Injectable conductive hydrogel for promoting cardiac repair and functional recovery after myocardial infarction and preparation method of injectable conductive hydrogel

Abstract

The invention relates to the technical field of medicine preparation, in particular to injectable conductive hydrogel for promoting cardiac repair and functional recovery after myocardial infarction and a preparation method of the injectable conductive hydrogel. The preparation method comprises the following steps: modifying fibroin with 4-carboxyphenylboronic acid to obtain PS; the preparation method comprises the following steps: functionalizing gelatin by using epigallocatechin gallate, and carrying out pyrrole-NH2 coupling reaction polymerization to generate GPE; then oxidized hyaluronic acid is introduced, an aldehyde group of oxidized hyaluronic acid and amino groups in PS and GPE form a pH response network through a Schiff base bond, a borate bond is formed between CBPA and EGCG to endow ROS with responsiveness, and a gelatin main chain is sensitive to degradation of MMP-9 and provides MMP-9 responsiveness, so that a triple-response hydrogel matrix PGO is formed. According to the method, Sal-loaded calcium alginate microspheres (CAM-Sal) are prepared through a microfluidic technology and introduced into PGO, the hydrogel is obtained, and stage-specific on-demand release of Sal in the MI disease course is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparation, in particular to an injectable conductive hydrogel for promoting heart repair and function recovery after myocardial infarction and a preparation method thereof. BACKGROUND

[0002] Myocardial infarction (MI) is one of the leading causes of death worldwide and has caused a serious burden on human health for a long time. It is usually caused by acute occlusion of the coronary artery, which triggers oxidative stress, inflammatory cascade and myocardial cell death. These pathological events lead to thinning of the ventricular wall, scarring, adverse remodeling and eventually development of heart failure. Although existing clinical strategies (such as drug therapy, percutaneous coronary intervention and coronary artery bypass grafting) can alleviate symptoms and delay disease progression to some extent, there are still obvious limitations in preventing adverse remodeling, restoring electrical-mechanical coupling and maintaining long-term cardiac function. Heart transplantation is the ultimate means for patients in the end stage, but its application is limited by donor scarcity and immune rejection. Therefore, there is an urgent need to develop new therapeutic strategies that can promote myocardial repair and functional recovery. SUMMARY

[0003] Based on the above, the present application provides an injectable conductive hydrogel for promoting heart repair and function recovery after myocardial infarction and a preparation method thereof. The hydrogel can achieve triple-responsive salidroside release and promote heart repair and function recovery after myocardial infarction.

[0004] To achieve the above-mentioned purpose, the present application provides the following solutions: One of the technical solutions of the present application is a preparation method of an injectable conductive hydrogel, comprising the following steps: Step 1: Using microfluidic technology, sodium alginate solution is used as the dispersed phase and mineral oil is used as the continuous phase to form uniform droplets in the microchannel, and then the droplets are contacted with calcium chloride solution to obtain CAM hydrogel microspheres through ion cross-linking reaction; The CAM hydrogel microspheres are soaked in a salidroside (Sal) solution for drug loading, and then centrifuged to obtain CAM@Sal microspheres; Step 2: 4-carboxyphenylboronic acid (CBPA) and NHS are dissolved in DMF, EDC·HCl is added under ice bath condition and stirred for reaction, and then washed, dried and solvent removed to obtain CBPA-NHS; The silk fibroin solution is added dropwise into MES buffer solution, CBPA-NHS solution is added and the pH of the reaction system is adjusted to neutral, and then the reaction is carried out, and PS is obtained after dialysis and centrifugal impurity removal; Step 3, pyrrole is added to the gelatin solution, and an oxidizing agent is added under an inert atmosphere to react, and then the product is obtained by dialysis and lyophilization; the gelatin is dissolved in the MES buffer solution, and then EGCG and EDC·HCl are added to react, and then the product is obtained by dialysis and lyophilization; the GP and the GE are mixed in a volume ratio of 1:1 and then lyophilized to obtain the GPE; Sodium periodate is added to the hyaluronic acid solution to react, and then the product is obtained by dialysis and lyophilization after the reaction is terminated; Step 4, the PS, the GPE and the OHA are respectively prepared into a PS solution, a GPE solution and an OHA solution; the PS solution, the GPE solution and the OHA solution are mixed, and then the CAM@Sal microspheres are uniformly dispersed and incubated to obtain the injectable conductive hydrogel; Steps 1, 2 and 3 are not in a specific order.

[0005] The second technical scheme of the present application is an injectable conductive hydrogel prepared by the preparation method.

[0006] The third technical scheme of the present application is the application of the injectable conductive hydrogel in the preparation of a drug for treating myocardial infarction.

[0007] The fourth technical scheme of the present application is a drug for promoting heart repair and functional recovery after myocardial infarction, and the effective component comprises the injectable conductive hydrogel.

