A formulation for sustained release of serelaxin based on functionalized self-assembling peptide nanofiber and application thereof

Abstract

The application relates to the field of biological medicine, and aims to solve the problems of short half-life of Serelaxin, difficulty in maintaining local concentration and poor safety of existing virus carriers, and provides a Serelaxin sustained-release preparation based on functional self-assembled peptide nanofibers and application, wherein the Serelaxin sustained-release preparation comprises self-assembled peptide RADA16-I, a functional modified peptide segment and a Serelaxin drug molecule; the self-assembled peptide RADA16-I serves as a basic unit of a nanofiber support, a three-dimensional nanofiber network is formed through self-assembly, the functional modified peptide segment is connected to the C terminal of the self-assembled peptide RADA16-I and serves as a specific binding domain of the Serelaxin, and the Serelaxin drug molecule is loaded in the three-dimensional nanofiber network by being combined with the functional modified peptide segment. The application can realize multiple repair effects of anti-fibrosis, promotion of angiogenesis and myocardial regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers and its application. Background Technology

[0002] Myocardial infarction (MI), commonly known as a "heart attack," is a life-threatening acute cardiovascular event caused by a sudden reduction or interruption of blood flow to the coronary arteries (the arteries supplying blood to the heart). This leads to the death of the myocardium supplied by the coronary arteries due to prolonged ischemia. The root cause of myocardial fibrosis is that the heart initiates an abnormal repair process under long-term stress (such as hypertension), ischemia (such as myocardial infarction), or inflammation. Simply put, myocardial infarction is one of the most common and important causes of myocardial fibrosis; while myocardial fibrosis is the consequence of the heart's "poor repair" after suffering various injuries such as infarction.

[0003] Clinically, there are no specific drugs that can directly reverse or combat myocardial fibrosis. Current treatments mainly target the underlying disease (such as controlling blood pressure and blood sugar) or use angiotensin-converting enzyme inhibitors (RAAS inhibitors). β Drugs such as beta-blockers can be used to slow the progression of the disease, but these methods have limited effectiveness and cannot stop or reverse the progression of fibrosis.

[0004] To date, Serelaxin is a small molecule peptide drug with anti-heart failure, anti-fibrotic and angiogenesis effects among existing drugs under development. However, it still has the following insurmountable defects, which has prevented Serelaxin from being successfully used in clinical practice: (1) Short half-life (about 1-2 hours); (2) It is difficult to maintain an effective concentration in the heart through conventional intravenous or local injection, requiring frequent administration and easily causing systemic side effects; (3) Although the use of viral vectors (such as adenovirus) to carry Serelaxin in the existing technology can prolong the duration of action, there are safety issues such as immunogenicity, potential carcinogenicity risk and unstable transfection efficiency; (4) After myocardial infarction (MI), cardiac fibroblasts (CF) are over-activated into myofibroblasts (MF), leading to myocardial fibrosis and seriously hindering myocardial regeneration and repair.

[0005] Therefore, there is an urgent need to develop a safe, efficient, and locally sustained-release Serelaxin delivery system to inhibit myocardial fibrosis, promote the transdifferentiation of CF into cardiovascular cells, promote myocardial regeneration after myocardial fibrosis, and improve cardiac function, thereby achieving the repair of myocardial structure and function. Summary of the Invention

[0006] The purpose of this invention is to provide a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers and its application. The application of the formulation is mainly reflected in inhibiting myocardial fibrosis, promoting myocardial regeneration and repairing myocardial infarction, effectively solving the problems of short half-life of Serelaxin, difficulty in maintaining local concentration and poor safety of existing viral vectors in the above-mentioned background technology.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention discloses a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers, comprising: The self-assembling peptide RADA16-I, with the sequence AcN-RADARADARADARADA-CONH2, serves as the basic unit of the nanofiber scaffold and forms a three-dimensional nanofiber network through self-assembly. The functionalized modified peptide is attached to the C-terminus of the self-assembled peptide RADA16-I as a specific binding domain for Serelaxin. And the Serelaxin drug molecule, which is loaded into a three-dimensional nanofiber network by binding to the functionalized modified peptide.

[0008] As a further preferred embodiment, the sequence of the functionalized modified peptide is GGCWIPIFVVKF, which is covalently linked to the C-terminus of the self-assembled peptide RADA16-I via an amino acid linker chain -GG-, to form the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I.

[0009] As a further preferred embodiment, the functionalized self-assembly peptide GGCWIPIFVVKF-RADA16-I is mixed with the self-assembly peptide RADA16-I in a 1:1 ratio under physiological conditions to self-assemble into a functionalized self-assembly peptide nanofiber hydrogel.

[0010] As a further preferred embodiment, the functionalized self-assembled peptide nanofiber hydrogel has nanofibers with a diameter of 10–15 nm and a length of 100–300 nm.

[0011] Secondly, this invention discloses a method for preparing a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers, comprising the following steps: (1) The functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I was prepared by solid-phase synthesis and purified by high performance liquid chromatography; (2) The self-assembled peptide RADA16-I and the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I were mixed in a 1:1 ratio, PBS buffer was added, and the self-assembly was triggered by sonication for 30 min to form a functionalized self-assembled peptide nanofiber hydrogel. (3) Mix Serelaxin with the precursor solution of functionalized self-assembled peptide nanofiber hydrogel and let stand for 30 min to allow Serelaxin to fully combine with functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I and load it into the three-dimensional nanofiber network.

[0012] Thirdly, this invention discloses the application of a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers in the preparation of drugs for myocardial repair.

[0013] A further preferred embodiment of the above scheme is that the formulation reduces the differentiation of cardiac fibroblasts into myofibroblasts by inhibiting the TGF-β1 / Smad2 signaling pathway, and / or promotes the transdifferentiation of cardiac fibroblasts into endothelial cells and cardiomyocytes by activating the PI3K / Akt pathway and upregulating Hoxa-9 and VEGF factors.

