Composite hydrogel for promoting hiPSC-CMs in-situ maturation and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to composite hydrogel for promoting hiPSC-CMs in-situ maturation and a preparation method and application thereof. According to the preparation method disclosed by the invention, a compound liposome modified by quaternary ammonium chitosan and loaded with Torrin 1 is constructed, a unique three-dimensional network structure (PDN) is constructed by compounding different chitosan, and the compound liposome and the PDN are combined to obtain the composite hydrogel for embedding and delivering hiPSC-CMs. Wherein residual amine of the quaternary ammonium chitosan can slowly release drugs in an acidic microenvironment after myocardial infarction, and the cell cycle is inhibited; the PDN structure enhances the retention rate of cell transplantation; the loaded hiPSC-CMs can restore the contraction function, reduce spontaneous depolarization and action potential, reduce the risk of lethal arrhythmia and promote in-situ maturation of transplanted cells. Experiments show that the gel loaded with the hiPSC-CMs can significantly promote myocardial tissue maturation after myocardial infarction, effectively slow down ventricular remodeling and improve heart functions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a composite hydrogel that promotes in-situ maturation of hiPSC-CMs, its preparation method, and its application. Background Technology

[0002] Myocardial infarction is a serious disease caused by insufficient blood supply to the coronary arteries, leading to irreversible necrosis and scarring of cardiomyocytes, which may eventually result in heart failure. Because the regenerative capacity of mature cardiomyocytes is extremely limited, damage to cardiac function is usually permanent. Currently, heart transplantation is the most effective treatment for end-stage heart failure, but its clinical application is severely limited by donor shortages, immune rejection, and high medical costs. Therefore, developing alternative therapies that can promote myocardial regeneration has become an urgent need in the field of cardiovascular medicine.

[0003] In recent years, therapies based on human induced pluripotent stem cells (hiPSCs) have emerged as a promising treatment option. Cardiomyocytes derived from these induced pluripotent stem cells (hiPSC-CMs) possess considerable cardiac regeneration potential and have shown great promise in the treatment of heart disease. hiPSC-CMs can be obtained through in vitro directed differentiation and possess contractile and electrophysiological activities, theoretically capable of repairing damaged myocardial tissue. However, hiPSC-CMs induced by current technologies are structurally and functionally closer to fetal cardiomyocytes, exhibiting electrophysiological immaturity, such as a tendency for spontaneous depolarization and unstable action potentials. When these cells are transplanted into large animal models (such as pigs or non-human primates), their abnormal electrical activity can competitively stimulate the host heart, leading to transient and lethal arrhythmias, severely limiting their clinical application.

[0004] Although researchers have developed various methods to promote the maturation of hiPSC-CMs in vitro (such as mechanical stretching, electrical stimulation, three-dimensional culture, or co-culture systems), these strategies still have significant drawbacks. First, cells matured in vitro require digestion and separation before transplantation, resulting in significant cell loss. Second, mechanical damage and the ischemic microenvironment during transplantation further reduce cell survival rates. Finally, even if cells are successfully implanted, their mature state may not be maintained long-term due to differences in the in vivo environment. Therefore, relying solely on in vitro maturation techniques is insufficient for achieving efficient and safe cell transplantation.

[0005] Therefore, there is an urgent need to develop a solution that ensures transplanted cells differentiate into electrophysiologically stable and functionally mature cardiomyocytes in situ in vivo without relying on in vitro pre-maturation. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art which requires in vitro promotion of hiPSC-CMs maturation and then transplantation, and to provide a composite hydrogel.

[0007] Another object of the present invention is to provide a composite hydrogel loaded with hiPSC-CMs.

[0008] Another object of the present invention is to provide the application of the composite hydrogel or the composite hydrogel loaded with hiPSC-CMs in the preparation of drugs for improving cardiac function.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects a method for preparing a composite hydrogel, comprising the following steps: S1. Mix the first chitosan and the second chitosan in an acidic reagent solution to obtain the precursor solution; S2. Mix the drug-loaded liposomes and the third chitosan solution to obtain composite liposomes; S3. Mix the precursor fluid obtained in step S1 and the composite liposome obtained in step S2, add glutaraldehyde crosslinking agent, and crosslink fully to obtain composite hydrogel. Wherein, the first chitosan is unmodified deacetylated chitin with a molecular weight of 200-400 kDa; the second chitosan is ethylene glycol-chitosan with a degree of polymerization ≥400 and a molecular weight of 200-300 kDa; the third chitosan is obtained by modifying the fourth chitosan with a quaternizing agent, with a degree of quaternization ≥65%; the fourth chitosan is unmodified deacetylated chitin with a molecular weight of 60-100 kDa; The drug in the drug-loaded liposomes is Torin 1; The mass ratio of the first chitosan to the second chitosan is (2.5~3.5):1.

[0010] This invention constructs anionic composite liposomes modified with quaternary ammonium chitosan and loaded with mTOR inhibitors, and uses different types of chitosan to construct a unique three-dimensional network structure (PDN). The two are combined to obtain a composite hydrogel, which can be used to embed and deliver hiPSC-CMs. The delivered hiPSC-CMs can mature in situ after myocardial infarction transplantation to avoid cell loss and death.

[0011] Preferably, the molecular weight of the fourth chitosan is 70-80 kDa.

[0012] Preferably, the degree of deacetylation of the fourth chitosan is ≥80%.

[0013] Preferably, the degree of quaternization substitution is 65% to 80%.

[0014] Preferably, the degree of deacetylation of the first chitosan is ≥80%.

[0015] Preferably, the mass ratio of the first chitosan to the second chitosan is (2.9~3.0):1.

[0016] Preferably, the mass ratio of the liposomes to the third chitosan is (9~10):1.

[0017] Preferably, the volume ratio of the drug-loaded liposomes to the third chitosan solution is 1:1.

[0018] Preferably, the concentration of the precursor solution is 32-35 mg / mL.

[0019] Preferably, the concentration of the third chitosan solution is 0.4~0.6 mg / mL.

[0020] Furthermore, the acidic reagent includes acetic acid.

[0021] Preferably, the concentration of the acetic acid solution is 0.1~0.2 mol / L.

[0022] Preferably, the volume ratio of the precursor fluid to the complex liposomes is (5~7):1.

[0023] Preferably, the quaternizing agent is iodomethane.

[0024] Specifically, the third chitosan is prepared by the following steps: dissolving the fourth chitosan in an alkaline reagent solution, adding dimethyl sulfoxide and iodomethane, reacting fully at 50~70 °C for 0.8~1.2 h, then adding alkaline reagent and iodomethane again, continuing the reaction for 0.8~1.2 h, and then performing post-treatment to obtain the final product.

[0025] Furthermore, the post-processing includes terminating the reaction, dialysis, and lyophilization. Specifically, after the reaction is completed, anhydrous ethanol is added to the system to terminate the reaction, dialysis is performed for 3 days to collect the white precipitate that appears, and lyophilization is performed to obtain quaternary ammonium chitosan.

[0026] Furthermore, the alkaline reagent is preferably sodium hydroxide.