[0008] Compared with the prior art, the present application has the following beneficial effects: Myocardial infarction can cause large-area myocardial cell death and adverse remodeling, and a single therapy is often difficult to reverse. The present application constructs an injectable conductive hydrogel (PGO / CAM@Sal), which can realize stage-specific on-demand release of salidroside (Sal) through triple response of pH, reactive oxygen species (ROS) and matrix metalloproteinase 9 (MMP-9). The hydrogel can effectively reduce myocardial fibrosis and hypertrophy and restore left ventricular structure and function by improving the microenvironment of the infarct area. BRIEF DESCRIPTION OF DRAWINGS

[0009] In order to more clearly illustrate the technical schemes in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0010] Figure 1 A schematic diagram of the injectable conductive hydrogel (pH / ROS / MMP-9 triple network stimulus-responsive hydrogel) for treating myocardial infarction.

[0011] Figure 2 SEM image of CAM.

[0012] Figure 3 SEM image of CAM@Sal.

[0013] Figure 4 Particle size distribution of CAM and CAM@Sal.

[0014] Figure 5 Zeta potential of CAM and CAM@Sal.

[0015] Figure 6 FTIR spectra of PGO, CAM@Sal and PGO / CAM@Sal.

[0016] Figure 7 SEM images of PGO and PGO / CAM@Sal.

[0017] Figure 8 Gelation images of PGO and PGO / CAM@Sal.

[0018] Figure 9 Responsive release curves of Sal from PGO / CAM@Sal hydrogels under different conditions.

[0019] Figure 10 Live / dead staining images of H9c2 (A) and HUVEC (B) treated with leaching solution of PGO, CAM@Sal and PGO / CAM@Sal.

[0020] Figure 11 H&E staining images of major organs (liver, spleen, lung, kidney) of rats treated with different hydrogels.

[0021] Figure 12 A: Representative echocardiography images of rats 28 days after hydrogel injection; B-E: Quantitative assessment of key cardiac function indicators by echocardiography: ejection fraction (EF), fractional shortening (FS), left ventricular internal dimension at end-systole (LVIDs), left ventricular internal dimension at end-diastole (LVIDd).

[0022] Figure 13 H&E and Masson trichrome staining images of infarcted myocardium of rats 28 days after treatment with different hydrogels. DETAILED DESCRIPTION

[0023] A number of exemplary embodiments of the present application will now be described in detail with reference to the drawings. Such embodiments are described herein with the intent to enable those skilled in the art to make and use them. However, these embodiments are not intended to limit the scope of the application, as one skilled in the art will recognize.

[0024] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an intermediate value of the parameter is understood to be specifically disclosed anywhere that the parameter is stated to have a range of values. Any smaller range of values of the parameter is understood to be specifically disclosed, even if not explicitly stated. The upper and lower limits of these smaller ranges of values are independently combinable with one another to constitute further smaller ranges of values within the ranges of values stated for the parameter.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials that are related to the present application. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0026] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0027] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0028] Injectable conductive hydrogels are considered as ideal candidates for post-myocardial infarction repair due to their good biocompatibility, tunable mechanical properties, and the ability to facilitate electrical signal conduction. For example, polypyrrole (PPy)-grafted conductive hydrogels can restore electrical conduction in infarcted myocardium and reduce ventricular remodeling; when combined with bioactive molecules, such materials not only provide structural support but also synergistically enhance myocardial repair. However, traditional materials are often difficult to adapt to the dynamic and heterogeneous changes of the MI pathological process, thus limiting the precision and timeliness of therapeutic intervention.

[0029] To this end, researchers have developed various stimuli-responsive hydrogels that can achieve adaptive drug release according to specific pathological signals. For example, pH-responsive systems can release drugs or exosomes in ischemic acidic environments to resist apoptosis, oxidation, and promote angiogenesis; ROS-responsive systems can induce macrophages to convert to the repair type M2 phenotype by delivering immune modulators; and MMP-responsive systems can improve cardiac function by stabilizing HIF-1α and up-regulating connexin 43 (Cx43) during myocardial remodeling. Although these studies have made important progress, most intelligent hydrogels only act on a single stage of the MI disease process, limiting the comprehensive therapeutic effect. Therefore, if a multifunctional conductive hydrogel can be developed that covers multiple stages of the pathological process, or a more optimal cardiac repair effect will be achieved.

[0030] The pathological evolution of myocardial infarction can generally be divided into three stages: the first stage is the acute injury period, a large number of cell death and lactic acid accumulation cause the local pH to drop to about 6.5-6.8; the second stage is the inflammatory response period, mainly immune cell infiltration and excessive ROS production, which exacerbates tissue damage; the third stage is the repair and remodeling period, M2 type macrophages and fibroblasts mediate the anti-inflammatory and repair process, at this time the expression of MMP-9 is increased, promoting extracellular matrix (ECM) remodeling and scar formation. The present application realizes dynamic and precise intervention on the changes of the disease course by corresponding the specific release mechanism to the different pathological stages of MI.

[0031] Salidroside (Sal) is a natural active glycoside small molecule (molecular weight about 300 Da) extracted from Rhodiolae Crenulatae, which has significant anti-inflammatory and antioxidant effects and shows good protective effect in ischemic heart injury. Loading Sal in metal organic frameworks (MOFs) or hydrogels can achieve sustained release of heart targeting, relieve oxidative stress and inflammatory response, and promote heart function recovery. However, its clinical translation is still limited by short half-life, low bioavailability, and easy degradation. Therefore, it is of great significance to develop a drug delivery platform that can achieve long-term retention, targeted accumulation, and multi-stage response release to maximize the therapeutic potential of Sal.

