A semeglutide-loaded hydrogel and a preparation method and application thereof

Abstract

The application belongs to the technical field of hydrogel preparation, and particularly relates to a hydrogel loaded with semaglutide and a preparation method and application thereof. The preparation method comprises the following steps: adding ferulic acid modified chitosan into deionized water to obtain a modified chitosan solution, adding oxidized dextran into deionized water, ultrasonic dissolving to obtain an oxidized dextran solution, adding semaglutide into the oxidized dextran solution to obtain an oxidized dextran mixed solution, and mixing the modified chitosan solution and the oxidized dextran mixed solution to obtain the hydrogel loaded with semaglutide. The hydrogel loaded with semaglutide has the advantages of short gelation time, good injection performance, non-toxicity and good biocompatibility. The hydrogel can significantly reduce myocardial interstitial fibrosis after myocardial infarction and improve the ejection capacity of the heart after myocardial infarction. The hydrogel can be used for preparing drugs for treating myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel preparation technology, specifically relating to a hydrogel loaded with smegglutinin, its preparation method, and its application. Background Technology

[0002] Currently, hydrogel drug delivery technology is mainly divided into natural hydrogel carriers and synthetic hydrogel carriers. The raw materials for natural hydrogels are primarily biological extracts (collagen, hyaluronic acid, gelatin, sodium alginate, chitosan, etc.). Crosslinking methods are divided into physical crosslinking: hydrogen bonds / ionic bonds (such as Ca²⁺, hydroxyl group monophosphate, and hydroxyl group monophosphate). 2+ Cross-linked hydrogels include sodium alginate and temperature-triggered cross-linking (thermally reversible gelatin); chemical cross-linking involves enzyme catalysis (transglutaminase cross-linking) and natural cross-linking agents (genipin). Major applications of natural hydrogels include myocardial patch injection and stem cell encapsulation and delivery (e.g., collagen gel-loaded mesenchymal stem cells).

[0003] The raw materials for synthesizing hydrogels mainly include: artificial polymers (polyethylene glycol / PEG, polyacrylic acid / PAA, poly(N-isopropylacrylamide / PNIPAM), etc.). Crosslinking methods include photocuring: UV-initiated polymerization of acrylate groups (e.g., PEG-DA); and click chemistry: highly specific reactions such as thiols-enes and tetraazine-norbornene. The main applications of synthetic hydrogels include: precision drug delivery systems (e.g., PEG-VEGF gels) and conductive myocardial repair hydrogels (polyaniline composite systems).

[0004] For example, the invention with application number 201811395263.1 discloses an injectable hydrogel, its preparation method and application; the invention with application number 202411008634.1 discloses a thermosensitive injectable controlled-release hydrogel and its application in the preparation of drugs for treating myocardial infarction; and the invention with application number 202411935292.8 discloses a composite hydrogel drug-eluting scaffold, its preparation method and application.

[0005] Existing drug-loaded hydrogels have the following main drawbacks: (1) insufficient biocompatibility; (2) uncontrollable degradation; and (3) drug burst release. Therefore, there is an urgent need to develop a hydrogel with good biocompatibility, controllable degradation, and long-term release. Summary of the Invention

[0006] Based on the above technical background, the main objective of this invention is to provide a hydrogel loaded with smegglutinin, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] The first aspect of this invention is to provide a method for preparing a hydrogel loaded with smegglutinin, the method comprising the following steps:

[0009] Step 1: Dissolve chitosan in a mixed solvent, stir well, and then add hydrochloric acid solution dropwise to obtain a chitosan solution;

[0010] Step 2: Dissolve ferulic acid in DMSO solution, stir well, blow high-purity nitrogen gas through it, and then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide dropwise for activation to obtain activated ferulic acid solution.

[0011] Step 3: Add the activated ferulic acid solution dropwise to the chitosan solution, react under stirring, then dialysis purification and vacuum freeze-drying to obtain ferulic acid modified chitosan;

[0012] Step 4: Dissolve dextran in deionized water, then add sodium periodate and stir magnetically. Then add ethylene glycol and continue stirring. Finally, dialyze and freeze dry under vacuum to obtain oxidized dextran.