[0027] Preferably, the liposomes comprise lecithin.

[0028] Preferably, the lecithin includes egg yolk lecithin and / or soybean lecithin.

[0029] Further, the drug-loaded liposomes are prepared by the following steps: dissolving liposomes in an organic solvent, treating them in a water bath at 35-40°C for 0.8-1.2 hours to form a thin film, adding a drug solution to the film, stirring in a water bath at 50-60°C, and then fully encapsulating the drug with ultrasound to obtain the drug-loaded liposomes.

[0030] Preferably, the mixing ratio of the liposomes and the organic solvent is (6~8) mg: 1 mL.

[0031] Preferably, the volume ratio of the organic solvent to the drug solution is (1.5~2.5):1.

[0032] Preferably, the concentration of the drug solution is 100-300 nM, more preferably 150-250 nM.

[0033] Preferably, the organic solvent is a mixture of chloroform and anhydrous ethanol.

[0034] Preferably, the volume ratio of chloroform to anhydrous ethanol is 1:1.

[0035] Furthermore, the fully cross-linked cross-linking is achieved by using glutaraldehyde as a cross-linking agent under alkaline conditions.

[0036] Furthermore, the alkaline conditions are provided by an alkaline reagent selected from sodium bicarbonate and / or sodium carbonate.

[0037] Furthermore, the glutaraldehyde crosslinking agent is used after being mixed with an alkaline reagent solution to provide an alkaline environment, which is beneficial for promoting crosslinking.

[0038] Specifically, the glutaraldehyde crosslinking agent is prepared by the following steps: mixing an alkaline reagent and glutaraldehyde in a buffer solution.

[0039] Preferably, the buffer solution is a phosphate solution.

[0040] Preferably, the volume ratio of glutaraldehyde to the buffer solution is (7~8)×10. -5 :1.

[0041] Preferably, when the alkaline reagent is selected from sodium bicarbonate, the mass-to-volume ratio of the alkaline reagent to the buffer solution is (3.5~4) g: 100 mL.

[0042] Preferably, the volume ratio of the precursor liquid to the glutaraldehyde crosslinking agent is 3:2.

[0043] The present invention discloses a composite hydrogel loaded with hiPSC-CMs, wherein the composite hydrogel is loaded with hiPSC-CMs cells.

[0044] This invention constructs anionic composite liposomes modified with quaternary ammonium chitosan and loaded with an mTOR inhibitor. A unique three-dimensional network structure (PDN) is constructed using different types of chitosan, and these are then mixed with hiPSC-CMs and fully cross-linked to obtain a composite hydrogel loaded with hiPSC-CMs. The residual amine of the quaternary ammonium chitosan acts as an acid-sensitive group, allowing for the sustained release of the mTOR inhibitor in the acidic microenvironment after myocardial infarction, inhibiting the cell cycle to improve cell maturation and survival. The PDN structure enhances cell transplantation retention. The hiPSC-CMs delivered by this composite gel can restore contractile function, reduce spontaneous depolarization and action potentials, lower the risk of lethal arrhythmias, and promote in situ cell maturation after transplantation. Experiments show that this composite gel loaded with hiPSC-CMs can significantly promote myocardial tissue maturation after myocardial infarction in mice, effectively slow ventricular remodeling, and improve cardiac function.

[0045] Furthermore, the injectable composite hydrogel loaded with hiPSC-CMs is prepared by the following steps: sI. Repeat steps S1 and S2 above to obtain the precursor fluid and the complex liposome solution, respectively; sII. Mix the precursor fluid, composite liposome solution, hiPSC-CMs cell suspension obtained in step sI with glutaraldehyde crosslinking agent and crosslink them thoroughly to obtain a composite hydrogel loaded with hiPSC-CMs.

[0046] Preferably, the cell content of the hiPSC-CMs cell suspension after mixing with the glutaraldehyde cross-linking agent is (0.5~20)×10⁻⁶. 4 per μL.

[0047] More preferably, the cell content of the hiPSC-CMs cell suspension after mixing with the glutaraldehyde cross-linking agent is (1~10)×10⁻⁶. 4 per μL.

[0048] The present invention also protects the use of the composite hydrogel or the composite hydrogel loaded with hiPSC-CMs in the preparation of drugs to improve cardiac function.

[0049] Preferably, the improvement of cardiac function includes one or more of the following: treatment of myocardial infarction, inhibition of cardiomyocyte hypertrophy, and inhibition of myocardial fibrosis.

[0050] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a Torin 1-loaded composite liposome modified with quaternary ammonium chitosan and utilizes different chitosan blends to construct a unique three-dimensional network structure (PDN). The combination of these two components yields a composite hydrogel for embedding and delivering hiPSC-CMs. Specifically, the residual amines of the quaternary ammonium chitosan can sustainably release drugs in the acidic microenvironment after myocardial infarction, inhibiting the cell cycle; the PDN structure enhances cell transplantation retention; and the loaded hiPSC-CMs restore contractile function while reducing spontaneous depolarization and action potentials, lowering the risk of lethal arrhythmias, and promoting in situ cell maturation after transplantation. Experiments show that this hiPSC-CM-loaded gel can significantly promote myocardial tissue maturation after myocardial infarction, effectively slow ventricular remodeling, and improve cardiac function. Attached Figure Description

[0051] Figure 1 This is a statistical graph showing the effect of the drug Torin 1 on the cell cycle of hiPSC-CMs. Simultaneous flow cytometry analysis of DNA and RNA was performed using Hoechst33342 and Pyronin Y to clarify the cell cycle. Figure A shows the percentage of hiPSC-CMs in the quiescent phase (G0) after drug intervention (three quadrants represent the proportion of cells in G0 phase). Figure B shows the statistical graph of the number of cells in G0 phase after drug intervention.* p <0.05, n=3 (only two of the three groups of cell cycle diagrams were actually shown), indicating that the difference was statistically significant.

[0052] Figure 2 This is a statistical graph showing the effect of the drug Torin 1 on the maturity of hiPSC-CMs. Graph A shows cell morphology, and graph B shows the statistical data on cell sphericity. p <0.0001 indicates that the difference is statistically significant.

[0053] Figure 3 This section presents statistical graphs of the effects of the drug Torin 1 on the electrophysiology of hiPSC-CMs. Figures A and B show representative extracellular potential maps and their statistical representations of hiPSC-CMs recorded by the ME-IDE device after intervention. Figure a shows the potential map before drug intervention, and figure b shows the potential map after drug intervention. Figure C shows the statistical graph of cell pulsation intervals. p <0.0001 indicates that the difference is statistically significant.

[0054] Figure 4 Scanning electron microscope images of drug-loaded liposome TL (left) and composite liposome TLT (right), scale bar 100 nm.

[0055] Figure 5The figures show statistical data for characterizing drug-loaded liposomes TL and composite liposomes TLT, where Figures A and B show the Zeta potentials of TL and TLT; and Figure C shows the hydrodynamic diameter of TLT measured by dynamic light scattering (DLS).