[0032] To this end, the present application constructs an injectable conductive hydrogel system PGO / CAM@Sal: silk fibroin (SF) is modified with 4-carboxyphenylboronic acid (CBPA) to obtain "PS"; gallic acid epigallocatechin gallate (EGCG) is functionalized with gelatin (G), and is polymerized by pyrrole-NH2 coupling reaction to obtain "GPE"; then oxidized hyaluronic acid (OHA) is introduced, and the aldehyde group thereof is connected with the amino group in PS and GPE through Schiff base bond to form a pH-responsive network, and CBPA and EGCG form a borate ester bond to endow ROS responsiveness, and the gelatin backbone is sensitive to MMP-9 degradation, providing MMP-9 responsiveness, thereby forming a triple-responsive (pH / ROS / MMP-9) hydrogel matrix "PGO". The calcium alginate microspheres (CAM@Sal) loaded with Sal are prepared by microfluidic technology, and are introduced into PGO to obtain the composite hydrogel PGO / CAM@Sal, and the stage-specific on-demand release of Sal in the MI process is realized. Figure 1 The multifunctional system is expected to serve as a precise post-MI treatment platform.

[0033] The present application provides a preparation method of an injectable conductive hydrogel, comprising the following steps: Step 1: using microfluidic technology, sodium alginate solution is used as the dispersed phase, mineral oil is used as the continuous phase, uniform droplets are formed in the microchannel, then the droplets are contacted with calcium chloride solution, and CAM hydrogel microspheres are obtained through ion cross-linking reaction; The CAM hydrogel microspheres are soaked in a salidroside solution for drug loading, and centrifuged to obtain CAM@Sal microspheres; Step 2: 4-carboxyphenylboronic acid and NHS are dissolved in DMF, EDC·HCl is added under ice bath condition, stirring reaction, washing, drying and removing the solvent to obtain CBPA-NHS; The silk fibroin solution is added dropwise into the MES buffer solution, the CBPA-NHS solution is added and the pH of the reaction system is adjusted to neutral, reaction is carried out, and PS is obtained after dialysis and centrifugal removal of impurities; Step 3: pyrrole is added to the gelatin solution, and an oxidizing agent is added under inert atmosphere for reaction, and GP is obtained after dialysis and freeze-drying; the gelatin is dissolved in the MES buffer solution, EGCG and EDC·HCl are added for reaction, and GE is obtained after dialysis and freeze-drying; GP and GE are mixed in a volume ratio of 1:1 and freeze-dried to obtain GPE; Sodium periodate is added to the hyaluronic acid solution for reaction, and OHA is obtained after termination of the reaction, dialysis and freeze-drying; Step 4: PS, GPE and OHA are respectively prepared into PS solution, GPE solution and OHA solution; the PS solution, GPE solution and OHA solution are mixed, and the CAM@Sal microspheres are dispersed uniformly and incubated to obtain the injectable conductive hydrogel; Step 1, Step 2 and Step 3 are not in the order.

[0034] In a preferred embodiment of the present application, in Step 1, the mass concentration of the sodium alginate solution is 2%; the mineral oil is a mineral oil containing Span 80 with a mass concentration of 5%; the mass concentration of the calcium chloride solution is 2%; The concentration of the salidroside solution is 1.0 mg / mL; the drug loading time is 12 h.

[0035] In a preferred embodiment of the present application, in Step 2, the ratio of the amounts of 4-carboxyphenylboronic acid, NHS, DMF and EDC·HCl is 12 mmol: 17 mmol: 20 mL: 18 mmol; the concentration of the silk fibroin solution is 6 wt%; the concentration of the CBPA-NHS solution is 40 mg / mL; the concentration of the MES buffer solution is 0.1 M; the concentration of the CBPA-NHS solution is 40 mg / mL; the volume ratio of the silk fibroin solution, the MES buffer solution and the CBPA-NHS solution is 4:6:1; and the reaction conditions for preparing PS are as follows: stirring at room temperature for 6 h.

[0036] In a preferred embodiment of the present application, in Step 3, the concentration of the gelatin solution is 2 wt%; the concentration of the pyrrole in the gelatin solution is 15 wt%; the oxidizing agent is FeCl3; the reaction conditions for preparing GP are as follows: reaction at 4 °C for 24 h; the concentration of the MES buffer solution is 0.05 M for preparing GE; the ratio of the amounts of gelatin, the MES buffer solution, EGCG and EDC·HCl is 1.0 g: 20 mL: 204 mg: 173 mg; and the reaction conditions for preparing GE are as follows: reaction at 27 °C for 3 h.

[0037] In a preferred embodiment of the present application, in Step 3, the concentration of the hyaluronic acid solution is 40 mg / mL; the mass ratio of hyaluronic acid to sodium periodate in the hyaluronic acid solution is 4:1.08; and the reaction conditions for preparing OHA are as follows: reaction at room temperature for 24 h.