[0014] A further preferred embodiment is that the formulation continuously releases Serelaxin in vivo for more than 28 days.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention mixes the functionalized self-assembling peptide GGCWIPIFVVKF-RADA16-I with Serelaxin drug molecules and triggers self-assembly under physiological conditions using ultrasound. This allows the Serelaxin drug molecules to bind to the functionalized modified peptides and be loaded onto a three-dimensional nanofiber network, forming an SPNF hydrogel. After implanting this SPNF hydrogel into the myocardial infarction area, it gradually degrades in the body fluid environment, and Serelaxin is slowly released, maintaining an effective drug concentration locally. By inhibiting the TGF-β1 / Smad2 signaling pathway, the Serelaxin drug molecules reduce the differentiation of CF into MF, while activating the PI3K / Akt pathway and upregulating factors such as Hoxa-9 and VEGF, promoting the transdifferentiation of CF into endothelial cells and cardiomyocytes, thereby achieving multiple repair effects of anti-fibrosis, angiogenesis, and myocardial regeneration.

[0016] 2. The SPNF hydrogel of the present invention can achieve continuous release of Serelaxin drug molecules for more than 28 days, with a cumulative release rate of 75%, which far exceeds the release effect of free Serelaxin and improves the inhibition effect of α-SMA expression by about 50%.

[0017] 3. This invention establishes a specific sustained-release system for the drug molecule Serelaxin, and for the first time studies and confirms that the drug molecule Serelaxin can induce CF to transdifferentiate into endothelial cells and cardiomyocytes. The sustained-release system increases the expression of CD31 and cTnT by 80-100%, and the SPNF hydrogel has good biocompatibility, the degradation products are amino acids, and it is non-immunogenic, so it can be administered locally and avoids the risk of viral vectors. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required in the embodiments will be briefly introduced below.

[0019] Figure 1 This is a high-performance liquid chromatography analysis report of the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I of the present invention; Figure 2 This is a mass spectrometry report of the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I of the present invention; Figure 3 The images are atomic force microscopy images of the SPNF hydrogel of the present invention; wherein: A shows the formation of nanofibers from self-assembled peptides after 30 min of ultrasonic treatment; B shows the increase in nanofiber length of self-assembled peptides after 2 h of ultrasonic treatment, with the gel forming a network scaffold. Figure 4 This invention relates to the formation of SPNF hydrogels from the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I and its degradation process; wherein: A is a molecular schematic diagram of the self-assembled peptide RADA16-I and the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I; B is a schematic diagram of the formation of SPNF after mixing the self-assembled peptide RADA16-I and the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I; C is the formation and degradation of SPNF hydrogels in PBS; and D is the degradation curve of the SPNF hydrogel. Figure 5 This is a graph showing the sustained-release serelaxin curve of the SPNF hydrogel of the present invention. Figure 6 This is an immunofluorescence staining image of the serelaxin sustained-release formulation of the present invention inhibiting CF differentiation into MF, with the scale bar indicating 100 μm; Figure 7The graph shows the results of the serelaxin sustained-release formulation of the present invention inhibiting CF differentiation into MF; where: B is the Western blot analysis, showing that serelaxin and the serelaxin sustained-release formulation can inhibit TGF-β1-induced CF expression of α-SMA; C is the statistical analysis graph of α-SMA expression, *P<0.05 compared with the control group, #P<0.05 compared with the serelaxin group, &P<0.05 compared with the serelaxin and SPNF groups, n = 4; Figure 8 The serelaxin sustained-release formulation of this invention promotes the transdifferentiation of CF cells into endothelial cells; Figure 9 The serelaxin sustained-release formulation of this invention promotes the expression of CD31, vWF, and Hoxa-9 mRNA and CD31 protein; wherein: A is CD31 expression analyzed by RT-PCR; B is a statistical analysis graph of CD31 mRNA expression; C is vWF expression analyzed by RT-PCR; D is a statistical analysis graph of vWF mRNA expression; E is Hoxa-9 expression analyzed by RT-PCR; F is a statistical analysis graph of Hoxa-9 mRNA expression; G is CD31 expression detected by Western blot; H is a statistical analysis graph of CD31 protein expression; *P<0.05 compared with the control group, #P<0.05 compared with the serelaxin group, &P<0.05 compared with the serelaxin and SPNF group, n=4; Figure 10 To promote VEGF expression in this invention; wherein: A is RT-PCR analysis of VEGF expression; B is a statistical analysis graph of VEGF mRNA; C is Western blot detection of VEGF protein expression; D is a statistical analysis graph of VEGF protein expression; E is ELISA detection of VEGF content in culture supernatant; *P<0.05 compared with the control group, #P<0.05 compared with the serelaxin group, &P<0.05 compared with the serelaxin and SPNF groups, n = 4; Figure 11 The serelaxin sustained-release formulation of the present invention promotes the transdifferentiation of CF into cardiomyocytes; Figure 12The serelaxin sustained-release formulation of this invention promotes the expression of Nkx2.5, GATA-4, and Mef2C mRNA; wherein: A represents the expression of Nkx2.5, GATA-4, and Mef2C; B is a statistical analysis graph of Nkx2.5 mRNA expression; C is a statistical analysis graph of GATA-4 mRNA expression; D is a statistical analysis graph of Mef2C mRNA expression; *P<0.05 compared with the control group, #P<0.05 compared with the serelaxin group, &P<0.05 compared with the serelaxin and SPNF groups, n = 4; Figure 13 The serelaxin sustained-release formulation of this invention promotes the expression of cTnT and Cx-43 mRNA; wherein: A is the expression of cTnT; B is a statistical analysis graph of cTnT mRNA expression; C is the expression of Cx-43; D is a statistical analysis graph of Cx-43 mRNA expression; *P<0.05 compared with the control group, #P<0.05 compared with the serelaxin group, &P<0.05 compared with the serelaxin and SPNF group, n = 4; Figure 14 The effect of the serelaxin sustained-release formulation of the present invention on the expression of p-Akt and Akt proteins; wherein: A represents the expression changes of p-Akt and Akt; B is a statistical analysis graph of p-Akt / Akt expression; *P<0.05 compared with the control group, #P<0.05 compared with the serelaxin group, &P<0.05 compared with the serelaxin and SPNF groups, n = 4; Figure 15 This invention relates to the improvement of cardiac function after intramyocardial injection of the serelaxin sustained-release formulation. A shows two-dimensional echocardiographic images 28 days after intramyocardial injection. In the control group, the left ventricular cavity was significantly enlarged, and the anterior wall of the left ventricle was significantly thinned. In the serelaxin, SPNF, and serelaxin sustained-release groups, the left ventricular cavity shrank, with the serelaxin sustained-release group showing the most significant shrinkage. In the control group, the systolic wave on the anterior wall of the left ventricle in the infarct area disappeared. In the serelaxin and SPNF groups, the systolic wave on the anterior wall of the left ventricle was visible, while in the serelaxin and SPNF groups, the systolic wave on the anterior wall of the left ventricle was most prominent. B and C show changes in EF (%) and FS (%). D and E show changes in LVDd and LVDs. F and G show changes in AWd and AWs. H and I show changes in LVEDV and LVESV. *P < 0.05, compared with Sham; #P < 0.05, compared with the control group; &P < 0.05, compared with the serelaxin and SPNF groups. Figure 16The changes in left ventricular wall scar area 28 days after intramyocardial injection of the serelaxin sustained-release formulation of the present invention are shown. Wherein: A represents the morphological changes in the short-axis cross-section of the heart as shown by Masson staining (scale bar indicates 1 mm); D represents the changes in scar area in the infarct region; E represents the changes in left ventricular wall thickness. Compared with the control group, *P<0.05; compared with the serelaxin group and SPNF group, #P<0.05; Figure 17 The figures show a comparison of the serelaxin sustained-release formulation of the present invention before and after treatment for myocardial fibrosis following infarction: In the control group, there was no significant difference in the scar area of ​​the infarcted area compared to the SPNF group. In the serelaxin group and the serelaxin sustained-release group, the scar area of ​​the infarcted area was significantly reduced. The scar area in the serelaxin sustained-release group was significantly smaller than that in the serelaxin group. In the control group, the left ventricular free wall was significantly thinned and expanded outwards, resulting in a significant enlargement of the left ventricular cavity. In the serelaxin, SPNF, and serelaxin sustained-release groups, the expansion of the left ventricular free wall was not significant, while the increase in left ventricular wall thickness was more pronounced in the serelaxin sustained-release group.