[0056] Figure 6 The following are statistical charts of rheological characterization data for injectable composite hydrogels TLT@PDN0.5, TLT@PDN, and TLT@PDN1.5, where Figure A is the dynamic time scan chart for each group; Figure B is the self-healing test chart for each group; and Figure C is the frequency scan chart for each group.

[0057] Figure 7 The images show the injectable composite hydrogel TLT@PDN (left image of A), scanning electron microscope image (right image of A, scale bars at 200 μm and 20 μm), injectability appearance (B), release performance (C), and stress-strain curve (D).

[0058] Figure 8 Figure A shows the morphological changes of hiPSC-CMs on the injectable composite hydrogel TLT@PDN. Figure B shows the solid image of the TLT@PDN hydrogel carrying hiPSC-CMs successfully cultured in a confocal dish. Figure B is a three-dimensional confocal scan image of hiPSC-CMs cultured in TLT@PDN for 7 days.

[0059] Figure 9 Figure 1 shows the retention rate of injectable composite hydrogel TLT@PDN and its hiPSC-CMs in mice. Figures A and B are schematic diagrams of the in situ retention of TLT@PDN in the heart of mice with myocardial infarction, as detected by in vivo imaging, and statistical graphs showing the changes over time. Figure C is a cross-sectional image of the heart of mice 7 days after myocardial infarction transplantation, showing fluorescence staining of TLT@PDN and TLT@PDN carrying hiPSC-CMs. HiPSC-CMs are labeled with CM Dil (red), sarcomere α-actin (α-SA, green), and cell nucleus (DAPI, blue). The scale bar is 20 μm. Figure D is a quantitative analysis of the in situ retention of hiPSC-CMs in the heart. p <0.0001, n=5, indicating a statistically significant difference; Figure E shows the quantitative analysis of cell volume of hiPSC-CMs preserved in situ in the heart.* p <0.05, **** p <0.0001, n=5, indicating that the difference is statistically significant.

[0060] Figure 10This is a quantitative analysis of cell maturation-related molecules in hiPSC-CMs implanted in TLT@PDN 7 days after orthotopic cardiac transplantation. ns indicates no significant difference. * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001, n=6, indicating a statistically significant difference.

[0061] Figure 11 This chart shows the postoperative assessment of cardiac function in mice. Figure A shows echocardiograms taken on day 28 after myocardial infarction in different groups of mice; Figures BE show the statistical data of EF and FS values ​​for each group from day 0 to 28 and on day 28 postoperatively.* p <0.05, # p <0.05, ## p <0.01 indicates a statistically significant difference (n=3).

[0062] Figure 12 Masson and WGA staining and ventricular remodeling analysis images of the heart were collected on day 28 after myocardial infarction. A is a cross-sectional image of the heart tissue stained with Masson staining, with scale bars of 2 mm and 150 μm, respectively; B is a statistical graph of the degree of myocardial fibrosis; C is a quantitative analysis image of left ventricular wall thickness; D is a cross-sectional image of the heart tissue stained with WGA staining, with a scale bar of 20 μm; E is a statistical graph of the relative cross-sectional area of ​​cardiomyocytes. ns indicates no significant difference, *p<0.05, **p<0.01, #p<0.05, ##p<0.01 indicate statistical significance, n=3. Detailed Implementation

[0063] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0064] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0065] Figure 1 A represents Figure 1 Figure A Figure 2 B indicates Figure 2 The naming of the other diagrams follows the same pattern, starting with diagram B.

[0066] Torin 1 was purchased from the Selleck website. It is an mTOR inhibitor, catalog number HY-13003, with a molecular weight of 607.62. The egg yolk lecithin was purchased from the Aladdin website, product number 8002-43-5; Medium molecular weight chitosan powder: molecular weight 200-400kDa, degree of deacetylation 80%; Ethylene glycol-chitosan: Sigma product, item number G7753, molecular weight 200~300kDa, degree of polymerization ≥400.

[0067] Low molecular weight chitosan powder: molecular weight 75kDa, degree of deacetylation 80%.

[0068] Example 1: Preparation of injectable composite hydrogels (TLT@PDN0.5, TLT@PDN, TLT@PDN1.5) The method for preparing the injectable composite hydrogel of the present invention includes the following steps: 1. Preparation of PDN precursor solution: S1. 600 μL of glacial acetic acid (C2H4O2) was added to 50 mL of deionized water to obtain an acetic acid solution with a concentration of 0.2 mol / L.

[0069] S2. Take 1 mL of 0.2 mol / L acetic acid solution at room temperature, add 25 mg of chitosan powder (medium molecular weight, 200-400 kDa, CS, degree of deacetylation 80%), and stir to form a homogeneous chitosan solution.

[0070] S3. Add different masses (4.2 mg, 8.4 mg, 16.8 mg) of ethylene glycol-chitosan (GC, 200-300 kDa, degree of polymerization ≥400) to the 1 mL chitosan solution obtained in step S2, and vortex thoroughly to obtain PDN precursor solutions of different concentrations (named PDN0.5, PDN (also known as PDN1, PDN in the following text is PDN1 unless otherwise specified), according to the content of different masses of ethylene glycol-chitosan added.

[0071] 2. Preparation of crosslinking agent: A phosphate solution (PS solution) was prepared by mixing 0.1 mol / L sodium diphosphate (Na2HPO4, 14.19588 g / L) and 0.1 mol / L sodium monophosphate (NaH2PO4, 11.99770 g / L) in a volume ratio of 50:3.

[0072] S2. Add 0.445 μL sodium bicarbonate (NaHCO3, 3.74 g) and 7.75 μL glyoxal to 100 mL of PS solution and mix thoroughly to obtain the crosslinking agent.

[0073] The preparation method of the composite liposome TLT of the present invention includes the following steps: Preparation of quaternary ammonium salt chitosan TMC: S1. Add 15g of sodium hydroxide (NaOH) to 100mL of deionized water and stir with a glass rod to prepare a 15% (m / v) NaOH solution.

[0074] S2. Add 4g of chitosan powder (low molecular weight, 75 kDa, degree of deacetylation 80%) to 10mL of 15% (m / v) NaOH solution, mix thoroughly, and then add 30mL of dimethyl sulfoxide (DMSO) and 10mL of iodomethane in sequence. React at 60℃ for 1h.

[0075] S3. After the reaction is complete, add 4 mg NaOH and 4 mL iodomethane, and continue the reaction for 1 h. After the reaction is complete, add anhydrous ethanol to the system to terminate the reaction, dialyze for 3 days to collect the white precipitate, and freeze-dry to obtain quaternary ammonium chitosan with a quaternization degree of 65%-80%.

[0076] Preparation of drug-loaded liposomes (TLs): S1. Weigh 70 mg of egg yolk lecithin (SPC) and add it to 10 mL of a mixed solution of chloroform and anhydrous ethanol in a ratio of 1:1 (v / v). Heat the solution in a water bath at 35°C for 1 h to form a uniform film on the inner wall of the beaker.

[0077] S2. Add 5 mL of 200 nM Torin 1 solution to the above reaction system, stir in a 55°C water bath for 30 min, and sonicate to obtain drug-loaded liposomes TL encapsulating Torin 1.

