimethylamide, 1,4-dioxane, mesitylene, glacial acetic acid, dichlorobenzene, and formaldehyde solution were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0033] (2) Experimental apparatus: Vacuum drying oven DZF-6020 Shanghai Yiheng Scientific Instruments Co., Ltd., high-pressure steam sterilizer MLS-3750 Shanghai Shenan Medical Instruments Co., Ltd., vacuum pump HP-01 Tianjin Hengao Technology Development Co., Ltd., fully automated cell counter SCC-M630 Wuhan Saiweier Biotechnology Co., Ltd., flow cytometer Cytoflex Beckman Coulter, Inc., solid-state NMR 600 MHz AVANCE III Bruker, atomic force microscope Dimension FastScan / Icon Bruker.

[0034] (3) Experimental cells: Caco-2 cells and Huh7 cells were purchased from Wuhan Sewell Biotechnology Co., Ltd. LO2 cells were donated by the School of Pharmacy of Shihezi University.

[0035] (4) Experimental animals: SPF-grade SD rats, 6 weeks old, weighing 80-100 g, were purchased from Henan SciBest Biotechnology Co., Ltd. (License No.: SCXK (Yu) 2020-0005). Caco-2 cells and LO2 cells were purchased from Shanghai CyberKang Biotechnology. This study strictly followed the relevant provisions of the "Guidelines for the Care and Use of Laboratory Animals" issued by the National Research Council of China, and all experimental protocols were approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Shihezi University School of Medicine (Ethics Approval No.: A2023-098-01).

[0036] A schematic diagram illustrating the design, synthesis, and oral insulin delivery of keratin-coated RT-COFs is shown below. Figure 1 As shown.

[0037] 1.1 Synthesis of RT-COF1 Trimethylbenzenesulfonaldehyde (TFB) and tris(4-aminophenyl)benzene (TAPB) were uniformly mixed, with a mass ratio of TFB to TAPB of approximately 1:2 to 1:2.5. The mixture was placed in a reaction vessel, and 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ([BMIm][NTf2]) was added as a solvent. The mixture was ultrasonically dispersed for 20–40 min. Subsequently, deionized water was added as a regulator, with the volume of water added accounting for 20% of the total volume of the reaction system. The reaction was allowed to stand at room temperature for 60 °C. After the reaction was completed, the precipitate was collected by centrifugation, washed several times successively with tetrahydrofuran (THF) and anhydrous ethanol, and dried under vacuum at 40–60 °C to obtain RT-COF1, with a yield of 92.62%.

[0038] 1.2 Synthesis of RT-COF2 Trialdehyde phloroglucinol (MOP) was dissolved in [BMIm][NTf2], and acetic acid was added as a catalyst, with the concentration of acetic acid controlled in the range of 0.1–2.0 M. Tris(4-aminophenyl)benzene (TAPB) was dissolved in N,N-dimethylformamide (DMF), and this solution was then added dropwise to the MOP mixture. The mass ratio of MOP to TAPB was approximately 1:1.75. After a pre-reaction at room temperature with stirring for 3 h, the reaction was allowed to continue for 72 h at room temperature. After the reaction was complete, the precipitate was collected by centrifugation, washed several times with THF and anhydrous ethanol, and dried under vacuum at 45 °C to obtain RT-COF2, with a yield of 93.73%.

[0039] 1.3 Synthesis of RT-COF3 Trialdehyde phloroglucinol (MOP) and diaminoterphenyl (DATP) were uniformly mixed at a mass ratio of 1:2. A mixed solvent of 1,4-dioxane and mesitylene (1:1 by volume) was added to the mixture, along with acetic acid as a catalyst at a concentration of 1.0 M. The reaction was allowed to proceed at room temperature for 84 h. After the reaction was complete, the precipitate was collected by centrifugation, washed several times with THF and anhydrous ethanol, and dried under vacuum at 45 °C to obtain RT-COF3, with a yield of 73.06%.

[0040] 2.1 Insulin Load (RT-ins) The drug-loaded system was prepared using an impregnation method. Approximately 10 mg of the RT-COFs material (RT1, RT2, RT3) prepared in Example 1 was accurately weighed and dispersed in CBS buffer, and an appropriate amount of insulin aqueous solution (concentration approximately 4 mg / mL) was added. The mixture was stirred continuously at 500 rpm for 12 h at room temperature to allow sufficient diffusion of insulin into the COFs channels. After centrifugation, the surface-adsorbed drug was removed by washing with deionized water, yielding the drug-loaded complex.

[0041] The loading results showed that the drug loading (LC) of RT1-ins, RT2-ins, and RT3-ins was approximately 25 mg / g, 70 mg / g, and 110 mg / g, respectively; the encapsulation efficiency (EE) was approximately 81.2%, 87.5%, and 88.4%, respectively. Among them, RT3-ins benefited from the matching effect between its larger pore size and the size of insulin molecules, resulting in a significantly higher drug loading than RT1-ins and RT2-ins.