[0020] Figure 18 The serelaxin sustained-release formulation of this invention promotes myocardial tissue regeneration when injected intramyocardially. In the control group, a small number of weakly cTnT-expressing (red) cardiomyocytes remained in the infarct area, and no Cx43 (green) fluorescence signal was detected. In the serelaxin, SPNF and serelaxin sustained-release groups, the number of cardiomyocytes expressing cTnT increased, and Cx43 expression was observed. The expression was most obvious in the serelaxin sustained-release group. Cx43 was mainly distributed at the junctions of cardiomyocytes (scale bar indicates 100 μm). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments, and not all embodiments. For those skilled in the art, other embodiments can be obtained without creative effort.

[0022] The following details the sources of the main experimental instruments and reagents used in the embodiments of this invention. The research on experimental rats, as well as the design and implementation of various animal experiments, all comply with animal welfare and ethical principles, namely, animal protection principles, animal welfare principles, and ethical principles. Furthermore, unless otherwise explicitly stated, all reagents mentioned in the following embodiments are commercially available products and can be purchased through legitimate channels.

[0023] The animal experiment was conducted at the Laboratory Animal Center of Shanghai University of Medicine & Health Sciences (Laboratory Animal Use License No. SYXK(Shanghai) 2016-0002), and the study was reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of the Laboratory Animal Center of Shanghai University of Medicine & Health Sciences (Approval Document No. 2021-JCKT-08-342901197510305830).

[0024] The following is combined with Figures 1-18 to describe in detail the Serelaxin sustained-release preparation based on functionalized self-assembling peptide nanofibers and the application thereof according to the present invention. Examples

[0025] The present invention provides a Serelaxin sustained-release preparation based on functionalized self-assembling peptide nanofibers, comprising self-assembling peptide RADA16-I, a functionalized modified peptide segment and Serelaxin drug molecule. The sequence of the self-assembling peptide RADA16-I is AcN-RADARADARADARADA-CONH₂, which serves as the basic unit of the nanofiber scaffold. The self-assembling peptide RADA16-I forms a three-dimensional nanofiber network through self-assembly. The sequence of the functionalized modified peptide segment is GGCWIPIFVVKF, and the functionalized modified peptide segment is linked to the C-terminus of the self-assembling peptide RADA16-I, serving as a specific binding domain for the Serelaxin drug molecule (recombinant human relaxin-2). The Serelaxin drug molecule is loaded in the three-dimensional nanofiber network by binding to the functionalized modified peptide segment.

[0026] Specifically, the functionalized modified peptide segment is covalently linked to the C-terminus of the self-assembling peptide RADA16-I via the amino acid linker -GG-, to form the functionalized self-assembling peptide GGCWIPIFVVKF-RADA16-I.

[0027] Under physiological conditions, the functionalized self-assembling peptide GGCWIPIFVVKF-RADA16-I can be mixed with the self-assembling peptide RADA16-I at a ratio of 1:1, and self-assembled to form a functionalized self-assembling peptide nanofiber (SPNF) hydrogel. The nanofibers of the SPNF hydrogel have a diameter of 10 to 15 nm and a length of 100 to 300 nm. The Serelaxin drug molecule is loaded in the SPNF hydrogel through specific binding, and is slowly released along with the degradation of the SPNF hydrogel. Examples

[0028] The present invention further provides a preparation method of the Serelaxin sustained-release preparation based on functionalized self-assembling peptide nanofibers, comprising the following steps: 1) Design and synthesis of self-assembling peptides: The self-assembled peptide RADA16-I was prepared by modifying the amino acid linker chain -GG- of the functionalized peptide sequence GGCWIPIFVVKF. Both the self-assembled peptide RADA16-I and the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I were synthesized and purified by Shanghai Taopu Biotechnology Co., Ltd. After synthesizing the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I using a solid-phase synthesis method, the purity of the product was detected by high-performance liquid chromatography (HPLC). Figure 1 As shown, the purity of the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I is 96.16%, which is greater than the 96% standard. The relative molecular weight of the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I was analyzed by mass spectrometry, as shown below. Figure 2 As shown, the mass spectrometer showed that the relative molecular mass of the self-assembled peptide was 3060.46, which is consistent with the theoretical design and meets the target molecular weight. The functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I was lyophilized into a sterile powder for later use. 2) Observation of the self-assembly process of self-assembling peptides under atomic force microscopy: Dissolve 1 mg of self-assembled peptide RADA16-I lyophilized powder in 0.1 ml of sterile distilled water, vortex for 1 min to prepare a 1% (w / v) self-assembled peptide RADA16-I stock solution, sonicate for 30 min, and store at 4℃. A 1% (w / v) functionalized self-assembly peptide stock solution of GGCWIPIFVVKF-RADA16-I was prepared using the same method. The self-assembly peptide RADA16-I and the functionalized self-assembly peptide GGCWIPIFVVKF-RADA16-I were mixed at a 1:1 ratio to form a functionalized peptide solution. 2 μl of the functionalized peptide solution was added to 38 μl of sterile deionized water, and the mixture was sonicated for 30 min to ensure uniform dispersion and bring it to the critical state of self-assembly. Take 2 μl of the above-mentioned critically self-assembled sample and spread it onto a freshly peeled mica sheet. After air drying, gently wash it twice with sterile deionized water. Observe and photograph the sample in Tapping mode of an atomic force microscope. The atomic force microscope probe parameters are: resonant frequency 75 kHz, spring constant 1-5 N / m, tip curvature radius 5-10 nm and 225 nm; the scanning parameters are: Tapping frequency 75 kHz, amplitude before pressure application 1-1.2 V, set point 0.7-0.9 V, image resolution 512×512 pixels, and scanning speed 1.51 Hz.