[0078] Preparation of complex liposome TLT: 1.5 mg of quaternary ammonium chitosan was added to 10 mL of deionized water to prepare a 0.5 mg / mL quaternary ammonium chitosan solution. 10 mL of this solution was slowly added to an equal volume of drug-loaded liposome TL suspension. The mixture was stirred at room temperature for 60 min and then sonicated to obtain composite liposome TLT.

[0079] The injectable composite hydrogel TLT@PDN includes the following steps: S1. Take 6 mL of the prepared PDN precursor solutions (PDN0.5, PDN, PDN1.5 precursor solutions) in different proportions, add 1 mL of the prepared composite liposome TLT suspension, and mix thoroughly to obtain TLT@PDN precursor solutions (TLT@PDN0.5, TLT@PDN, TLT@PDN1.5 precursor solutions) in different proportions.

[0080] S2. Add 4 mL of crosslinking agent to the TLT@PDN precursor solutions of different proportions and mix thoroughly to obtain three different proportions of injectable composite hydrogels TLT@PDN (TLT@PDN0.5, TLT@PDN, TLT@PDN1.5 hydrogels). The mixture can gel at room temperature for 1 min.

[0081] The injectable composite hydrogel TLT@PDN described in this invention is used for orthotopic cardiac transplantation and maturation of hiPSC-CMs.

[0082] Unless otherwise specified, TLT@PDN mentioned below refers to TLT@PDN prepared using PDN1 precursor solution.

[0083] Example 2: Detection of the efficacy of drug Torin 1 in altering the maturity of hiPSC-CMs cells. Culture and intervention of human induced pluripotent stem cells (hiPSC-CMs) 1) Thaw human induced pluripotent stem cells (hiPSC-CMs) and use 1×10 6 / cm 2 Cells were seeded at the specified density onto culture flasks coated with Help99A03 at 37°C for 1 hour. Beating cells were observed 1 to 2 days after seeding.

[0084] 2) Two days after the onset of pulsation, the cells were treated with 200 nM Torin 1 (dissolved in cardiomyocyte maintenance medium containing 0.02% DMSO) for 7 days.

[0085] Cell cycle identification of HiPSC-CMs by flow cytometry 1) HiPSC-CMs were cultured for 7 days in cardiomyocyte maintenance medium with or without Torin 1, and then centrifuged at 300g for 5 minutes after trypsin digestion for 1 minute to collect the cells.

[0086] 2) Wash three times with pre-cooled DPBS at 4°C and collect the precipitate.

[0087] 3) Resuspend cells in 1 mL of cell culture medium containing 10 μg / mL Hoechst 33342 and incubate at 37°C for 30 min. Add 5 μL of 100 μg / mL Pyronin Y directly to the cells and incubate at 37°C for another 15 min. Transfer cells to ice. Analyze using flow cytometry with an LSR II analyzer (BD). Analyze data using Flowjo software (TreeStar, USA).

[0088] Morphological changes of hiPS-CMs after Torin1 intervention 1) HiPSC-CMs were seeded on confocal dishes. The culture and intervention methods for hiPSC-CMs were as described above.

[0089] 2) Cell sample fixation: Wash three times with PBS, 5 min each time. At room temperature, add 200 μL of 4% paraformaldehyde to each dish and let stand for 20 min to fix the cells. Wash three times with PBS, 5 min each time.

[0090] 3) Cell permeabilization: Add 200 μL of 0.1% Triton X100 (diluted with PBS) to each dish and incubate at room temperature for 20 min. Wash three times with PBS, 5 min each time.

[0091] 4) Blocking: Add 200 μL of immunofluorescence blocking solution to each dish and block at room temperature for 1 h.

[0092] 5) Incubation with primary antibody: Wash twice with PBS, 5 min each time. Add 100 μL of rabbit polyclonal CD206 to each dish (the antibody is diluted with primary antibody dilution buffer at a ratio of 1:200), and incubate overnight at 4°C.

[0093] 6) Incubate at room temperature for 1 hour. Wash three times with PBS, 5 minutes each time. Add goat anti-rabbit Cy3 secondary antibody (diluted with secondary antibody dilution buffer, dilution ratio 1:500) to a confocal dish. Incubate at room temperature in the dark for 2 hours.

[0094] 7) Nuclear staining and mounting: Discard the secondary antibody, wash three times with PBS for 5 min each time. Add DAPI staining working solution to stain the cell nuclei, adding 50 μL of working solution to each dish. Incubate at room temperature for 3 min. Wash three times with PBS for 5 min each time. Add 100 μL of anti-fluorescence quencher to each confocal dish for mounting.

[0095] 8) Observe and photograph under a confocal microscope.

[0096] Electrophysiological changes in hiPSC-CMs after Torin 1 intervention The pulsation frequency and extracellular potential of hiPS-CMs after Torin 1 intervention were measured using integrated microelectrodes (ME-IDE).

[0097] 1) HiPSC-CMs were seeded on ME-IDE chips. The culture and intervention methods for hiPSC-CMs were as described above.

[0098] 2) The chip was incubated at 37°C in an incubator containing 5% CO2 for 7 days.

[0099] 3) Insert the chip into the ME-IDE workstation, and then click the Start / Continue icon to start the measurement. Perform a scan measurement every two days.

[0100] The results are as follows Figures 1-3 As shown, Torin 1 intervention successfully altered the cell cycle, cell morphology, and function of hiPSC-CMs. Figure 1 As shown, compared with the control, Torin 1 treatment of hiPSC-CMs successfully induced cell cycle arrest, with the percentage of quiescent cells (G0 cells) increasing from 37% to 48%. Figure 2 As shown, this invention performed immunofluorescence staining on hiPSC-Ms to further observe the morphology and arrangement of cells after Torin 1 intervention. It was observed that the control group cells had irregular arrangement, higher cell sphericity, and smaller cell volume. In contrast, the hiPSC-CMs treated with Torin 1 were large cells with an approximately cuboid shape, and the cell sphericity was significantly reduced. P <0.05) significantly increased cell size ( P <0.05), with a clearly visible longitudinal axis, and as shown by α-actin staining, the myofibrils of Torin 1-treated hiPSC-CMs are arranged in parallel and orderly fashion. Figure 3 As shown in A~3C, Torin 1 intervention successfully altered the electrophysiological function of hiPSC-CMs. Specifically... Figure 3 As shown in Figure B, the extracellular potential of hiPSC-CMs significantly increased after Torin 1 treatment. P <0.05), increasing from 53.95±17.15 μV in the control group to 116.73±4.95 μV. Meanwhile, as Figure 3 As shown in Figure C, compared with the control group, the mechanojugation interval of hiPSC-CMs recorded by the ME-IDE device increased from 0.43±0.14 ms to 2.77±0.25 ms, as the inventors expected. These results validate that Torin 1 treatment induces maturation-like changes in cell morphology, a significant decrease in mechanojugation rate, and a maturation-like increase in extracellular potential amplitude. This means that Torin 1 successfully promotes morphological and electrophysiological changes and maturation of hiPSC-CMs by inducing cell cycle quiescence.