[0013] Step 5: Under stirring conditions, ferulic acid-modified chitosan is added to deionized water to obtain a modified chitosan solution. Oxyglucan is added to deionized water and dissolved by sonication to obtain an oxidized dextran solution. Smegglutinin is added to the oxidized dextran solution to obtain an oxidized dextran mixed solution. The modified chitosan solution and the oxidized dextran mixed solution are mixed to obtain a hydrogel loaded with smegglutinin.

[0014] The steps described above are described in detail below.

[0015] In step 1, the mixed solvent is obtained by mixing deionized water and dimethyl sulfoxide.

[0016] Preferably, the mixed solvent is obtained by mixing deionized water and dimethyl sulfoxide in a volume ratio of (4-6):2.

[0017] More preferably, the mixed solvent is obtained by mixing deionized water and dimethyl sulfoxide in a volume ratio of 5:2.

[0018] The ratio of chitosan to mixed solvent added is 1g:(50-90)mL.

[0019] Preferably, the ratio of chitosan to mixed solvent is 1g:70mL.

[0020] Add hydrochloric acid solution dropwise until the solution becomes transparent, wherein the molar concentration of the hydrochloric acid solution is 0.5–2 mol / L.

[0021] Preferably, the molar concentration of the hydrochloric acid solution is 1 mol / L. This invention uses chitosan as the main raw material to prepare the hydrogel, which can improve the biocompatibility of the hydrogel.

[0022] In step 2, the ratio of ferulic acid to DMSO (dimethyl sulfoxide) solution is (350-400) mg: 20 mL.

[0023] Preferably, the ratio of ferulic acid to DMSO solution is 388 mg: 20 mL.

[0024] The blowing time with high-purity nitrogen is 20 to 45 minutes, preferably 30 minutes.

[0025] Blowing the solution with high-purity nitrogen can prevent the reactants from undergoing oxidation with oxygen in the air, thus avoiding the formation of byproducts.

[0026] The mass ratio of ferulic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is (350-400):(750-800):460.

[0027] Preferably, the mass ratio of ferulic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is 388:766:460.

[0028] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 460 mg N-hydroxysuccinimide were added dropwise to activate the carboxyl group on ferulic acid.

[0029] In step 3, the reaction conditions are: at 50–70°C, the reaction is stirred for 20–30 h.

[0030] Preferably, the reaction conditions are: stirring at 60°C for 24 h.

[0031] Under the above reaction temperature and reaction time, ferulic acid can be fully grafted.

[0032] The dialysis purification conditions are as follows: dialysis is performed using a dialysis bag, the dialysate is a weak acid solution, the weak acid solution is used for dialysis for the first 1 to 2 days, and deionized water is used for dialysis for the 2nd to 4th days. The dialysate is changed every 6 to 10 hours.

[0033] Preferably, the dialysis purification conditions are as follows: dialysis purification is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa, the dialysate is a weak acid solution, the weak acid solution is used for dialysis for the first 2 days, and deionized water is used for dialysis on the third day, with the dialysate being changed every 8 hours.

[0034] The vacuum freeze-drying temperature is -90 to -70°C, preferably -80°C.

[0035] In step 4, the molecular weight of the dextran is 60-80 kDa, preferably 70 kDa.

[0036] The mass ratio of dextran to sodium periodate is (1-3):1, preferably, the mass ratio of dextran to sodium periodate is 2:1.

[0037] After adding sodium periodate, an oxidation reaction occurs, and the solution turns orange-red.

[0038] The magnetic stirring conditions are as follows: magnetic stirring for 10 to 13 hours, preferably 12 hours, under room temperature and light-protected conditions.

[0039] The purpose of adding ethylene glycol is to terminate the oxidation reaction. After adding ethylene glycol, continue stirring for 1 to 3 hours, preferably for 2 hours.

[0040] The dialysis conditions are as follows: dialysis is performed using a dialysis bag, the dialysis solution is deionized water, the dialysis time is 2 to 4 days, and the dialysis solution is changed every 6 to 10 hours.

[0041] Preferably, the dialysis conditions are as follows: dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa, the dialysate is deionized water, the dialysis time is 3 days, and the dialysate is changed every 8 hours.

[0042] In step 5, the concentration of the modified chitosan solution is 1-3% (w / v).