[0029] like Figure 3As shown in the figure, observations from atomic force microscopy indicate that after 30 minutes of ultrasonic treatment, self-assembled peptides form nanofibers. Figure 3 (A) After ultrasonic treatment for 2 h, the self-assembled peptides further self-assembled, the nanofiber length increased, and they intertwined to form a network scaffold with a diameter of 10 ~ 15 nm and a length of 100 ~ 300 nm. Figure 3 (B)

[0030] 3) Properties and degradation detection of functionalized self-assembled peptide nanofiber (SPNF) hydrogels: Take 50 μl each (1:1) of the 1% self-assembled peptide RADA16-I stock solution and the 1% functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I stock solution after sonication and inject them into a 2 ml EP tube. Then add 100 μl of PBS and gently pipette to mix the peptide solution and PBS thoroughly to obtain a peptide solution with a final concentration of 0.5%. After ultrasonic treatment for 30 min, self-assembly was triggered to form SPNF hydrogel. After standing at room temperature for 30 min, a transparent gel was visible at the bottom of the EP tube. The SPNF hydrogel was removed and weighed using a precision electronic balance. It was then immersed in PBS solution and placed in a 37°C incubator, with the PBS solution changed daily. The gel was removed after 1, 3, 5, 7, 14, 21, and 28 days, rinsed three times with deionized water, and then freeze-dried and weighed. Five samples were weighed at each time point, and the weight loss rate of the SPNF hydrogel at different times was compared. The degradation performance of the SPNF hydrogel was evaluated by the change in mass. The degradation rate of the SPNF hydrogel was calculated as follows: hydrogel degradation rate (%) = (weight of gel before immersion - weight of gel after immersion) / weight of gel before immersion × 100%.

[0031] The in vivo biodegradation of SPNF hydrogels occurs through natural hydrolysis under physiological conditions, with the decomposition products being absorbed by the body, such as... Figure 4 As shown, SPNF hydrogel was observed to degrade in PBS (0.02 mol / L, pH=7.4), with degradation rates of 20%, 73%, and 85% on days 7, 21, and 28, respectively, indicating that SPNF hydrogel degrades slowly.

[0032] 4) Detection of sustained-release formulations of Serelaxin: To evaluate the feasibility of SPNF hydrogel for sustained release of serelaxin, the duration of serelaxin efficacy maintained by the SPNF hydrogel was determined. 15 μl each of 1% (w / v) self-assembled peptide RADA16-I and 1% (w / v) functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I were mixed in a 1:1 ratio, and 30 μl of PBS was added. The mixture was sonicated for 30 min to obtain a functionalized peptide solution. 1 μl of serelaxin (100 ng / μl) was added, and the mixture was gently pipetted to mix. The solution was incubated at 37°C for 30 min to allow self-assembly into a colorless, semi-transparent hydrogel. Then, 200 μl of sterile PBS was slowly added, and the solution was incubated at 37°C for 10 min. 200 μl of the supernatant was aspirated and stored at -80°C. After washing the hydrogel with 200 μl of PBS, 200 μl of the supernatant was immediately aspirated, injected into a new EP tube, and stored. Add 200 μl of PBS and place in a constant temperature incubator at 37°C. At 1 d, 3 d, 5 d, 7 d, 14 d, 21 d and 28 d, take out 200 μl of the supernatant and keep it for later use.

[0033] The first supernatant was collected to detect the binding of serelaxin drug molecules to the SPNF hydrogel, and the second supernatant was collected to detect the retention of serelaxin in the liquid before the sustained-release experiment. Supernatant samples were collected at 1, 3, 5, 7, 14, 21, and 28 days, and the release of serelaxin was detected according to the ELISA reagent instructions, as follows: (1) Prepare all necessary reagents and working concentration standards.

[0034] (2) Add 300 μl of 1× washing solution, let stand and soak for 30 seconds, then discard the washing solution.

[0035] (3) Add 100 μl of standard solution of each dilution to the standard wells in sequence, and set up blank control wells (without adding sample).

[0036] (4) Add 50 μl of 1× detection buffer and 50 μl of sample to the sample well. Add the sample to the bottom of the well of the microplate. After adding the sample, gently shake the microplate to mix the detection buffer.

[0037] (5) Add 50 μl of diluted detection antibody per well. Seal the ELISA plate with sealing film, gently shake, and incubate at room temperature for 2 h.

[0038] (6) Discard the detection antibody, add washing buffer to wash the plate, 300 μl / well, wash 6 times.

[0039] (7) Add 100 μl of horseradish peroxidase-labeled detection antibody to each well.

[0040] (8) Seal the plate with a new sealing film, shake, and incubate at room temperature for 45 min.

[0041] (9) Repeat step 8; add chromogenic substrate TMB (100 μl / well) and incubate at room temperature in the dark for 5–30 min.

[0042] (10) Add stop solution (100 μl / well). Gently tap the plate frame to mix the liquid thoroughly. The liquid color will gradually change from blue to yellow.

[0043] (11) Within 30 min, use an ELISA reader to perform dual-wavelength detection, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the wavelength of 570 nm. The calibrated OD value is the measured value at 450 nm minus the measured value at 570 nm.

[0044] The serelaxin content in the supernatant of samples at different time points was determined using ELISA. The average OD values ​​of the samples and standards were subtracted from the OD value of the zero-concentration standard. The x-axis represented the standard concentration, and the y-axis represented the OD value. A serelaxin standard curve was generated using computer software through regression fitting. By fitting the logarithm of the concentration and OD values, the amount of serelaxin released at each sampling was calculated. The cumulative release amount was fitted with an equation over time to create an in vitro sustained-release serelaxin curve for SPNF.