[0101] Example 3: Characterization of morphology, rheology, mechanical properties and release performance of injectable composite hydrogel TLT@PDN The preparation methods of TLT@PDN0.5, TLT@PDN, and TLT@PDN1.5 are as described in Example 1, and will not be repeated here.

[0102] The results are as follows Figures 4-7 As shown, this invention successfully prepared an injectable composite hydrogel, TLT@PDN. (The text appears to be incomplete and requires further context.) Figure 4As can be seen, both uncoated and TMC-coated liposomes are dispersed throughout the aqueous phase and are approximately spherical. Furthermore, some liposomes aggregate with TMC, resulting in a significant increase in diameter. These imaging results support the inventors' previous hypothesis that cationic TMC can be linked to multiple liposomes and nanoclusters.

[0103] Potential results as follows Figure 5 As shown, the zeta potential of TL is approximately -0.54 mV ( Figure 5 A), the zeta potential of TLT reversed to +14.99 mV ( Figure 5 B), further indicating that the TMC coating was successful.

[0104] Further measurements were taken to determine the effect of TMC on bead diameter, and the results are as follows: Figure 5 As shown in C, the particle diameters of TL and TLT were measured using dynamic light scattering. Without TMC, the diameter of the liposome core was approximately 117.5 nm (PDI = 0.4717), while the diameter of the liposomes significantly increased after TMC coating (258.3 nm, PDI = 0.3396).

[0105] Simultaneously, rheological recovery tests were performed on TLT@PDN under 1% and 100% stress, with the two stresses alternating every 120 seconds for a total of 1200 seconds. Figure 6 As shown in Figure B, TLT@PDN exhibits rapid stress relaxation behavior. At low shear strain (1%), the hydrogel's G' is higher than G'', indicating that TLT@PDN has a solid-like state and elasticity. Conversely, at high strain (100%), G' drops sharply to below G'', indicating a liquid state. This shows that the collapse and remodeling behavior of TLT@PDN can be repeated alternately multiple times, indicating its rapid and efficient self-repair ability and excellent resilience, which is conducive to cell proliferation and migration. In contrast, the collapse and remodeling behavior of TLT@PDN0.5 and TLT@PDN1.5 cannot be repeated alternately, indicating poor self-repair ability and resilience. Meanwhile, through... Figure 6A compared the stability of TLT@PDN0.5, TLT@PDN, and TLT@PDN1.5 with 6C. The modulus of different groups of hydrogels was detected in time and frequency scanning modes. All groups remained solid and relatively stable over a long period of time. However, with the increase of frequency, TLT@PDN0.5 transitioned from a solid-like state to a liquid state. At low frequencies, the storage modulus (G') of TLT@PDN0.5 was higher than the loss modulus (G''), exhibiting elastic behavior. As the frequency gradually increased, G'' exceeded G', and TLT@PDN0.5 gradually transitioned to a viscous state, while TLT@PDN remained a solid-like state. This indicates that TLT@PDN is not affected by frequency changes and is more suitable as a support material for cardiac tissue. Considering its self-repair ability, resilience, and time and frequency stability, TLT@PDN was the preferred material.

[0106] To further evaluate the state of the injectable composite hydrogel TLT@PDN, this invention tested its injectability and observed its microstructure. As shown in 7B, the hydrogel can gel after injection and placement at 37°C, and under SEM (… Figure 7 A) The physical morphology of the hydrogel and the spatial distribution of TLT in the PDN1 matrix were observed. The cross-sectional image showed that TLT@PDN formed a well-structured cell network (500×). At a higher magnification (5000×), TLT was successfully observed to be uniformly distributed in PDN1.

[0107] To further measure the mechanical properties of the TLT@PDN hydrogel, the stiffness of the hydrogel was evaluated by compression testing, such as... Figure 7 As shown in Figure D, the results indicate that the Young's modulus of the hydrogel (the linear slope of the stress-strain curve is the Young's modulus) is approximately 49.6 kPa. This stiffness spans the range of adaptations for various biological tissues, such as the heart and gastrointestinal tract, suggesting that the hydrogel can be used as a support material for myocardial tissue and cell proliferation.

[0108] Finally, this invention tested the Torin 1 release capacity of the biological system under acidic conditions. The inventors stored the prepared TLT and TLT@PDN hydrogels in Tris-HCl solution at pH 5.5, respectively, and collected the supernatant at fixed time points (6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days). The absorbance at 240 nm was measured using a Varioskan LUX multi-functional microplate reader. The results are as follows: Figure 7As shown in Figure C, after mixing with PDN1, the percentage of drug release on day 1 decreased from 80.81% to 56.42%, avoiding a burst of drug release. Meanwhile, the final release on day 7 increased from 92.34% to 100%, reflecting that the addition of PDN did not reduce the total amount of drug released.

[0109] Example 4: Preparation of TLT@PDN hydrogel loaded with hiPSC-CMs and the effect of hydrogel on the morphology of hiPSC-CMs 1) Prepare TLT@PDN precursor solution, the specific method is as described above.

[0110] 2) Thaw human induced pluripotent stem cells (hiPSC-CMs) and use 1×10 6 / cm 2 Cells were seeded at the specified density onto culture flasks coated with Help99A03 at 37°C for 1 hour. Beating cells were observed 1 to 2 days after seeding.

[0111] 3) The day after HiPSC-CMs began to pulsate, they were digested with trypsin and centrifuged at 300g for 5 minutes to collect the cells.

[0112] 4) Wash three times with pre-cooled DPBS at 4°C and collect the precipitate.

[0113] 5) Powder the cell pellet at 1×10⁻⁶ 6 The cells were resuspended in the crosslinking agent at 100 μL and gently blown into the agent using a Pasteur tube to prepare a crosslinking agent containing hiPSC-CMs cells.

[0114] 6) Mix the TLT@PDN precursor solution with the cross-linking agent containing hiPSC-CMs cells at a volume ratio of 3:2 and spread it evenly on the bottom of the confocal dish, 100 μL per well.

[0115] 7) Place the confocal dish in a 37°C incubator for 30 seconds to gel the TLT@PDN containing hiPSC-CMs (i.e., obtain TLT@PDN hydrogels loaded with hiPSC-CMs), and quickly add cardiomyocyte maintenance medium, 100 μL per well after gelation.

[0116] 8) After 7 days of culture, fix the samples: wash three times with DPBS, 5 min each time. At room temperature, add 100 μL of 4% paraformaldehyde to each dish and let stand for 30 min to fix the cells. Wash three times with DPBS, 5 min each time.

[0117] 9) Permeabilization: Add 100 μL of 0.1% Triton X100 (diluted with PBS) to each dish and incubate at room temperature for 30 min. Wash three times with DPBS, 5 min each time.

[0118] 10) Blocking: Add 100 μL of immunofluorescence blocking solution to each dish and block at room temperature for 2 hours.