[0043] Preferably, the concentration of the modified chitosan solution is 2% (w / v).

[0044] The concentration of the oxidized dextran solution is 3-7% (w / v).

[0045] Preferably, the concentration of the oxidized dextran solution is 5% (w / v).

[0046] The ratio of the amount of oxidized dextran solution to smegglutinin added is 1 mL: (2-3) mg.

[0047] Preferably, the ratio of the added oxidized dextran solution to smegglutinin is 1 mL: 2.4 mg.

[0048] The modified chitosan solution and the oxidized dextran mixed solution were mixed at a volume ratio of 1:(1~2), and the mixture was observed to gradually gel at room temperature.

[0049] Preferably, the modified chitosan solution and the oxidized dextran mixed solution are mixed at a volume ratio of 1:1.

[0050] After the modified chitosan solution (FACS) and oxidized dextran (ODex) are mixed, the -NH groups of FACS react with ODex to form a Schiff base (C=N) structure. The hydrogel described in this invention is a hydrogel loaded with smegglutinin based on Schiff base bond crosslinking.

[0051] The hydrogel described in this invention has the advantages of short gelation time and good injectability.

[0052] A second aspect of the present invention is to provide a hydrogel loaded with smegglutinin prepared by the preparation method described in the first aspect of the present invention.

[0053] The present invention relates to the use of the hydrogel loaded with smegglutinin as described in the second aspect of the present invention in the preparation of drugs for improving or treating myocardial infarction.

[0054] The beneficial effects of this invention are as follows:

[0055] (1) The preparation method described in this invention can successfully load semaglutide into a hydrogel, and the semaglutide is uniformly doped into the three-dimensional network of the hydrogel. Compared with the blank hydrogel (unloaded hydrogel), the semaglutide-loaded hydrogel prepared by this invention has a shorter gelation time, higher stability, and excellent shear-thinning behavior and rapid recovery ability. The hydrogel does not experience phase separation or jet instability during injection extrusion and has good injectability.

[0056] (2) The hydrogel loaded with semaglutide described in this invention can achieve long-term release of peptide drugs. The hydrogel releases semaglutide at a relatively fast rate in the early stage, and the cumulative release reaches 60% around the 8th day. The fast release rate in the early stage meets the pH change in the microenvironment during the acute phase of myocardial infarction. The hydrogel loaded with semaglutide described in this invention can achieve the goal of pH-based targeted therapy.

[0057] (3) The hydrogel loaded with smegglutinin described in this invention is non-toxic and has good biocompatibility. It can significantly reduce myocardial interstitial fibrosis after myocardial infarction. At the same time, the hydrogel can improve the heart's ejection capacity after myocardial infarction. It has a good therapeutic effect on myocardial infarction. The hydrogel can be used to prepare drugs for treating or improving myocardial infarction. Attached Figure Description

[0058] Figure 1 The image shows an inverted experimental photograph of the hydrogel loaded with smegglutinin prepared in Example 1, and a gelation time test graph of the hydrogels prepared in Comparative Example 1 and Example 1.

[0059] Figure 2 The Fourier transform infrared spectrum of the hydrogel loaded with smegglutinin prepared in Example 1 is shown.

[0060] Figure 3 The elemental composition of the hydrogel loaded with smegglutinin prepared in Example 1 and the hydrogel prepared in Comparative Example 1 are shown.

[0061] Figure 4 The curve showing the relationship between the cumulative release of smegglutinin and time in the hydrogel loaded with smegglutinin prepared in Example 1 is shown.

[0062] Figure 5 The bar chart shows the biocompatibility of the hydrogel loaded with smegglutinin prepared in Example 1 and the hydrogel prepared in Comparative Example 1.

[0063] Figure 6 Masson staining images of paraffin sections of rat hearts are shown, where: (a) is a schematic diagram of the staining results; (b) is a bar chart of the quantitative results (**P<0.01; ****P<0.0001).

[0064] Figure 7 Echocardiography and gross specimen of the heart of a rat are shown. Detailed Implementation

[0065] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0066] Example

[0067] The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention are commercially available.