[0045] ELISA results showed that no free serelaxin was detected in the supernatant from the first and second washes, indicating that serelaxin was completely bound to the SPNF hydrogel. The release at different time points was detected by ELISA, and the results are as follows: Figure 5 As shown, serelaxin is released slowly and uniformly, reaching 44% at 7 days and approximately 75% at 28 days. Fitting the cumulative release rate against time indicates that the SPNF hydrogel continuously releases serelaxin through degradation.

[0046] Experimental Case: I. Experimental Procedure: 1.1 Experimental Grouping: SD rats were euthanized by cervical dislocation, and ventricular tissue was harvested under aseptic conditions. The tissue was washed twice with pre-cooled PBS, then cut into 1 mm³ pieces using ophthalmic scissors. 0.125% trypsin (Gibco, CA, USA) was added, and the tissue was digested with shaking in a 37°C water bath for 6 min. The tissue pieces were then allowed to settle. The supernatant was discarded, and fresh digestion solution was added, with digestion continuing for 6 min. After standing on ice, the supernatant was collected in centrifuge tubes, and DMEM containing 10% FBS was added to terminate the digestion. The above digestion process was repeated 4 times with fresh digestion solution added to the remaining tissue pieces. All collected supernatant was filtered through a sieve and centrifuged (1000 r / min, 6 min). The supernatant was discarded, and the cells were resuspended in DMEM containing 10% FBS. The cells were seeded in plastic culture flasks (Corning, CA, USA) and incubated at 37°C in a 5% CO₂ incubator for 60 min. Unattached cells were aspirated, and fresh culture medium was added for further culture. When cells reached 80% confluence, they were passaged. TGF-β1 was used to induce the transformation of cardiac fibroblasts (CFs) into myofibroblasts (MFs). Second-generation CFs were seeded in 6-well plates, and when 70% confluence was reached, the medium was replaced with DMEM containing 2% FBS and cultured for 24 h. Then, 5 ng / ml TGF-β1 was added to the culture medium, and the induction treatment was continued for 72 h. The induced CFs were seeded onto coverslips, and immunofluorescence staining was used to detect changes in α-SMA expression in each group of cells, thus establishing a model for induced CF to MF transdifferentiation.

[0047] The experiment was divided into a control group, an SPNF group, a serelaxin group, and an SPNF-serelaxin group. In the control group, 30 μl of PBS was added to DMEM medium containing 10% FBS; in the SPNF group, 30 μl of SPNF was added; in the serelaxin group, 100 ng / ml serelaxin was added; and in the SPNF-serelaxin group, 30 μl of SPNF and 100 ng / ml serelaxin were added. The mixtures were cultured for 2 or 4 weeks.

[0048] 1.2 Immunofluorescence staining: To evaluate the expression changes of related proteins during CF phenotypic transformation, cells treated according to the above group were cultured for 4 weeks, then seeded onto coverslips, washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, and cell membranes were ruptured with 0.3% Triton-X 100 for 10 min. Cells were washed 3 times with PBS for 5 min each time, and blocked with 10% goat serum at room temperature for 20 min.

[0049] To detect MF phenotypic transformation, mouse anti-α-SMA (1:400) and rabbit anti-vimentin (1:200) were added.

[0050] To detect CF differentiation into endothelial cells, mouse anti-CD31 (1:200) and rabbit anti-vimentin (1:200) were added, respectively.

[0051] To detect the differentiation of CF cells into cardiomyocytes, mouse anti-cTnT (1:200) and rabbit anti-vimentin (1:200) were added, respectively.

[0052] The above treatments were incubated overnight at 4°C, then warmed to room temperature, washed three times with PBS, and incubated for 1 h at room temperature in the dark with DyLight 594-labeled goat anti-mouse IgG (1:400) and / or Alexa Fluor 488-labeled goat anti-rabbit IgG (1:200). After washing with PBS, the cell nuclei were counterstained with DAPI (1:1000). After mounting with anti-fluorescence quenching agent, the cells were observed and photographed under an Olympus fluorescence microscope.

[0053] 1.3 RT-PCR detection: (1) Primer design: The relevant gene sequence information of the required primers was retrieved from http: / / www.ncbi.nlm.nih.gov. Primer sequences were designed using Primer Primer 5.0 software. The PCR primer sequences are shown in Table 1.

[0054] Table 1 Primer Sequences (2) Total RNA extraction and reverse transcription: (I) RT-PCR detection: 1) Take 1 × 10⁷ cells from each group in Table 1 and place the culture dish on ice. Wash twice with PBS pre-cooled to 4°C, add 1 ml of Trizol lysis buffer, and mix thoroughly by pipetting.

[0055] 2) Transfer the cell lysate from each group to a 1.5 ml Eppendorf tube and incubate at room temperature for 5 min.

[0056] 3) Add 0.2 ml of chloroform pre-cooled at 4°C, vortex vigorously for 15 seconds, and let stand at room temperature for 10 minutes.

[0057] 4) Centrifuge at 4℃ (12000 rpm, 20 min).

[0058] 5) Carefully aspirate approximately 0.5 ml of the supernatant into a new 1.5 ml Eppendorf tube, add 0.5 ml of isopropanol, mix well, and incubate on ice at 4°C for 15 min.

[0059] 6) Centrifuge at 4°C (12000 rpm, 10 min), discard the supernatant, and retain the white precipitate at the bottom of the tube, which is RNA.

[0060] 7) Add 1 ml of 75% ethanol pre-cooled at 4℃, invert to suspend, and wash the RNA precipitate.

[0061] 8) Centrifuge at 4°C (7500 rpm, 10 min), discard the supernatant; repeat steps 7) to 8) once. Carefully discard the ethanol, invert onto absorbent paper, and dry at room temperature for 10 min.

[0062] 9) Add 20 μl of DEPC water to dissolve the precipitate.

[0063] (II) Determination of total RNA quality: Dilute the dissolved RNA sample appropriately and use a Nano-100 micro spectrophotometer to read the values ​​at wavelengths of 230 nm, 260 nm, and 280 nm, calculating the OD 260 / 280 ratio. The purity of total RNA is determined by the ratios of A260 / A280 and A260 / A230. An A260 / A280 ratio > 1.8 indicates no protein residue, and an A260 / A230 ratio > 2.0 indicates no guanidine isothiocyanate residue contamination.