[0119] 11) Incubation with primary antibody: Wash 3 times with DPBS, 5 min each time. Add 100 μL of rabbit polyclonal CD α-actinin to each dish (the antibody is diluted with primary antibody dilution buffer at a ratio of 1:200), and incubate overnight at 4°C.

[0120] 12) Rewarm to room temperature for 1 hour. Wash three times with DPBS, 5 minutes each time. Add goat anti-rabbit Cy3 secondary antibody (diluted with secondary antibody dilution buffer, dilution ratio 1:500) to a confocal dish. Incubate at room temperature in the dark for 2 hours.

[0121] 13) Phalloidin staining: Discard the secondary antibody, wash 3 times with PBS, 5 min each time. Add phalloidin working solution (FITC-labeled phalloidin stock solution: DPBS = 1:200) and incubate in the dark for 1 h.

[0122] 14) Mounting: Discard the phalloidin working solution, wash three times with DPBS for 5 min each time. Add 100 μL of anti-fluorescence quencher to each confocal dish for mounting.

[0123] 15) Observe under a confocal microscope and use 3D confocal microscopy to scan the hiPSC-CMs encapsulated in TLT@PDN to observe their morphology and arrangement.

[0124] The results are as follows Figure 8 As shown in Figure A, TLT@PDN containing hiPSC-Ms was successfully gelled in a confocal dish, and the hydrogel did not float or lyse after the addition of cardiomyocyte maintenance medium. The cells were then cultured in the confocal dish for another 7 days (culture protocol as previously shown), and the results were as follows. Figure 8 As shown in Figure B, hiPSC-Ms can grow well in a 3D porous hydrogel matrix, specifically exhibiting cell elongation, sarcomere elongation, and a morphology closer to cuboids. These morphological characteristics indicate that the TLT-containing PDN hydrogel successfully induced a specialized morphological transformation of hiPSC-Ms towards cardiomyocytes.

[0125] Example 5: Detection of in vivo transplantation rate and maturity changes of hiPSC-CM loaded in injectable composite hydrogel TLT@PDN Retention rate of injectable composite hydrogel TLT@PDN and its hiPSC-CMs in mice. The preparation method of the TLT@PDN hydrogel precursor solution is as described in Example 1.

[0126] 1) Indocyanine green (ICG) was dissolved in a crosslinking agent at a concentration of 1 μM / 1 μL and physically mixed with TLT@PDN precursor liquid to obtain ICG / TLT@PDN hydrogel.

[0127] 2) Establishment of a mouse model of myocardial infarction and transplantation of ICG / TLT@PDN hydrogel: Mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (0.1 mL / 20 g dose). After anesthesia, the hair on the right chest of the mice was removed with a razor and depilatory cream. The head and limbs were then fixed on the operating table. The tongue of the mice was grasped with forceps to fully expose the epiglottis. An indwelling intravenous catheter was gently inserted into the trachea. After removing the needle core, the catheter was fixed with tape, and the other end was connected to the ventilator. The ventilator was turned on, and the respiratory parameters of the mice were set (tidal volume: 3.3 mL; respiratory ratio: 5:4; 110 breaths / min). After ventilation, the chest wall movement of the mice was observed to determine whether the indwelling catheter tubing had been successfully inserted into the trachea. After successful tracheal intubation, the mice were placed in a right lateral decubitus position. The skin was cut in the 3rd and 4th intercostal spaces on the lateral chest, and the muscles were separated layer by layer to expose the heart. The ligation point is located 2-3 mm below the left atrial appendage. An 8 / 0 suture is inserted at this point and quickly tied. Successful establishment of the myocardial infarction model is confirmed when the entire anterior wall of the left ventricle below the ligation line turns pale. ICG / TLT@PDN is injected around the infarct site using a 1 mL syringe, employing a two-point injection method, with 10 μL at each injection point. The tissue is sutured layer by layer, the thoracic cavity is closed, and the mice are marked with ear tags. After spontaneous breathing resumes, the indwelling needle is removed, the ventilator is turned off, and the mice are placed under a suitable temperature physiotherapy lamp until the anesthetic wears off. Once the mice can move normally, they are transferred to a regular animal enclosure for observation. Specific groupings are as follows: ① 20 μL DPBS; ② 20 μL TLT@PDN hydrogel containing ICG (20 μM) (i.e., ICG / TLT@PDN). NIR fluorescence images of the mice are acquired on days 3, 7, 14, and 28 after the establishment of the myocardial infarction model.

[0128] 3) Establishment of a mouse myocardial infarction model and transplantation of hiPSC-CM / TLT@PDN hydrogel: The method for establishing the mouse myocardial infarction model was as described above. hiPSC-CMs collected on the second day after myocardial infarction were labeled with CM Dil. The CM Dil was dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution (1 mg / mL). Before labeling, the stock solution was diluted to 2 μg / mL with PBS and incubated with hiPSC-CMs at 4℃ and 37℃ for 10 minutes respectively. After washing, digestion, and centrifugation, the samples were collected and stored at 1×10⁻⁶. 5The cells / μL were resuspended in the cross-linking agent and gently blown into a Pasteur tube. The TLT@PDN precursor solution and the cross-linking agent containing hiPSC-CM were mixed at a volume ratio of 3:2 to obtain hiPSC-CM / TLT@PDN hydrogel. The obtained hiPSC-CM / TLT@PDN hydrogel was injected into the area around the myocardial infarction site using a 1mL syringe, using a two-point injection method, with 10μL at each injection point. The specific groups are as follows: ① 20μL of DPBS containing hiPSC-CM ( Duchenne phosphate buffer ); ② 20 μL hiPSC-CM / TLT@PDN hydrogel. Heart tissue from mice was obtained on day 7 after MI for section immunofluorescence staining.

[0129] 4) Immunofluorescence staining of cardiac tissue sections: ① Fixation: After collection, the heart tissue was immersed in paraformaldehyde and placed in a 4°C freezer for 24 hours. ② After 24 hours, it was removed and dehydrated with a gradient sucrose solution (PBS as solvent). It was then incubated overnight in a 15% sucrose solution (w / v). After the tissue settled, the solution was replaced with 30% sucrose and incubated overnight again until settled. OCT embedding was then possible. ③ Frozen sections of the heart were selected at a thickness of 20 micrometers. The sections were mounted on glass slides (avoiding wrinkling) and stored at -80°C. Staining was performed on the slides. Specific staining procedures were as described in the previous immunofluorescence staining procedure, using α-SA to label myocardial tissue and DAPI to label cell nuclei.