[0038] In a preferred embodiment of the present application, in Step 4, the concentration of the PS solution is 3 wt%; the concentration of the GPE solution is 8 wt%; the concentration of the OHA solution is 0.5 wt%; the volume ratio of the PS solution, the GPE solution and the OHA solution is 2:1:3; and the concentration of the CAM@Sal microspheres in the reaction system is 10 mg / L.

[0039] In a preferred embodiment of the present application, in Step 4, the incubation conditions are set as follows: incubation in a 37 °C water bath for 30-60 min.

[0040] The second aspect of the present application provides an injectable conductive hydrogel prepared by the preparation method described above.

[0041] The third aspect of the present application provides use of the injectable conductive hydrogel described above in the preparation of a medicament for treating myocardial infarction.

[0042] The fourth aspect of the present application provides a medicament for promoting heart repair and functional recovery after myocardial infarction, and the effective component includes the injectable conductive hydrogel described above.

[0043] The hydrogel of the present application has excellent biocompatibility, injectability, self-healing and conductivity. In vivo and in vitro experiments show that the material can reduce the level of reactive oxygen species, inhibit the expression of pro-inflammatory cytokines, promote M2 macrophage polarization and angiogenesis, and enhance myocardial electrical coupling. By improving the microenvironment of the infarct area, the system effectively reduces myocardial fibrosis and hypertrophy, restores left ventricular structure and function. The present study provides a multifunctional, intelligent and controllable precise myocardial infarction treatment strategy.

[0044] The meanings of the English abbreviations in the present application are as follows: Sal: salidroside NHS: N-hydroxysuccinimide; DMF: N,N-dimethylformamide; EDC·HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EGCG: epigallocatechin gallate; MMP-9: matrix metalloproteinase 9.

[0045] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed if not specifically stated.

[0046] The dialysis described in the embodiments of the present application is water-phase dialysis using a molecular weight cut-off (MWCO) dialysis membrane. The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0047] Example 1 1.1 Synthesis of Sal-loaded calcium alginate microspheres (CAM@Sal) Sal-loaded calcium alginate microspheres were prepared by microfluidic combination with post-soaking method. 2% (w / v) sodium alginate solution (solvent: deionized water) was used as the dispersed phase, 5% (w / v) Span 80 mineral oil as the continuous phase, and 2% (w / v) calcium chloride solution (solvent: deionized water) as the collection / crosslinking phase. In the T-shaped microfluidic chip, the flow rates of the dispersed phase and the continuous phase were set to 1.0 μL / min and 450 μL / min, respectively. The uniform "water-in-oil" droplets were formed by the shearing action of the continuous phase on the dispersed phase, and the droplets flowed along the outlet channel of the chip and were injected into the collection / crosslinking phase with a flow rate of 50 μL / min to form uniform alginate droplets. The calcium alginate microspheres were formed by crosslinking with Ca 2+ The microspheres were washed with distilled water to remove residual oil, and then soaked in a 1.0 mg / mL Sal solution (solvent: deionized water) for 12 h to complete drug loading. The excess solution was removed by centrifugation (3000 x g, 5 min), and the obtained CAM@Sal microspheres were stored at 4 °C in the dark for later use.

[0048] 1.2 Preparation of PS CBPA-NHS was prepared by EDC / NHS activation method. 4-carboxyphenylboronic acid (2 g, 12 mmol) and NHS (2 g, 17 mmol) were dissolved in 20 mL DMF, and EDC-HCl (3 g, 18 mmol) was added under ice bath conditions. The reaction was stirred overnight. The reaction solution was washed with 0.1 M HCl solution (solvent: deionized water) and 0.1 M NaHCO3 solution (solvent: deionized water) in sequence, dried, and solvent was removed under reduced pressure to obtain CBPA-NHS. Subsequently, 6 wt% silk fibroin solution (solvent: ultrapure water) 4.0 mL was added to 0.1 M MES buffer (pH 5.5, 6.0 mL) (4 °C), CBPA-NHS dissolved in DMSO (40 mg CBPA-NHS dissolved in 1 mL DMSO) was added, and the pH of the system was adjusted to 7.0 by adding 1.0 M NaHCO3 dropwise. The system was stirred at room temperature for 6 h. After dialysis and centrifugation, PS was obtained.

[0049] 1.3 Preparation of GPE and OHA Preparation of GPE included two steps: first, 15% w / w pyrrole was added into 2 wt% gelatin solution (solvent: deionized water) and reacted with FeCl3(0.2 M, 17 mL) as oxidant at 4 °C for 24 h under nitrogen protection, and then dialyzed and lyophilized to obtain GP; second, 1.0 g gelatin was dissolved in 20 mL 0.05 M MES buffer (pH 5.5), 204 mg EGCG and 173 mg EDC·HCl were added, and stirred at 27 °C for 3 h, then dialyzed and lyophilized to obtain GE. GP and GE were mixed at a volume ratio of 1:1 and lyophilized to obtain the composite product GPE.