[0064] The formula for calculating RNA concentration is as follows: RNA (μg / μl) = A260 × 40 × dilution factor / 1000.

[0065] (III) Reverse transcription of cDNA: 1) Prepare the RT reaction solution as follows: Oligo dT Primer (50 μM) 1 μl Total RNA 2 μl <![CDATA[RNae Free dH2O]]> 13 μl 2) The conditions for the reverse transcription reaction are as follows: Reverse transcription reaction: 37℃, 15 min; Inactivation reaction of reverse transcriptase: 85℃, 5 s; The reverse transcription product was diluted 10-fold and aliquoted, then stored at -20°C.

[0066] (IV) PCR reaction: PCR was performed using the StepOnePlus Real-Time PCR System.

[0067] 1) The PCR reaction system is as follows: DEPC water 8.5 μl template cDNA 0.5 μl Primer (F) 0.5 μl Primer (R) 0.5 μl Total volume 20 μl 2) Reaction conditions: Pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing (annealing temperatures are shown in Table 1) for 45 s, extension at 72℃ for 45 s, 35 cycles, extension at 72℃ for 10 min.

[0068] 3) Electrophoretic analysis of RT-PCR products Add 0.2 g agarose to 20 ml of 1× TAE electrophoresis buffer, heat in a microwave oven for 2 min, cool slightly, add 2 μl EB, and pour into a comb plate. Allow it to cool naturally to a gel state. Take 5 μl of marker and amplification product from each tube, mix evenly with 1 μl of DNA loading buffer, and then load the samples. Start electrophoresis at 110 mV. When the indicator bands migrate to 2 / 3 of the gel, perform gel imaging using a bioelectrophoresis imaging system (Furi Technology, Shanghai). Analyze the absorbance values ​​of the PCR bands using image analysis software. Calculate the relative mRNA expression levels of the relevant target genes using β-actin as an internal reference.

[0069] (3) Western blot analysis of the expression of CD31, cTnT, VEGF, Smad2, p-Smad2, Akt, and p-Akt proteins: Incubate the primary antibodies by diluting the following monoclonal antibodies according to the ratio: mouse anti-CD31 monoclonal antibody (1:1000), mouse anti-cTnT monoclonal antibody (1:1000), rabbit anti-VEGF polyclonal antibody (1:1000), rabbit anti-Smad2 polyclonal antibody (1:1000), rabbit anti-p-Smad2 polyclonal antibody (1:1000), rabbit anti-Akt polyclonal antibody (1:1000), rabbit anti-p-Akt polyclonal antibody (1:1000), and mouse anti-β-actin monoclonal antibody (1:1000). Incubate overnight at 4°C. The next day, recover the primary antibodies.

[0070] Add 1× TBST and wash slowly on a side-shaking shaker for 6 minutes, 5 times.

[0071] Dilute horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) with Western blotting secondary antibody dilution buffer (1:2000). Incubate slowly with shaking at room temperature for 1 h, then recover the secondary antibody.

[0072] Add 1 × TBST and wash slowly on a side-swinging shaker for 6 minutes, 5 times.

[0073] Add ECL luminescent solution (Bio-Rad, USA) and react for 1-2 minutes.

[0074] Imaging was performed using the Bio-Rad imaging system (Bio-Rad, USA). The gray values ​​of the target protein bands were analyzed using the image analysis software ImageJ. The relative expression level of the target protein was calculated using β-actin as an internal reference.

[0075] (4) ELISA method for detecting VEGF released by CF: Collect cell culture supernatant from each group and strictly follow the ELISA kit instructions to detect VEGF concentration. The detection steps are as follows: 1) Standard curve: Set up standard wells. Add 100 μl of standard sample diluent to each well. Add 100 μl of standard to the first well, mix well, and then aspirate 100 μl to transfer to the second well. Repeat this dilution up to the seventh well. Aspirate 100 μl from the seventh well and discard it, so that each well contains 100 μl of liquid. The eighth well is a blank control well.

[0076] 2) Antigen capture: Dilute the test sample 5 times and add 100 μl of each sample to the well. Mix thoroughly, seal the reaction well with sealing film, and incubate at 37°C for 60 min. Wash the plate 3 times, 5 min each time.

[0077] 3) Incubation of secondary antibodies: Except for the blank control wells, add 100 μl of biotinylated secondary antibody to each well, seal the wells with sealing film, incubate at 37℃ for 30 min, and wash the plate 3 times with 300 μl of 1× washing buffer, 5 min each time.

[0078] 4) Horseradish peroxidase labeling: Except for the blank control wells, add 100 μl of enzyme conjugate working solution per well, seal the wells with sealing film, incubate at 37°C for 60 min, and wash the plate 3 times, 5 min each time.

[0079] 5) Color rendering: Add 100 μl of TMB colorimetric reagent to each well and develop the color at 37°C in the dark for 5 min. 6) Termination: Add the stop solution (100 μl / well), mix well, and immediately measure the absorbance (OD value) at 450 nm on a microplate reader. Calculate the corresponding VEGF concentration on the curve based on the OD value of the sample.

[0080] Data were processed using SPSS 20.0 statistical analysis software. Quantitative data were expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for inter-group comparisons, and P < 0.05 was considered statistically significant.

[0081] II. Experimental Results: 2.1 Serelaxin sustained-release formulations inhibit the differentiation of cardiac fibroblasts (CF) into myofibroblasts (MF); like Figures 6-7 As shown, myofibrils (MF) characteristically express α-SMA. Immunofluorescence staining revealed that the control group cells were significantly larger in size, with high expression of α-SMA and dense, thick myofibrils. Figure 6 Compared with the control group, the serelaxin group showed decreased cell volume and reduced α-SMA expression, while the SPNF group showed no significant changes. The serelaxin sustained-release group significantly inhibited α-SMA expression and significantly reduced cell volume. Western blot results indicated that, compared with the control group, the serelaxin group showed decreased α-SMA expression, the serelaxin sustained-release group showed significantly reduced α-SMA expression, and the SPNF group showed no significant changes. Figure 7 (P < 0.05), indicating that serelaxin sustained release can inhibit CF differentiation into MF.