[0068] Example 1

[0069] Dissolve 1g of chitosan powder in a mixture of 50ml deionized water and 20ml dimethyl sulfoxide (DMSO). Place the resulting mixture on a magnetic stirrer and stir until homogeneous. Slowly add 1mol / L hydrochloric acid solution (HCl) until the solution becomes transparent to obtain a chitosan solution.

[0070] Dissolve 388 mg of ferulic acid powder in 20 ml of DMSO solution, stir well at room temperature, and purge the solution with high-purity nitrogen for 30 min to prevent oxidation of the reactants with oxygen in the air. After purging, slowly add 766 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide, EDC) and 460 mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group on ferulic acid, thus obtaining an activated ferulic acid solution.

[0071] The activated ferulic acid solution was slowly added dropwise to the chitosan solution, and the mixture was stirred thoroughly on a magnetic stirrer at a constant temperature of 60°C for 24 hours to ensure sufficient grafting of ferulic acid. The ferulic acid-chitosan mixture was then placed in a dialysis bag (molecular weight cutoff of 3.5 kDa) for dialysis purification. A weak acid solution was used for dialysis for the first two days, and pure deionized water was used for dialysis on the third day. The dialysis solution was changed every 8 hours. The purified mixture was then rapidly frozen in a -80°C freezer, and the solid ice was placed in a vacuum freeze dryer for vacuum freeze drying to obtain a white solid substance of ferulic acid-modified chitosan.

[0072] Weigh 4g of dextran and dissolve it in 50ml of deionized water. Then add 2g of sodium periodate. The solution turns orange-red. Stir magnetically overnight (12h) at room temperature in the dark to carry out the oxidation reaction. Then add 900mL of ethylene glycol and continue stirring for 2h to terminate the reaction. Finally, pour the solution into a dialysis bag and dialyze with deionized water for 3 days, changing the dialysate every 8h. After dialysis, freeze-dry under vacuum to obtain the white substance oxidized dextran for later use.

[0073] Weigh 2g of lyophilized ferulic acid-modified chitosan solid and dissolve it in 100mL of deionized water under magnetic stirring to obtain a 2% (w / v) modified chitosan solution; weigh 5g of lyophilized oxidized dextran solid and dissolve it in 100mL of deionized water under ultrasonic assistance to obtain a 5% (w / v) oxidized dextran solution; add 2.4mg of smegglutinin powder to 1mL of oxidized dextran solution to obtain a mixed oxidized dextran solution; take 1mL each of the modified chitosan solution and the mixed oxidized dextran solution, and physically mix them at room temperature to observe gelation of the solution; after vacuum freeze-drying, a white solid (hydrogel loaded with smegglutinin) is obtained.

[0074] Example 2

[0075] Dissolve 1g of chitosan powder in a mixture of 40ml of deionized water and 20ml of dimethyl sulfoxide (DMSO). Place the resulting mixture on a magnetic stirrer and stir until homogeneous. Slowly add 1mol / L hydrochloric acid solution (HCl) until the solution becomes transparent to obtain a chitosan solution.

[0076] Dissolve 350 mg of ferulic acid powder in 20 ml of DMSO solution, stir well at room temperature, and purge the solution with high-purity nitrogen for 20 min to prevent oxidation of the reactants with oxygen in the air. After purging, slowly add 750 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide, EDC) and 460 mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group on ferulic acid, thus obtaining an activated ferulic acid solution.

[0077] The activated ferulic acid solution was slowly added dropwise to the chitosan solution, and the mixture was stirred thoroughly on a magnetic stirrer at a constant temperature of 50°C for 30 hours to ensure sufficient grafting of ferulic acid. The ferulic acid-chitosan mixture was then placed in a dialysis bag (molecular weight cutoff of 3.5 kDa) for dialysis purification. A weak acid solution was used for dialysis for the first two days, and pure deionized water was used for dialysis on the third day. The dialysis solution was changed every 6 hours. The purified mixture was then rapidly frozen in a -80°C freezer, and the solid ice was placed in a vacuum freeze dryer for vacuum freeze drying to obtain a white solid substance of ferulic acid-modified chitosan.