[0050] Preparation of OHA: 4.0 g hyaluronic acid (HA) was dissolved in 100 mL deionized water, and 1.08 g sodium periodate (NaIO4) was added after complete dissolution. The reaction was carried out at room temperature for 24 h, followed by the addition of ethylene glycol to terminate the reaction and stirring for 2 h. The product was dialyzed and lyophilized to obtain OHA.

[0051] 1.4 Preparation of PGO and PGO / CAM@Sal composite hydrogel Prepare PS (3 wt%), GPE (8 wt%) and OHA (0.5 wt%) solutions (solvent: deionized water) respectively, mix them at a volume ratio of 2:1:3 to form PGO matrix hydrogel, add CAM@Sal microspheres (5% w / v), and gently stir at 50 rpm at room temperature for 5 min to ensure uniform dispersion, then incubate in a 37 °C water bath for 60 min to complete the formation of the hydrogel. The obtained PGO / CAM@Sal composite hydrogel was stored at 4 °C in the dark for later use.

[0052] 1.5 Scanning Electron Microscopy (SEM) The PGO (hereinafter PGO refers to the same preparation method as above) and PGO / CAM@Sal composite hydrogel samples prepared above were cut into small pieces of about 3 × 3 × 1 mm 3 , frozen in liquid nitrogen for 30 min and lyophilized for 24 h. The dried samples were fixed on conductive tape and gold-coated using a metal sputter coater (Quorum SC7620, UK) at a current of 10 mA for 45 s. The microstructure of the samples was observed using a scanning electron microscope (Sigma 500, Zeiss, Germany), and the results are as follows.

[0053] Scanning electron microscope observation, particle size and Zeta potential test were performed on CAM@Sal and CAM (CAM is calcium alginate microspheres, and CAM@Sal is obtained after loading Sal; the difference between the preparation method of CAM and CAM@Sal is that the addition of Sal is omitted; the CAM appearing hereinafter is prepared by the same method as this place), and the results are as follows.

[0054] Figure 2 The scanning electron micrograph of CAM; from Figure 2 It can be seen that the surface of the blank microspheres CAM is smooth and dense.

[0055] Figure 3 The scanning electron micrograph of CAM@Sal; from Figure 3 It can be seen that the drug-loaded microspheres CAM@Sal have slight surface wrinkles.

[0056] Figure 4 The particle size graph of CAM and CAM@Sal; particle size analysis shows that the average diameter of CAM is 104.58 μm, and the average diameter of CAM@Sal is 118.82 μm, indicating that the particle size increases slightly after drug encapsulation.

[0057] Figure 5 The Zeta potential graph of CAM and CAM@Sal; the Zeta potential changes from -68.60 mV to -58.57 mV, suggesting that Sal has partially neutralized the surface charge of the microspheres.

[0058] Figure 7 The scanning electron micrograph of PGO and PGO / CAM@Sal; from Figure 7 It can be seen that PGO and PGO / CAM@Sal have a porous, cross-linked three-dimensional network structure, and microspheres are embedded in PGO / CAM@Sal.

[0059] Figure 8 The gel formation real object graph of PGO and PGO / CAM@Sal; the inverted bottle test shows that PGO / CAM@Sal can form a gel in about 20 seconds, which is faster than PGO alone (30 seconds), and can achieve rapid in-situ formation.

[0060] 1.6 Fourier Transform Infrared ectrometry (FTIR) About 20 mg of freeze-dried sample (PGO, CAM@Sal and PGO / CAM@Sal) was sandwiched between two glass slides and pressed into a thin film. Fourier transform infrared spectrometer (Nicolet iS10, Thermo Fisher, USA) was used to scan the sample at 4000-500 cm-1.-1 wavenumber range, at 4 cm -1 The resolution, the infrared spectra of PGO, CAM@Sal and PGO / CAM@Sal were recorded by attenuated total reflection (ATR).

[0061] Figure 6 The infrared spectra of PGO, CAM@Sal and PGO / CAM@Sal; the spectra of PGO / CAM@Sal retained the characteristic peaks of PGO at 1620, 1406 and 1034 cm Figure 6 It can be seen that the spectrum of PGO / CAM@Sal retained the characteristic peaks of PGO at 1620, 1406 and 1034 cm -1 and the peaks of CAM@Sal at 1516 and 1234 cm -1 , indicating that the composite hydrogel had been successfully formed.

[0062] 1.7 In vitro drug release experiment of hydrogel The drug release behavior of PGO / CAM@Sal hydrogel was evaluated under different stimulation conditions. The hydrogel samples were incubated in 3 mL buffer solution at 37 °C with slight shaking. The experimental groups included: PBS (pH 7.4), acetic acid buffer (pH 5.0), PBS (pH 7.4) containing H2O2 (200 μM), PBS (pH 7.4) containing MMP-9 (1 μg / mL), PBS (pH 7.4) containing H2O2 (200 μM) and MMP-9 (1 μg / mL), triple stimulation conditions (pH 5.0 + 200 μM H2O2 + 1 μg / mL MMP-9).