[0130] The results are as follows Figure 9 As shown, where Figure 9 A~9B show the in situ retention of TLT@PDN in the heart of mice with myocardial infarction and its changes over time as detected by in vivo imaging. The in situ retention of TLT@PDN hydrogel in infarcted myocardium was verified by encapsulation with the near-infrared (NIR) dye indocyanine green (ICG) and in vivo imaging. Quantitative fluorescence intensity showed that TLT@PDN had a strong retention effect and remained in the infarcted myocardium for more than 14 days. Figure 9C~9E show cross-sectional images of mouse hearts stained with fluorescence chromatograms 7 days after myocardial infarction transplantation using TLT@PDN and TLT@PDN loaded with hiPSC-CMs (i.e., hiPSC-CM / TLT@PDN hydrogel). hiPSC-CMs are labeled with CM Dil (red), sarcomere α-actin (α-SA, green), and cell nuclei (DAPI, blue). The inventors found that hiPSC-CMs rapidly disappeared when transplanted alone, while the retention rate of hiPSC-CMs delivered by TLT@PDN hydrogel was significantly increased at every time point (P<0.0001). This invention measured the area of ​​retained cells in different groups and found that the average cell volume delivered by TLT@PDN hydrogel was significantly increased (P<0.05), as observed in cells within the hydrogel after 7 days of in vitro culture. These results suggest that the hydrogel not only promotes in situ transition of cells to maturity but also provides cells with suitable stiffness and surface morphology to improve cell survival.

[0131] Example 6: Quantitative analysis of cell maturation-related molecules of TLT@PDN-loaded hiPSC-CMs 7 days after orthotopic cardiac transplantation The establishment of the mouse myocardial infarction model and the transplantation method of hiPSC-CM / TLT@PDN hydrogel were as described above. Specific groupings were: ① individual injection of hiPS-CMs; ② hiPS-CMs encapsulated in TLT@PDN hydrogel (i.e., hiPSC-CM / TLT@PDN hydrogel). Mouse hearts were harvested on day 7 post-myocardial infarction.

[0132] 1) RNA Extraction: Total RNA was extracted from the transplant site of the heart. 1 mL of TRIzol reagent was added per 100 mg of fresh tissue. The tissue was minced on ice using a sterile scalpel and then homogenized using a sterile homogenizer or other equipment. For each 1 mL of TRIzol used, 0.2 mL of chloroform was added, and the mixture was shaken vigorously for 15 seconds. The mixture was centrifuged at 10000 × g for 10 minutes at 4°C, and the supernatant was discarded. The RNA precipitate was washed with 75% ethanol. At least 1 mL of 75% ethanol was added for each 1 mL of TRIzol used. The mixture was centrifuged at 7000 × g for 5 minutes at 4°C, and the supernatant was discarded. 25–200 μL of RNase-free water or 0.5% SDS was added, and the mixture was pipetted several times using a pipette tip. The mixture was then incubated at 55–60°C for 10 minutes to dissolve the RNA.

[0133] 2) Add reverse transcription reagents sequentially according to the reaction system, obtain cDNA on the instrument, and detect the concentration. Perform PCR cDNA amplification on maturation-related genes according to the instructions.

[0134] The results are as follows Figure 10As shown, this invention found a significant increase in the expression of TNNI3 mRNA, a phenotypic marker of mature sarcomeres (P<0.05), along with increased expression of MYH6, MYH7, TNNI1, and other sarcomere-related mRNAs (P<0.05). Simultaneously, the expression of ACTN2, an important marker of the Z-line structure of myoblast sarcomeres, and the mature gap junction marker (Cx43; encoded by GJA1) increased significantly as expected (P<0.05; P<0.001). In terms of electrophysiology, this invention found a significant increase in the expression of electrophysiological markers similar to those in mature cardiomyocytes, such as KCNJ2 (P<0.05), CACNA1c (P<0.001), and SCN5A mRNA (P<0.05) (including loss of automaticity, acquisition of a more negative resting membrane potential, increased duration and amplitude of action potentials, and improved calcium treatment). In terms of metabolism, the expression of molecules involved in fatty acid transport (CD36; P<0.01) was significantly increased, and the expression of myocardial mitochondrial activation-related molecules (PPARGC1A, ESRRA; P<0.001, P<0.05) was also significantly increased, indicating that the cell has achieved a metabolic transformation from glycolysis to oxidative phosphorylation.

[0135] In summary, morphological, functional, and metabolic analyses indicate that hiPSC-CMs transplanted with TLT@PDN possess mature cellular characteristics, exhibiting better sarcomeres and transverse tubules, more mature contractile and electrical properties, and more mature metabolic functions.

[0136] Example 7: Effect of TLT@PDN loaded with hiPSC-CMs on improving cardiac function after acute myocardial infarction (AMI) The establishment of the mouse myocardial infarction model was as described above. Specific groupings were as follows: sham operation group (Sham group, undergoing open-chest surgery but without coronary artery ligation), DPBS (20 μL DPBS injected into the peri-infarction site after myocardial infarction modeling), TLT@PDN hydrogel group (20 μL TLT@PDN hydrogel injected into the peri-infarction site after myocardial infarction modeling), and TLT@PDN hydrogel + hiPSC-CMs group (20 μL hiPSC-CM / TLT@PDN hydrogel injected into the peri-infarction site after myocardial infarction modeling).

[0137] Mouse Cardiac Function Assessment: Cardiac function was assessed in mice on days 7 and 28 after myocardial infarction modeling. Mice were anesthetized with sodium pentobarbital, their limbs were fixed to the ultrasound operating table, and the fur on their chests was shaved to facilitate ultrasound probe examination. The ventricular short axis was detected using two-dimensional ultrasound, and ventricular wall motion was observed using M-mode ultrasound. Data were processed, with each indicator derived from the average of three consecutive cardiac cycles. Left ventricular ejection fraction (LVEF) = (LVEDV - LVESV) / ​​LVEDV × 100%. Left ventricular fractional shortening (LVFS) = (LVDd - LVDs) / LVDd × 100%.

[0138] The results are as follows Figure 11 As shown, echocardiography at 7 days post-MI showed significantly reduced EF (left ventricular ejection fraction) and FS (left ventricular shortening fraction) in the TLT@PDN hydrogel + hiPSC-CMs group, with no significant difference observed between the TLT@PDN hydrogel and PBS groups. Echocardiography at 28 days post-MI showed significantly increased EF and FS in the TLT@PDN hydrogel + hiPSC-CMs group (P < 0.0001), while there was no significant difference between the TLT@PDN hydrogel and PBS groups. Simultaneously, cardiac function in the TLT@PDN hydrogel + hiPSC-CMs group continued to recover until day 28 (P < 0.05), while EF and FS remained the same on day 14 in the TLT@PDN hydrogel group (P > 0.05). This indicates that TLT@PDN hydrogel + hiPSC-CMs can, to some extent, slow down the adverse adaptive remodeling of the ventricles after myocardial infarction, and this improvement can persist up to 28 days post-MI.