[0042] 2.2 Keratin coating (RTK-ins) Take the above RT-ins dispersion, add an appropriate amount of keratin aqueous solution (5% by mass), and continue stirring for 2 hours. The keratin layer is coated on the carrier surface by electrostatic self-assembly. The resulting product is freeze-dried and stored at -20℃, which is the keratin-coated insulin-loaded nanodelivery system (RT1K-ins, RT2K-ins and RT3K-ins).

[0043] 3.1 Structural analysis of RT-COFs Two possible COF models were constructed using Materials Studio 2019 software: an AA-stacked layered configuration and an AB-stacked staggered configuration. The Forcite module was then used for geometric optimization of the model's cell parameters, atomic coordinates, and total energy. Finally, Pawley refinement was performed using the Reflex module, iteratively optimizing the lattice parameters until the RWP value converged. During the refinement process, the Pseudo-Voigt peak shape function was used for full-spectrum fitting, and the Berr-Baldinozzi function was used for asymmetry correction. The results are shown in the appendix. Figure 2 As shown, the experimental PXRD peak positions of RT-COF1, RT-COF2, and RT-COF3 are basically consistent with the AA stacking simulation spectra.

[0044] 3.2 Phase and Morphology Analysis PXRD testing: A Cu-Kα radiation source was used, with a tube voltage of 40 kV and a tube current of 130 mA. Powder X-ray diffraction data were acquired in the range of 5°–40° (2θ) at a scan rate of 0.3° / min. Figure 2 As shown in Figure A, the sharp diffraction peaks in the diffraction pattern confirm that RT-COFs are highly crystalline organic porous materials.

[0045] Microstructure: The microstructure was observed using a field emission scanning electron microscope (SEM) at an appropriate accelerating voltage; lattice fringes and selected area electron diffraction were observed using a field emission transmission electron microscope (TEM). (See attached image) Figure 3 SEM images of A show that all three COFs and three RTK-ins exhibit relatively uniform micron-sized spherical shapes; (attached) Figure 3The TEM image of B further shows that all three COFs monomers have an approximately circular morphology.

[0046] 3.3 Chemical Structure Characterization The chemical bonding and functional groups of the material were confirmed using Fourier transform infrared spectroscopy (FT-IR) and solid-state ¹³C cross-polarized magic angle rotating nuclear magnetic resonance spectroscopy (¹³CCP-MAS NMR). (See attached image.) Figure 4 As shown in Figure A, the FT-IR spectrum shows a characteristic absorption peak of an imine bond (C=N), and... Figure 4 The ¹³C NMR spectrum of B showed a corresponding characteristic chemical shift at approximately 158 ppm, which together confirmed the successful construction of the COF framework. 3.4 Pore Structure and Thermal Stability Gas adsorption: Using a fully automated specific surface area analyzer, after high-temperature degassing, nitrogen adsorption-desorption tests were performed at 77 K. Specific surface area was calculated based on the Brunauer-Emmett-Teller (BET) model. Thermogravimetric analysis (TGA): Using a thermogravimetric analyzer, under a nitrogen atmosphere (appropriate flow rate) (30 mL / min), the temperature was increased from room temperature to 900 °C at a rate of 5 °C / min to examine the thermal stability of the material. The results show that the RT-COFs framework exhibits good thermal stability below 300 °C. Figure 5 The pore size distribution was analyzed using nonlocal density functional theory (NLDFT). Figure 6 ).

[0047] 4.1 In vitro release and stability Release behavior was investigated using dialysis. RTK-ins were placed in dialysis bags and immersed in simulated gastric fluid (SGF, pH 2.0), simulated intestinal fluid (SIF, pH 6.8), and PBS buffer containing different concentrations of glucose (pH 7.4), respectively, and incubated at 37°C with shaking. Samples were taken periodically and an equal volume of fresh medium was added. The concentration of insulin in the release medium was determined by HPLC.

[0048] As attached Figure 7 As shown, within 24 h of sequential SGF / SIF incubation, the cumulative insulin release of RTK-ins was consistently below 20%, demonstrating excellent gastric acid stability. In a physiological environment (PBS, pH 7.4), when glucose concentration was at a physiologically high level (approximately 3 mg / mL), the cumulative release rate of RT3K-ins reached 95.66% within 48 h. Furthermore, circular dichroism (CD) analysis confirmed that the secondary structure of the released insulin remained unchanged, indicating that this delivery system effectively protects the biological activity of insulin.

[0049] 4.2 Cellular Experiments Cytotoxicity: The effect of RTK-ins on the viability of Caco-2, LO2, and Huh7 cells was detected using the CCK-8 assay. Experimental setup: 5 × 10⁶ cells were seeded in 96-well plates. 4 Single-cell suspensions of 100 μL per well were prepared and incubated with different concentrations of RTK-ins for 6 h. Results are attached. Figure 8 The results showed that even at a high concentration of 1000 μg / mL, the cell survival rate was still higher than 85%.

[0050] Cellular uptake and transport: Apparent permeability coefficient (P) determined using the Transwell model. app (Attached) Figure 9 The display shows that RT3K-ins' P app The value increased to 5.6 times that of free insulin.