[0078] Weigh 4g of dextran and dissolve it in 50ml of deionized water. Then add 2g of sodium periodate. The solution turns orange-red. Stir magnetically overnight (12h) at room temperature in the dark to carry out the oxidation reaction. Then add 900mL of ethylene glycol and continue stirring for 2h to terminate the reaction. Finally, pour the solution into a dialysis bag and dialyze with deionized water for 3 days, changing the dialysate every 6h. After dialysis, freeze-dry under vacuum to obtain the white substance oxidized dextran for later use.

[0079] Weigh 2g of lyophilized ferulic acid-modified chitosan solid and dissolve it in 100mL of deionized water under magnetic stirring to obtain a 2% (w / v) modified chitosan solution; weigh 5g of lyophilized oxidized dextran solid and dissolve it in 100mL of deionized water under ultrasonic assistance to obtain a 5% (w / v) oxidized dextran solution; add 2mg of smegglutinin powder to 1mL of oxidized dextran solution to obtain a mixed oxidized dextran solution; take 1mL of modified chitosan solution and 1mL of oxidized dextran mixed solution, and physically mix them at room temperature to observe gelation of the solution; after vacuum freeze-drying, a white solid (hydrogel loaded with smegglutinin) is obtained.

[0080] Example 3

[0081] Dissolve 1g of chitosan powder in a mixture of 60ml of deionized water and 20ml of dimethyl sulfoxide (DMSO). Place the resulting mixture on a magnetic stirrer and stir until homogeneous. Slowly add 1mol / L hydrochloric acid solution (HCl) until the solution becomes transparent to obtain a chitosan solution.

[0082] Dissolve 400 mg of ferulic acid powder in 20 ml of DMSO solution, stir well at room temperature, and purge the solution with high-purity nitrogen for 45 min to prevent oxidation of the reactants with oxygen in the air. After purging, slowly add 800 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide, EDC) and 460 mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group on ferulic acid, thus obtaining an activated ferulic acid solution.

[0083] The activated ferulic acid solution was slowly added dropwise to the chitosan solution, and the mixture was stirred thoroughly on a magnetic stirrer at a constant temperature of 70°C for 20 hours to ensure sufficient grafting of ferulic acid. The ferulic acid-chitosan mixture was then placed in a dialysis bag (molecular weight cutoff of 3.5 kDa) for dialysis purification. A weak acid solution was used for dialysis for the first two days, and deionized water was used for dialysis on the third day. The dialysis solution was changed every 10 hours. The purified mixture was then rapidly frozen in a -80°C freezer, and the solid ice was placed in a vacuum freeze dryer for vacuum freeze drying to obtain a white solid substance of ferulic acid-modified chitosan.

[0084] Weigh 4g of dextran and dissolve it in 50ml of deionized water. Then add 2g of sodium periodate. The solution turns orange-red. Stir magnetically overnight (12h) at room temperature in the dark to carry out the oxidation reaction. Then add 900mL of ethylene glycol and continue stirring for 2h to terminate the reaction. Finally, pour the solution into a dialysis bag and dialyze with deionized water for 3 days, changing the dialysate every 10h. After dialysis, freeze-dry under vacuum to obtain the white substance oxidized dextran for later use.

[0085] Weigh 2g of lyophilized ferulic acid-modified chitosan solid and dissolve it in 100mL of deionized water under magnetic stirring to obtain a 2% (w / v) modified chitosan solution; weigh 5g of lyophilized oxidized dextran solid and dissolve it in 100mL of deionized water under ultrasonic assistance to obtain a 5% (w / v) oxidized dextran solution; add 3mg of smegglutinin powder to 1mL of oxidized dextran solution to obtain a mixed oxidized dextran solution; take 1mL of modified chitosan solution and 2mL of oxidized dextran mixed solution, and physically mix them at room temperature. The solution gels, and after vacuum freeze-drying, a white solid (hydrogel loaded with smegglutinin) is obtained.

[0086] Comparative Example

[0087] Comparative Example 1

[0088] The preparation was carried out in a similar manner to Example 1, except that: 2g of lyophilized ferulic acid-modified chitosan solid was weighed and dissolved in 100mL of deionized water under magnetic stirring to obtain a 2% (w / v) modified chitosan solution (FACS solution); 5g of lyophilized oxidized dextran solid was weighed and dissolved in 100mL of deionized water under ultrasonic assistance to obtain a 5% (w / v) oxidized dextran solution (ODex solution). The obtained 2% FACS solution and 5% ODex solution were physically mixed at a 1:1 volume ratio, and the solution was observed to gradually gel at room temperature. The gel was frozen into ice cubes in a -80°C freezer and then placed in a vacuum freeze dryer to obtain a porous white blank deionized hydrogel solid.