[0082] 2.2 Serelaxin sustained-release formulation promotes transdifferentiation of CF into endothelial cells; 1) Immunofluorescence staining results: like Figure 8 As shown, no CD31 expression was observed in the control group, a small number of cells in the SPNF group expressed CD31, more cells in the serelaxin group than in the SPNF group, and the number of CD31-expressing cells in the serelaxin sustained-release group was significantly increased. CD31-positive cells were elongated spindle-shaped, oval, etc.

[0083] 2) Expression of CD31, vWF, and Hoxa-9 mRNA and CD31 protein: like Figure 9 As shown, CD31 and vWF are endothelial cell markers. RT-PCR results indicated that no CD31 and vWF mRNA expression was observed in the control group, weak expression was observed in the SPNF group, enhanced expression was observed in the serelaxin group, and more significant expression was observed in the serelaxin sustained-release group. Figure 9 Hoxa-9 is involved in endothelial cell transcriptional regulation. RT-PCR results showed that Hoxa-9 mRNA was weakly expressed in the control and SPNF groups. Compared with the control and SPNF groups, Hoxa-9 expression was enhanced in the serelaxin group, and the expression was more obvious in the serelaxin sustained-release group. Figure 9 (EF). Western blot results showed that no CD31 protein was expressed in the control group, weakly expressed in the SPNF group, enhanced in the serelaxin group, and even more pronounced in the serelaxin sustained-release group. Figure 9 (Zhong GH).

[0084] 3) Serelaxin sustained-release formulation promotes VEGF expression and release: like Figure 10 As shown, compared with the control group, the expression levels of VEGF mRNA and protein in the serelaxin, SPNF, and serelaxin sustained-release groups were significantly increased, with the increase being more pronounced in the serelaxin sustained-release group. Figure 10 (P < 0.05). ELISA results indicated that, compared with the control group, the VEGF levels in the culture supernatant of the serelaxin, SPNF, and serelaxin sustained-release groups were significantly increased (AD) (P < 0.05). Figure 10 (E). Serelaxin and serelaxin extended-release groups were higher than SPNF, with the increase being more significant in the serelaxin extended-release group (P < 0.05).

[0085] 2.3 Serelaxin sustained-release formulations promote the transdifferentiation of CF cells into cardiomyocytes; 1) Immunofluorescence staining results: like Figure 11 As shown, no cTnT expression was observed in the control group and SPNF group, a small number of cells in the serelaxin group expressed cTnT, and the number of cTnT-expressing cells in the serelaxin sustained-release group was significantly increased, with obvious striations.

[0086] 2) Expression of early transcription factors Nkx2.5, GATA-4, and Mef2C mRNA in cardiomyocytes: like Figure 12 As shown, the control group and SPNF group showed weak expression of Nkx2.5 and GATA-4, while the serelaxin group showed enhanced expression of Nkx2.5, GATA-4 and Mef2C mRNA, and the serelaxin sustained-release group showed significantly enhanced expression.

[0087] 3) Expression of myocardial-specific genes cTnT and Cx-43 mRNA: like Figure 13 As shown, no cTnT mRNA expression was detected in the control group and SPNF group. The Serelaxin group showed weak cTnT mRNA expression, while the cTnT expression was significantly increased in the serelaxin sustained-release group. Cx-43 mRNA expression was weak in the control group and SPNF group, but increased in the serelaxin group, and significantly increased in the serelaxin sustained-release group.

[0088] 4) Serelaxin sustained-release formulation upregulates p-Akt protein expression: like Figure 14As shown, there was no significant difference in Akt protein expression among the groups. Compared with the control group, p-Akt expression was enhanced in the serelaxin group and the SPNF group, and p-Akt expression was significantly enhanced in the sustained-release serelaxin group.

[0089] 2.4 Recovery of cardiac function after drug treatment; 1) Establishment of a myocardial infarction model and drug treatment: Before surgery, the animal was weighed and anesthetized by intraperitoneal injection of ketamine (80 mg / kg). The animal was then fixed in a supine position on a rodent board. The hair in the precordial region was shaved. An 18-gauge intravenous catheter was inserted into the endotracheal tube. After confirmation, the catheter was removed. The small animal ventilator was then connected and turned on. The respiratory rate was set to 80 breaths / min, the tidal volume to 7 ml, and the expiratory-to-inspiratory ratio to 5:4.

[0090] After disinfecting the skin on the rat's chest, make a longitudinal incision of about 2 cm in length using ophthalmic straight scissors about 1 cm away from the apex of the heart in the direction of the neck. Bluntly separate the muscles with sterile hemostatic forceps and open the chest wall in the third and fourth intercostal spaces. After fully exposing the heart, open the pericardium with ophthalmic straight scissors to fully expose the great cardiac vein. At the lower edge of the left atrial appendage, use a 6-0 suture needle to suture obliquely from the anterior inferior to the posterior superior to the posterior superior, to a depth of about 0.5 to 1.0 mm.

[0091] The left anterior descending coronary artery was ligated, and the color change of the myocardium in the area below the ligation line was observed. The establishment of the rat MI model was confirmed if the pale myocardium below the ligation line did not turn bright red again. In the sham operation group (Sham group), the left anterior descending coronary artery of rats was only threaded through the ligation line without ligation.

[0092] The chest cavity is sealed with a needle-stitched silk thread, then the chest muscles are sutured layer by layer from the inside out, and finally the chest skin is sutured with a purse-string suture; the chest cavity is squeezed to expel the residual air in the chest cavity to allow the lungs to re-expand, and the endotracheal tube is removed.

[0093] After the surgery, the rats were returned to their cages, and penicillin was administered intramuscularly to prevent infection.

[0094] One week after the establishment of the rat myocardial infarction (MI) model, surviving rats were randomly divided into five groups: sham-operated group (6 Sham rats), control group (8 MI rats), serelaxin group (10 rats), SPNF group (10 rats), and SPNF-serelaxin group (11 rats). Rats were anesthetized with ketamine (80 mg / kg) via intraperitoneal injection and then underwent thoracotomy. In the MI group, PBS was injected at four points along the infarct margin, 20 μl at each point. In the serelaxin group, 3 μl serelaxin + 77 μl PBS was injected at four points along the infarct margin, 20 μl at each point. In the SPNF group, 20 μl SPNF + 60 μl PBS was administered. In the SPNF-serelaxin group, a mixture of 20 μl SPNF + 3 μl serelaxin + 57 μl PBS (serelaxin concentration 100 ng / μl; SPNF was sonicated for 30 min before being added to PBS) was injected at four points along the infarct margin, 20 μl at each point. All injections were single-dose. Postoperatively, penicillin was administered via intramuscular injection to prevent infection.