[0051] 5.1 Establishment of animal models SPF-grade SD rats were selected and acclimatized for 7 days before a type 1 diabetes model was established by intraperitoneal injection of streptozotocin (STZ). Modeling method: After fasting for an appropriate period, rats were given a single intraperitoneal injection of STZ (approximately 50–70 mg / kg). Fasting blood glucose was measured 5 days after intervention. Rats with significantly higher than normal blood glucose levels (e.g., >16.7 mmol / L) and accompanied by typical diabetic symptoms were considered to have successfully established the model.

[0052] 5.2 Pharmacodynamic Evaluation Diabetic rats that successfully developed the model were randomly divided into three groups: oral free insulin (oral control group), subcutaneous insulin injection (positive control group), and oral RTK-ins (experimental group, dose of 75 IU / kg). Blood glucose levels were monitored at different time points after administration.

[0053] As attached Figure 10 and Figure 11 As shown, the oral RT3K-ins group maintained a sustained blood glucose-lowering effect for approximately 12 hours, with a stable and prolonged effect. Compared to the subcutaneous injection group, the relative bioavailability of RT3K-ins was significantly improved, reaching 15.90%. Furthermore, RT3K-ins significantly promoted hepatic glycogen synthesis, restoring liver glycogen content to approximately 96.28% of the normal group level, demonstrating good liver-targeted accumulation and bioefficacy.

[0054] As shown in the above embodiments, this invention provides a keratin-modified covalent organic framework material with controllable pore size and its oral insulin delivery system. This invention successfully constructed a series of covalent organic framework materials using a room-temperature synthesis strategy and systematically studied the influence of carriers with different pore sizes on insulin loading capacity. Drug loading experiments showed that the degree of matching between pore size and insulin molecule size is a key factor determining drug loading efficiency. Release experiments showed that keratin coating effectively inhibited the burst release behavior of the carrier in an acidic environment, while endowing the delivery system with glucose concentration-dependent intelligent release characteristics, and the released insulin maintained its complete secondary structure. Cell experiments confirmed that the delivery system has good biocompatibility and can effectively promote insulin crossing the intestinal epithelial barrier, improving the efficiency of transmembrane drug transport. Animal experiments showed that after oral administration, the delivery system can achieve a sustained and stable hypoglycemic effect, and form drug accumulation in liver tissue, promoting liver glycogen synthesis, demonstrating good liver-targeted delivery capability. Safety evaluation results showed that no significant liver and kidney function damage or intestinal tissue lesions were observed after continuous administration. This invention provides a feasible technical solution for the efficient delivery of oral protein drugs.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a keratin-modified covalent organic framework material, characterized in that, Includes the following steps: The condensation reaction of aldehyde monomers and amino monomers in a solvent is followed by washing, separation, and drying to obtain a covalent organic framework. The covalent organic framework was dispersed in a buffer solution, and then an insulin solution was added for drug loading to obtain a drug-loaded complex. A keratin aqueous solution was added to the drug-loaded complex, and the complex was coated by electrostatic self-assembly. After stirring, the complex was freeze-dried to obtain a keratin-modified covalent organic framework material.

2. The preparation method according to claim 1, characterized in that, The solvent is selected from ionic liquids or organic mixed solvents; the ionic liquid is 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imine salt, and the organic mixed solvent is a mixture of 1,4-dioxane and mesitylene; deionized water or acetic acid is added as a regulator in the condensation reaction, the volume of deionized water added accounts for 0% to 50% of the total volume of the reaction system, and the concentration of acetic acid is 0.1 to 2.0 M.

3. The preparation method according to claim 1, characterized in that, The aldehyde monomer is selected from pyromellitic pyrogallol or trialdehyde pyrogallol, and the amino monomer is selected from tris(4-aminophenyl)benzene or diaminoterphenyl; the mass ratio of the aldehyde monomer to the amino monomer in the condensation reaction is 1:1.5 to 1:2.5, and the condensation reaction time is 12 to 96 hours.

4. The preparation method according to claim 1, characterized in that, The concentration of the insulin solution is 2-6 mg / mL; the mass concentration of the keratin aqueous solution is 4%-6%; and the stirring time is 1-4 hours.

5. A keratin-modified covalent organic framework material, characterized in that, The material is prepared by the method according to any one of claims 1 to 4, and the material has a core-shell structure, wherein the covalent organic framework is the core and the keratin is the shell.

6. The material according to claim 5, characterized in that, The covalent organic framework has a pore size of 0.5~5 nm.

7. The material according to claim 5, characterized in that, The material has a particle size of 100 nm to 500 nm; the zeta potential of the material at pH 7.4 is -6.7 mV to -7.27 mV, and the zero charge point of the material is at pH... pzc It is 6.

5.

8. An oral insulin delivery system, characterized in that, The system comprises a keratin-modified covalent organic framework material as described in any one of claims 5 to 7 and insulin loaded therein, the system having glucose concentration-dependent release characteristics.

9. The oral insulin delivery system according to claim 8, characterized in that, After incubating the system in simulated gastric juice at pH 2.0 for 2 hours, the insulin release was less than 20%.

10. Use of the oral insulin delivery system of claim 8 or 9 in the preparation of a medicament for treating diabetes.

Images