[0063] At predetermined time points, 1 mL of release medium was removed and replaced with an equal volume of fresh PBS. The concentration of Sal in the solution was determined at the characteristic absorption wavelength of salidroside using a UV-Vis spectrophotometer, and the cumulative release rate was calculated. Each condition was repeated three times to ensure data reliability.

[0064] Figure 9 The responsive release curves of Sal in PGO / CAM@Sal hydrogel under different conditions; the drug release experiment showed that the system had significant triple responsiveness to pH, reactive oxygen species (ROS) and MMP-9. Under the conditions of pH 5.0, 200 μM hydrogen peroxide (H2O2) and 1 μg / mL MMP-9, the cumulative release amount reached 77.72%, 68.01% and 57.49% within 7 days, respectively; and under the combined stimulation conditions, the cumulative release amount reached as high as 88.43%, confirming its stage-specific delivery characteristics.

[0065] 1.8 Cell survival / death staining Cell viability was evaluated by live / dead staining. H9c2 cardiomyocytes and human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates and cultured for 24 h at 37 °C in a 5% CO2 humidified environment. Subsequently, the culture medium was replaced with the leaching solution containing PGO, CAM@Sal and PGO / CAM@Sal (100 μL / well; the leaching solution was prepared as follows: according to the ISO 10993-12 standard, an equal amount of hydrogel sample was placed in a sterile centrifuge tube, and serum-free complete culture medium was added at a mass / volume ratio of 0.2 g / mL, and leaching was performed at 37 °C and 150 rpm for 24 h. The supernatant was collected and sterilized by 0.22 μm filter membrane, and stored at 4 °C in the dark for standby use). After incubation for 24, 48 and 72 h, respectively, PBS was added for washing and Calcein-AM / PI staining solution (100 μL, Beyotime, China) was added, and incubation was performed at 37 °C in the dark for 10 min. A fluorescence microscope (Olympus, Japan) was used to obtain fluorescence images to observe the live / dead distribution of cells.

[0066] Figure 10 Fig. 9 is a live / dead staining diagram of H9c2 (A) and HUVEC (B) treated with the leaching solution of PGO, CAM@Sal and PGO / CAM@Sal; the live / dead staining results show that, after treatment with different leaching solutions, H9c2 cells (A) and HUVEC cells (B) both have high cell survival rate and normal morphology.

[0067] 1.9 SD rat myocardial infarction (MI) model construction and in vivo verification experiment 1.9.1 Model construction method A total of 60 male Sprague-Dawley (SD) rats weighing 200 ± 20 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All experiments were performed under sterile conditions.

[0068] After the rats were anesthetized by inhaling 2% isoflurane, tracheal intubation and mechanical ventilation were performed, and electrocardiogram (ECG) was continuously monitored. The left chest was opened at the 4th intercostal space, and after the heart was exposed, the left anterior descending branch (LAD) of the left coronary artery was ligated with a 6-0 suture to establish a myocardial infarction (MI) model. When the left ventricular anterior wall of the heart apex region appeared pale and gray, and the ECG ST segment was elevated, it was considered that the infarction model was successfully established. The myocardial infarction model of the present application was successfully constructed.

[0069] Immediately after MI induction, 100 μL of normal saline or different hydrogel solutions were injected into the infarcted area and its surrounding edges by multiple points using a 30G injection needle. The sham group only underwent thoracotomy operation without coronary ligation. After operation, the thoracic cavity was sutured and disinfected with iodophor. All rats were allowed to freely eat normal pellet feed and drink water after operation.

[0070] Animals were randomly divided into five groups (n = 12 for each group): (1) Sham group, (2) MI group, (3) PGO group, (4) CAM@Sal group, (5) PGO / CAM@Sal group.

[0071] 1.9.2 Echocardiography To reduce bias, echocardiography was performed by a professional technician who was unaware of the experimental grouping. Vevo2100 ultrasound imaging system (VisualSonics, Canada) equipped with MS250 rat linear array probe (13-24 MHz) was used to evaluate left ventricular function. Rats were anesthetized with isoflurane, and examinations were performed at the 14th day and the 28th day after treatment. The main parameters measured included: ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-systole (LVIDs), left ventricular internal diameter at end-diastole (LVIDd). The average value of three consecutive cardiac cycles was taken for each index for analysis.

[0072] 1.9.3 Pathological tissue staining examination (1) Slice preparation method After the end of the in vivo experiment, the rat's heart was quickly removed and perfused through the aortic sinus opening with heparin sodium saline until the heart turned white; then it was repeatedly washed with PBS solution to completely remove residual blood. The auricular tissue was retained and placed in a 10 mL centrifuge tube containing 4% cell tissue fixative, and the tube was placed on a shaker to shake evenly at room temperature overnight for subsequent pathological tissue staining analysis.

[0073] Preparation of paraffin tissue sections: after fixation, the tissue sample was taken out and sequentially subjected to gradient dehydration (50%, 70%, 80%, 90%, 95%, 100%), xylene and alcohol (1:1) mixed solution transparency treatment, immersion in wax, embedding, sectioning and mounting. During sectioning, 10 sections were continuously cut from each wax block, and the thickness of each section was controlled at 5 μm.