[0139] Example 8: Effects of TLT@PDN loaded with hiPSC-CMs on ventricular wall thickness and fibrosis degree after AMI Preparation of cardiac tissue sections for each group: 1) Preparation of paraffin-embedded blocks: After sampling, the heart was fixed in 4% paraformaldehyde solution for 24 hours. The heart was then placed in a tissue embedding cassette and rinsed overnight in a glass tank with running water. Following the tissue dehydration and clearing process, the tissue was treated with a gradient of 70% anhydrous ethanol for 1 hour, 80% anhydrous ethanol for 1 hour, 90% anhydrous ethanol for 1 hour, 95% anhydrous ethanol for 1 hour, 100% anhydrous ethanol I for 1 hour, 100% anhydrous ethanol II for 1 hour, xylene I for 10 minutes, and xylene II for 10 minutes. The fully dehydrated and cleared heart tissue was then immersed in paraffin I at 60°C for 1 hour and paraffin II for 1 hour. The paraffin blocks were then melted in a machine, and the melted paraffin was dripped into the embedding mold. Using preheated forceps, the tissue block was placed into the embedding mold with the cut side of the tissue facing down and in close contact with the mold surface. After simple cooling and fixation, the paraffin was poured out again until the mold was nearly full. Transfer the mold filled with paraffin wax to a cooling table for solidification. After it has completely cooled and solidified, remove the embedded paraffin wax block, trim off any excess paraffin wax around the edges with a blade, and store it at room temperature.

[0140] 2) Paraffin section preparation: Remove the paraffin-embedded block and trim its edges. Beforehand, adjust the microtome, setting the section thickness to 8 μm. After installing the blades, lock the sectioning assembly to avoid accidental cutting. Rotate the handwheel for continuous sectioning. Float the prepared, continuous, and complete tissue sections in 40°C warm water to gradually flatten them. Gently lift the tissue sections with a glass slide. Use filter paper to absorb excess moisture around the paraffin sections. Place them on a 60°C baking machine for baking, ready for subsequent staining. Masson staining: 1) Dewax the paraffin sections of the heart with xylene three times, 10 min each time. Then, immerse the sections in ethanol of different concentrations for 5 min each (100%, 95%, 85%, and 75%) for gradient rehydration. Finally, immerse the sections in double-distilled water for 5 min.

[0141] 2) Add hematoxylin dye to the myocardial tissue sections and let them stand for 20 minutes. Then place the sections in double-distilled water and gently shake them on a shaker for 5 minutes to remove unbound dye.

[0142] 3) After absorbing the moisture from the slices, add 1% hydrochloric acid and place them under a microscope for color separation for 30 seconds.

[0143] 4) Next, rinse the slide with running water for 10 minutes. After the water on the slide is absorbed, the staining solution can be dropped onto the tissue for staining for 10 minutes.

[0144] 5) Wash off excess staining solution from the sections with double-distilled water, blot dry, and then add 1% phosphomolybdic acid solution to the tissue for reaction. After differentiation for 5 minutes, add aniline blue solution and react for another 5 minutes.

[0145] 6) Soak the tissue sections in glacial acetic acid solution (0.2%) and place them on a shaker to react slowly for 20 seconds.

[0146] 7) Soak the slices again in two portions of xylene for 10 minutes each time.

[0147] 8) Neutral resin sealing.

[0148] WGA staining: 1) Dewax the paraffin sections of the heart with xylene three times, 10 min each time. Then, immerse the sections in ethanol of different concentrations for 5 min each (100%, 95%, 85%, and 75%) for gradient rehydration. Finally, immerse the sections in double-distilled water for 5 min.

[0149] 2) Add WGA dye to the myocardial tissue sections and let them stand for 20 minutes. Then place the sections in PBS and gently shake on a shaker for 5 minutes to remove unbound dye. Repeat the washing process 3 times.

[0150] 3) Add an appropriate amount of anti-fluorescence quenching mounting medium to the glass slide, and gently cover it with the coverslip from one side. Avoid air bubbles during the sealing process.

[0151] This invention assesses the left ventricular wall thickness and the degree of myocardial fibrosis in rats 28 days after myocardial infarction using Masson staining. The results are as follows: Figure 12 In Figure A, all groups of mice showed varying degrees of thinning and fibrous tissue deposition in the left ventricle. Compared with the DPBS group, the ventricular wall thickness was significantly increased in the TLT@PDN hydrogel + hiPSC-CMs group (P<0.0001). The area of ​​fibrotic myocardial tissue (blue after Masson staining) was calculated using ImageJ software. Statistical results of fibrosis area showed that, compared with the DPBS group, the TLT@PDN hydrogel + hiPSC-CMs group significantly reduced the degree of myocardial fibrosis (P<0.01) and improved cardiomyocyte hypertrophy (P<0.0001). These results indicate that implantation of TLT@PDN hydrogel + hiPSC-CMs can alleviate ventricular fibrosis after myocardial infarction in mice and largely preserve the thickness of the left ventricular wall, thus slowing down adverse adaptive remodeling of the ventricle.

[0152] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composite hydrogel, characterized in that, The preparation process includes the following steps: S1. Mix the first chitosan and the second chitosan in an acidic reagent solution to obtain the precursor solution; S2. Mix the drug-loaded liposomes and the third chitosan solution to obtain composite liposomes; S3. Mix the precursor fluid obtained in step S1 and the composite liposome obtained in step S2, add glutaraldehyde crosslinking agent, and crosslink fully to obtain composite hydrogel. Wherein, the first chitosan is unmodified deacetylated chitin with a molecular weight of 200-400 kDa; the second chitosan is ethylene glycol-chitosan with a degree of polymerization ≥400 and a molecular weight of 200-300 kDa; the third chitosan is obtained by modifying the fourth chitosan with a quaternizing agent, with a degree of quaternization ≥65%; the fourth chitosan is unmodified deacetylated chitin with a molecular weight of 60-100 kDa; The drug in the drug-loaded liposomes is Torin 1; The mass ratio of the first chitosan to the second chitosan is (2.5~3.5):

1.

2. The composite hydrogel according to claim 1, characterized in that, The mass ratio of the first chitosan to the second chitosan is (2.9~3.0):

1.

3. The composite hydrogel according to claim 1, characterized in that, The quaternizing agent includes iodomethane.

4. The composite hydrogel according to claim 1, characterized in that, The liposomes of the drug-loaded liposomes include lecithin.

5. The composite hydrogel according to claim 4, characterized in that, The lecithin includes egg yolk lecithin and / or soybean lecithin.

6. The composite hydrogel according to claim 1, characterized in that, The acidic reagent includes acetic acid.

7. A composite hydrogel loaded with hiPSC-CMs, characterized in that, hiPSC-CMs cells were obtained by loading the composite hydrogel according to any one of claims 1 to 6.

8. The composite hydrogel loaded with hiPSC-CMs according to claim 7, characterized in that, Specifically, it is prepared by the following steps: sI. Repeat steps S1 and S2 in claim 1 to obtain the precursor fluid and the composite liposome solution, respectively; sII. Mix the precursor fluid, composite liposome solution, hiPSC-CMs cell suspension obtained in step sI with glutaraldehyde crosslinking agent and crosslink them thoroughly to obtain a composite hydrogel loaded with hiPSC-CMs.

9. The use of the composite hydrogel according to any one of claims 1 to 6 or the composite hydrogel carrying hiPSC-CMs according to claim 7 or 8 in the preparation of drugs for improving cardiac function.

10. The application according to claim 9, characterized in that, The improvement of cardiac function includes one or more of the following: treatment of myocardial infarction, inhibition of myocardial cell hypertrophy, and inhibition of myocardial fibrosis.

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