[0089] Experimental Example

[0090] Experimental Example 1: Gel formation time and injectability test of hydrogels

[0091] The gelation time of the hydrogels prepared in Example 1 and Comparative Example 1 was tested, and the test results are as follows: Figure 1 As shown, the test results indicate that the gelation time of the blank hydrogel (Gel) prepared in Comparative Example 1 was 108.33±4.50s (n=3), while the gelation time of the hydrogel (Gel@Sema) loaded with semaglutide prepared in Example 1 was significantly shortened to 90.33±2.05s (n=3), and the gelation rate was increased by 16.62%.

[0092] The injectability of the hydrogels prepared in Example 1 and Comparative Example 1 was tested using a dynamic extrusion test conducted with a 26G needle (0.45 mm inner diameter) at an injection rate of 0.5 mL / min. During extrusion, Gel@Sema exhibited excellent shear-thinning behavior and rapid structural recovery. High-speed imaging analysis confirmed that no phase separation or jet instability occurred in Gel@Sema during extrusion. This indicates that the semaglutide-loaded hydrogel described in this invention has a short gelation time, excellent shear-thinning behavior, rapid recovery ability, and high stability.

[0093] Experimental Example 2: Fourier Transform Infrared Spectroscopy Test

[0094] Fourier transform infrared spectroscopy was performed on the modified chitosan solution, the oxidized dextran solution, the hydrogel loaded with smegglutinin prepared in Example 1, and the blank hydrogel prepared in Comparative Example 1, respectively. The test results are as follows: Figure 2 As shown.

[0095] from Figure 2 It can be seen that after FACS and ODex are crosslinked, the blank hydrogel (Gel) sample mainly exhibits the main characteristics of ODex, but the sample at 1000 cm⁻¹... -1 The stretched CO absorption peak shape at [location missing] indicates successful cross-linking between FACS and ODex. The reaction between FACS and ODex mainly occurs on the NH groups of chitosan, and the cross-linked sample shows peaks in the 3000–3600 cm⁻¹ range. -1 The relative intensity of the absorption peak at 1600 cm⁻¹ is significantly reduced. -1 The relative intensity of the absorption peak at this point increased significantly, which is due to the -NH group of FACS participating in the cross-linking reaction to form a Schiff base (C=N) structure, further confirming the successful cross-linking of FACS and ODex. The infrared characteristics of the hydrogel sample loaded with semaglutide (Gel@Sema) did not change significantly, mainly due to the low peptide loading and the overlap of characteristic peaks with gel. However, the 3000~3600 cm⁻¹... -1 The wavelength range is highly sensitive to changes in the hydrogen bonding system and is most valuable for studying hydrogen bond changes. After cross-linking, the absorption peak of the sample in this wavelength range shifted significantly, indicating that peptide doping disrupted the original hydrogen bonding system and formed a new, more compact hydrogen bond network. Furthermore, the hydrogel loaded with smegglutinin showed a significant absorption peak shift at 1520 cm⁻¹. -1 The increase in the intensity of the absorption peak (amide II band) is mainly related to the NH bending and CN stretching vibration modes in the primary and secondary amine groups (-NH-CO-) of the polypeptide; at 1640 cm⁻¹ -1The increase in the intensity of the absorption peak (amide I band) is related to the C=O stretching vibration mode in the peptide amide group (-NH-CO-), and the above characteristics indicate that the peptide is successfully loaded into the hydrogel mainly through hydrogen bonding.

[0096] Experimental Example 3: Analysis of the Microstructure and Elemental Composition of Hydrogels

[0097] Energy dispersive spectroscopy (EDS) was performed on the hydrogel loaded with smegglutinin prepared in Example 1 and the blank hydrogel prepared in Comparative Example 1, respectively. The test results are as follows: Figure 3 As shown.