[0074] (2) H&E staining The paraffin sections were placed in a 60°C oven for drying overnight, and the next day H&E staining was performed, with the specific steps as follows: (I) De-waxing and hydration: The sections were sequentially immersed in xylene I (15 min), xylene II (10 min), and then in absolute ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol (10 s each), and finally in distilled water (1 min); (II) Hematoxylin staining: The sections were immersed in hematoxylin staining solution for 5 min, and then rinsed with tap water for 2 min; (III) Differentiation and counterstaining: After continuously rinsing with tap water for 2 min, the sections were immersed in 1% hydrochloric acid alcohol for 10 s, and then continuously rinsed with tap water for 2 min for counterstaining; (IV) Eosin staining: The sections were immersed in eosin staining solution for 1 min, and then rinsed with tap water for 30 s; (V) Dehydration, transparency and mounting: The sections were sequentially dehydrated in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol and absolute ethanol (10 s each). Then the sections were sequentially immersed in xylene II and xylene (15 s) and naturally air-dried, and then mounted with neutral balsam; (VI) Microscopic observation and image acquisition.

[0075] (3) Masson staining The paraffin sections were dried in a 60°C oven overnight, and then the next day, the Masson three-color staining kit was used for staining, and the specific steps were as follows: (I) De-waxing and hydration of the sections: The method was the same as that of H&E staining; (II) Masson composite staining: Masson composite staining solution (A solution) was added dropwise to cover the tissue, and incubated for 5 min, and then washed with distilled water; (III) Phosphomolybdic acid treatment: Phosphomolybdic acid solution (B solution) was added dropwise to cover the tissue, and incubated for 5 min, and then poured off and spun dry; (IV) Aniline blue staining: Aniline blue solution (C solution) was added dropwise to cover the tissue, and incubated for 5 min, and then rinsed with distilled water; (V) Acetic acid differentiation: Acetic acid differentiation solution (D solution) was added dropwise to cover the tissue, and incubated for 30-60 s, and then poured off; (VI) Dehydration, transparency and mounting: The method was the same as that of H&E staining; (VII) Microscopic observation and image acquisition; (VIII) Calculation of fibrosis proportion: ImageJ (version 1.8.0, National Institutes of Health) software was used to calculate the collagen volume fraction (CVF) CVF = collagen area / total area x 100%.

[0076] Figure 11H&E staining images of major organs (liver, spleen, lung, kidney) of rats in different hydrogel treatment groups; in vivo biocompatibility evaluation (by histological H&E staining) showed that no obvious inflammation or tissue damage was observed in the major organs (liver, spleen, lung, kidney) of rats 28 days after hydrogel injection.

[0077] Figure 12 Fig. 13 shows representative echocardiograms of rats 28 days after hydrogel injection (A); quantitative evaluation of key cardiac function indicators by echocardiography 14 and 28 days after hydrogel treatment, including ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) (B-E); based on the good in vitro biocompatibility of the hydrogel, the present application evaluated its therapeutic potential for relieving pathological left ventricular remodeling in a rat myocardial infarction (MI) model. A myocardial infarction model was constructed by ligating the left anterior descending coronary artery. Rats were randomly divided into a sham operation group (Sham), a myocardial infarction group (MI), a PGO hydrogel group, a CAM@Sal hydrogel microsphere group, and a PGO / CAM@Sal hydrogel group. The hydrogel was injected at multiple sites in the infarct area using a 30G needle. Echocardiography was performed 2 and 4 weeks after treatment, and the results showed that there were differences in cardiac function among the groups (A). At 2 weeks of treatment, the ejection fraction (EF) of the MI group decreased significantly to 30.70%, the fractional shortening (FS) decreased to 15.26%, the left ventricular end-systolic diameter (LVIDs) increased to 8.70 mm, and the left ventricular end-diastolic diameter (LVIDd) increased to 10.03 mm, indicating severe cardiac dysfunction and left ventricular remodeling (B-E). Figure 12 Figure 12

[0078] Figure 13 Fig. 14 shows H&E staining and Masson's trichrome staining results of infarct myocardium 28 days after treatment with different hydrogels; from the results of H&E staining and Masson's trichrome staining of infarct myocardium 28 days after treatment with different hydrogels, it can be seen that the infarct myocardium of the PGO / CAM@Sal hydrogel treatment group has the least inflammatory cell infiltration and the least collagen deposition, indicating that the PGO / CAM@Sal hydrogel treatment group has the best therapeutic effect on pathological left ventricular remodeling (A-B). Figure 13 ​​As can be seen, the histological analysis after 28 days of treatment confirmed the long-term benefits of the hydrogels. Hematoxylin-eosin (H&E) staining showed that the ventricle of the MI group rats was enlarged and the ventricular wall was thinned; while the ventricular wall thickness of all hydrogel-treated rats was increased, among which the PGO / CAM@Sal group improved most significantly. This structural protection effect can be attributed to the borate ester bond of the hydrogel and the sustained release of Sal - both of which synergistically scavenge reactive oxygen species (ROS) in the infarct area, thereby improving the local microenvironment and effectively inhibiting the expansion of the infarct area.