[0098] from Figure 3 As can be seen, energy dispersive spectroscopy (EDS mapping) shows that C, O, N, and S elements are uniformly distributed on the honeycomb structure of the hydrogel. A small amount of S element is visible in the Gel@Sema hydrogel, but no S element is observed in the Gel hydrogel. This clearly indicates that semaglutide is uniformly distributed in the hydrogel, demonstrating that the preparation method described in this invention can successfully load semaglutide into the hydrogel. Quantitative energy dispersive spectroscopy (Q-EDS) analysis shows that the S element content in the Gel@Sema hydrogel is 0.14%, with a Sigma value of 0.03 (Wt / % ≥ 3*Sigma, indicating the presence and significance of the element), further confirming that in this invention, semaglutide is uniformly doped into the three-dimensional network of the hydrogel through non-covalent interactions.

[0099] Experiment Example 4: In vitro drug sustained-release performance test

[0100] To simulate the microenvironmental changes during myocardial infarction, the hydrogel loaded with semaglutide prepared in Example 1 was placed in phosphate buffer at pH 6.5 to study the release of semaglutide from the hydrogel. The drug release curve is shown below. Figure 4 As shown.

[0101] from Figure 4 As can be seen, the hydrogel releases semaglutide rapidly in the first half of the process, reaching a cumulative release of 60% around day 8; the release rate gradually flattens in the second half, with a cumulative release of approximately 70% around day 15. The rapid release rate in the early stage also corresponds to the pH changes in the microenvironment during the acute phase of myocardial infarction, indicating that the hydrogel loaded with semaglutide described in this invention achieves the goal of pH-based targeted therapy.

[0102] Experimental Example 5: Application of injectable hydrogel loaded with smegglutinin in myocardial infarction

[0103] (1) Biocompatibility test of hydrogels

[0104] The extracts of the blank hydrogel (Gel) prepared in Comparative Example 1 and the extracts of the semaglutide-loaded hydrogel (Gel@Sema) prepared in Example 1 were used to culture H9c2 cells separately. Cytotoxicity was compared with that of cells cultured in normal high-glucose medium containing 10% serum. Cell viability was detected using a CCK-8 assay kit. The test results are as follows: Figure 5 As shown.

[0105] from Figure 5 As can be seen, the cell viability of each group was as follows: control group (100.00±0.00)%, Gel group (92.26±4.79)%, and Gel@Sema group (94.78±3.56)%. There were no statistically significant differences between the Gel group and the control group (P>0.05), indicating that the extract of the hydrogel prepared in this invention has no cytotoxicity to cardiomyocyte culture, and the hydrogel loaded with smegglutinin has good biocompatibility in H9c2 cells.

[0106] (2) Post-myocardial infarction interstitial fibrosis test

[0107] The blank hydrogel prepared in Comparative Example 1 and the semaglutide-loaded hydrogel prepared in Example 1 were used to test myocardial interstitial fibrosis after myocardial infarction. To detect the degree of myocardial interstitial fibrosis in rats, tissue samples were taken from rats 28 days after myocardial infarction, and Masson staining was performed on paraffin sections. The percentage of interstitial fibrosis area was measured using ImageJ software. Statistical analysis results are as follows: Figure 6 As shown, (a) shows a schematic diagram of the staining results; (b) is a bar chart of the quantification results (**P<0.01; ****P<0.0001).

[0108] from Figure 6 As shown in Figure (b), the percentages of interstitial fibrosis area in the sham-operated group (Sham group), myocardial infarction group (MI group), semaglutide group (Sema group), blank hydrogel group (Gel group), and semaglutide-loaded hydrogel group (Gel@Sema group) were (1.40±0.48)%, (25.69±1.64)%, (16.41±1.06)%, (18.08±1.15)%, and (10.40±1.89)%, respectively. The percentage of myocardial interstitial fibrosis area in the MI group was significantly larger than that in the other four groups (P<0.05). The area of ​​myocardial interstitial fibrosis in the Sema, Gel, and Gel@Sema groups was reduced by 36.12%, 29.62%, and 59.52% compared to the MI group, respectively (P<0.001). The results indicate that semaglutide-loaded hydrogel can significantly reduce myocardial interstitial fibrosis after myocardial infarction in rats.

[0109] (3) Blood Ejection Capacity Test

[0110] The blank hydrogel prepared in Comparative Example 1 and the hydrogel loaded with smegglutinin prepared in Example 1 were used to test the ejection capacity after myocardial surgery. The test procedure was as follows: on the 28th day after myocardial infarction, the heart of rats was scanned using a small animal ultrasound imaging system, and the left ventricular ejection fraction (LVEF) was measured to evaluate changes in cardiac structure and function. Figure 7 The image shows schematic diagrams of rat hearts in B-mode and M-mode ultrasound. In the M-mode ultrasound, thinning of the ventricular wall and weakening of ventricular wall motion can be clearly observed in the myocardial infarction group, a change consistent with the morphology of the heart in the gross specimen.

[0111] The specific analysis is as follows. Figure 7 The changes in various indicators reflecting cardiac systolic and diastolic function, and left ventricular ejection fraction were analyzed: Sham group (78.79±5.42)%, MI group (38.11±4.36)%, Sema group (52.11±2.70)%, Gel group (50.88±2.31)%, and Gel@Sema group (59.34±2.43)%. The differences between the myocardial infarction group and the three treatment groups were statistically significant (P<0.05). Among the three treatment groups, Gel@Sema showed the best therapeutic effect. This indicates that the hydrogel loaded with semaglutide prepared in this invention can improve the cardiac ejection capacity after myocardial infarction in rats, and has a good ameliorative and therapeutic effect on myocardial infarction.

[0112] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing a hydrogel loaded with smegglutinin, characterized in that, The preparation method includes the following steps: Step 1: Dissolve chitosan in a mixed solvent, stir well, and then add hydrochloric acid solution dropwise to obtain a chitosan solution; Step 2: Dissolve ferulic acid in DMSO solution, stir well, blow high-purity nitrogen gas through it, and then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide dropwise for activation to obtain activated ferulic acid solution. The ratio of ferulic acid to DMSO solution added is (350-400) mg: 20 mL; The blowing time with high-purity nitrogen is 20–45 minutes; The mass ratio of ferulic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is (350-400):(750-800):460; Step 3: Add the activated ferulic acid solution dropwise to the chitosan solution, react under stirring, then dialysis purification and vacuum freeze-drying to obtain ferulic acid modified chitosan; The reaction conditions are: at 50–70°C, with stirring for 20–30 hours; Step 4: Dissolve dextran in deionized water, then add sodium periodate and stir magnetically. Then add ethylene glycol and continue stirring. Finally, dialyze and freeze dry under vacuum to obtain oxidized dextran. Step 5: Under stirring conditions, ferulic acid-modified chitosan is added to deionized water to obtain a modified chitosan solution. Oxyglucan is added to deionized water and dissolved by sonication to obtain an oxidized dextran solution. Smegglutinin is added to the oxidized dextran solution to obtain an oxidized dextran mixed solution. The modified chitosan solution and the oxidized dextran mixed solution are mixed to obtain a hydrogel loaded with smegglutinin.

2. The preparation method according to claim 1, characterized in that, In step 1, The mixed solvent is obtained by mixing deionized water and dimethyl sulfoxide; and / or, The ratio of chitosan to mixed solvent added is 1g:(50-90)mL.

3. The preparation method according to claim 1, characterized in that, In step 4, The molecular weight of the dextran is 60-80 kDa; The mass ratio of the dextran to sodium periodate is (1-3):

1.

4. The preparation method according to claim 1, characterized in that, In step 4, The conditions for magnetic stirring are: magnetic stirring for 10–13 hours at room temperature in the dark; and / or, After adding ethylene glycol, continue stirring for 1–3 hours.

5. The preparation method according to claim 1, characterized in that, In step 5, The concentration of the modified chitosan solution is 1-3% (w / v); and / or, The concentration of the oxidized dextran solution is 3-7% w / v; and / or, The ratio of the added oxidized dextran solution to smegglutinin is 1 mL : (2-3) mg; and / or, The modified chitosan solution and the oxidized dextran mixed solution were mixed at a volume ratio of 1:(1~2).

6. A hydrogel loaded with smegglutinin prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the hydrogel loaded with smegglutinin as described in claim 6 in the preparation of a drug for treating or improving myocardial infarction.

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