[0079] Myocardial fibrosis is a key link of left ventricular remodeling. Masson trichrome staining results show that the degree of fibrosis in the PGO group and the CAM@Sal group is reduced compared with the MI group, and this effect can be attributed to the anti-fibrosis effect of gallic acid and Sal. Notably, the degree of fibrosis in the PGO / CAM@Sal group is reduced most significantly, which is consistent with the phased release of Sal and the slow degradation characteristics of the hydrogel.

[0080] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method of preparing an injectable conductive hydrogel, characterized by, Comprise the following steps: Step 1, using microfluidic technology, sodium alginate solution as the dispersed phase, mineral oil as the continuous phase, form uniform droplets in the microchannel, then the droplets contact with calcium chloride solution, through the ion crosslinking reaction, obtain CAM hydrogel microspheres; CAM hydrogel microspheres are soaked in salidroside solution for drug loading, centrifuged, and CAM@Sal microspheres are obtained; Step 2, 4-carboxyphenylboronic acid and NHS are dissolved in DMF, EDC·HCl is added under ice bath condition, stirring reaction, washing, drying and removing solvent, CBPA-NHS is obtained; The silk fibroin solution is added dropwise into the MES buffer solution, the CBPA-NHS solution is added and the pH of the reaction system is adjusted to neutral, the reaction is carried out, and PS is obtained after dialysis and centrifugal impurity removal; Step 3, pyrrole is added to the gelatin solution, and an oxidizing agent is added under inert atmosphere for reaction, and GP is obtained after dialysis and freeze-drying; the gelatin is dissolved in the MES buffer solution, EGCG and EDC·HCl are added for reaction, and GE is obtained after dialysis and freeze-drying; GP and GE are mixed in a volume ratio of 1:1 and freeze-dried to obtain GPE; Sodium periodate is added to the hyaluronic acid solution for reaction, and OHA is obtained after dialysis and freeze-drying; Step 4, PS, GPE and OHA are respectively prepared into PS solution, GPE solution and OHA solution; the PS solution, GPE solution and OHA solution are mixed, and the CAM@Sal microspheres are uniformly dispersed and incubated to obtain the injectable conductive hydrogel; Step 1, step 2 and step 3 are not in the order.

2. The production method according to claim 1, characterized by, In step 1, the mass concentration of the sodium alginate solution is 2%-5%; the mineral oil is a mineral oil containing Span 80 with a mass concentration of 2%-5%; the mass concentration of the calcium chloride solution is 2%-5%; the concentration of the salidroside solution is 1.0 mg / mL; and the drug loading time is 12 h.

3. The method of claim 1, wherein, In step 2, the amount ratio of 4-carboxyphenylboronic acid, NHS, DMF and EDC·HCl is 12 mmol:17 mmol:20 mL:18 mmol; the concentration of the silk fibroin solution is 6 wt%; the concentration of the CBPA-NHS solution is 40 mg / mL; the concentration of the MES buffer solution is 0.1 M; the concentration of the CBPA-NHS solution is 40 mg / mL; the volume ratio of the silk fibroin solution, the MES buffer solution and the CBPA-NHS solution is 4:6:1; and when preparing PS, the reaction conditions are as follows: stirring at room temperature for 6 h.

4. The method of claim 1, wherein, In step 3, the concentration of the gelatin solution is 2 wt%; the concentration of pyrrole in the gelatin solution is 15% w / w; the oxidizing agent is FeCl3; when preparing GP, the reaction conditions are as follows: reaction at 4 °C for 24 h; when preparing GE, the concentration of the MES buffer solution is 0.05 M, and the amount ratio of gelatin, the MES buffer solution, EGCG and EDC·HCl is 1.0 g:20 mL:204 mg:173 mg; and when preparing GE, the reaction conditions are as follows: reaction at 27 °C for 3 h.

5. The preparation method according to claim 1, characterized in that, In step 3, the concentration of the HA solution was 40 mg / mL; the mass ratio of HA to NaIO4 in the HA solution was 4:1.08; and the reaction conditions for preparing OHA were as follows: room temperature for 24 h.

6. The method of claim 1, wherein, In step 4, the concentration of the PS solution was 3 wt%; the concentration of the GPE solution was 8 wt%; the concentration of the OHA solution was 0.5 wt%; the volume ratio of the PS solution, the GPE solution and the OHA solution was 2:1:3; and the concentration of the CAM@Sal microspheres in the reaction system was 10 mg / L.

7. The preparation method according to claim 1, characterized in that, In step 4, the incubation conditions were set as follows: 37 °C water bath for 30-60 min.

8. An injectable conductive hydrogel prepared by the preparation method according to any one of claims 1-7.

9. Use of the injectable conductive hydrogel according to claim 8 in the preparation of a medicament for treating myocardial infarction.

10. A medicament for promoting cardiac repair and functional recovery after myocardial infarction, characterized by, The effective component comprises the injectable conductive hydrogel according to claim 8.

Images