[0095] The sham surgery group here is a special control group in medical experiments. Its core purpose is to eliminate the influence of non-therapeutic factors such as surgical trauma, anesthesia, and psychological factors on the experimental results by simulating the complete surgical process, thereby ensuring that the observed effect truly comes from the treatment method being tested.

[0096] 2) Cardiac function testing: Changes in cardiac function in rats were assessed using small animal ultrasound 1 day before myocardial infarction (MI), 7 days after infarction (i.e., before intramyocardial injection), and 28 days after intramyocardial injection. The rats were anesthetized using an animal anesthesia machine, with 2% isoflurane inhaled via the mouth and nose. The heart rate was maintained between 350-400 beats / min. The rats were fixed in a supine position on the Vevo ultrasound system operating platform, and the temperature was maintained appropriately. The fur on the anterior chest was shaved, and an appropriate amount of ultrasound coupling agent was applied to the hairless area of ​​the precordial region. Ultrasound scanning was performed after the M-type probe contacted the coupling agent. The probe frequency was set to 15 MHz, with a sampling volume of 0.6 mm and a scanning speed of 100 mm / s. The papillary muscles were located on the image, and M-type sampling lines were set at the interventricular septum and the posterior wall of the left ventricle to acquire two-dimensional echocardiographic images. The left ventricular structure of rats was evaluated by measuring the anterior wall thickness of the systolic wall (AWs) and the anterior wall thickness of the diastolic wall (AWd), as well as the left ventricular end-diastolic diameter (LVDd) and the left ventricular end-systolic diameter (LVDs) using standard small animal ultrasound methods.

[0097] Left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were measured in rats to evaluate cardiac function; the average value of each measurement was calculated after three consecutive cardiac cycles.

[0098] like Figure 15 The results showed that compared with the Sham group, the control group had significantly increased LVDd, LVDs, LVEDV, and LVESV, and significantly decreased EF%, FS%, AWd, and AWs, indicating a significant decrease in cardiac function after myocardial infarction in rats. Compared with the control group, the serelaxin group and SPNF group showed decreased LVDd and LVDs, while EF% and FS% showed an increasing trend. Compared with the control group, serelaxin group, and SPNF group, the serelaxin sustained-release group showed significantly decreased LVDd, LVDs, LVEDV, and LVESV, and significantly increased EF%, FS%, AWd, and AWs, with statistically significant differences (P < 0.05), indicating that sustained-release serelaxin significantly reduced left ventricular dilation and significantly improved cardiac function.

[0099] 2.5 Changes in chamber wall structure after drug treatment; like Figure 16 As shown, after Masson staining, blue represents fibrous tissue, and red represents myocardial tissue. In the control group, the infarct area mostly appeared blue, indicating dense fibrous tissue, with only a small amount of residual red myocardium scattered near the endocardium. In the Serelaxin, SPNF, and serelaxin extended-release groups, myocardial tissue was visible under the epicardium in the infarct area. Compared to the SPNF group, the Serelaxin and serelaxin extended-release groups showed more myocardial tissue under the epicardium, and the serelaxin extended-release group had significantly more myocardial tissue than the serelaxin group. Figure 16 (A). Statistical analysis was performed using Image Pro Plus 6.0 image software. The results showed that compared with the control group (56.02±0.012), there was no significant difference in the infarct area scar area of ​​the SPNF group (52.59±0.009), while the serelaxin group (37.62±0.008, P<0.05) and the serelaxin sustained-release group (14.48±0.007, P<0.05) showed significantly smaller infarct area scar area. The scar area in the serelaxin sustained-release group was significantly smaller than that in the serelaxin group (P<0.05). Figure 16 In the control group (D), the left ventricular free wall was significantly thinned and expanded outward, resulting in a significant enlargement of the left ventricular cavity. In the Serelaxin, SPNF, and serelaxin sustained-release groups, the expansion of the left ventricular free wall was not significant. Figure 16 (A) Compared with the control and SPNF groups, serelaxin increased left ventricular wall thickness. The increase in left ventricular wall thickness was more pronounced in the serelaxin sustained-release group compared with the serelaxin group (P<0.05). Figure 16 (E).

[0100] 2.6 Serelaxin sustained-release formulation for anti-post-infarction myocardial fibrosis; Immunofluorescence staining was performed on the infarct area and the infarct margin area using α-SMA and vimentin dual staining. The results are as follows: Figure 17 As shown, 28 days after intramyocardial injection, the number of double-positive cells in the infarct border area and infarct area was significantly increased in the control group; the number of double-positive cells in the infarct border area and infarct area was decreased in the Serelaxin, SPNF and Serelaxin sustained-release groups. Compared with the SPNF group, the number of Serelaxin cells was reduced, while the number of Serelaxin sustained-release cells was less than that in the Serelaxin group. Figure 17(AB). qRT-PCR and Western blot results showed that, compared with the Sham group, the expression of α-SMA mRNA and protein in the control group was significantly enhanced; compared with the control group, the serelaxin, SPNF, and serelaxin sustained-release groups all inhibited the expression of α-SMA mRNA and protein. Figure 17 The CE (P < 0.05) was not significantly different between the Serelaxin and SPNF groups. Compared with the Serelaxin and SPNF groups, the Serelaxin sustained-release group showed a more significant inhibitory effect.

[0101] 2.7 Serelaxin sustained-release formulations promote myocardial regeneration in infarcted myocardial tissue; Myocardial regeneration in the infarct border zone and infarct area was detected using cTnT and Cx43 immunofluorescence dual staining. The results are as follows: Figure 18 As shown, the expression of cTnT and Cx43 in cardiomyocytes in the infarct border area was significantly reduced in the control group. A few cells in the subepicardial region of the infarct area expressed cTnT, but no Cx43 expression was observed. In contrast, the serelaxin, SPNF, and serelaxin sustained-release groups showed more cTnT and Cx43-positive cells in the infarct border area, and more cells expressing cTnT and Cx43 in the subepicardial region of the infarct area compared to the control group. Compared to the serelaxin and SPNF groups, the serelaxin sustained-release group showed more cTnT and Cx43-positive cells in the infarct area, with higher fluorescence intensity; Cx43 was mainly expressed at cell junctions.

[0102] It should be understood that the above detailed description of the embodiments of the present invention provided in the accompanying drawings is only for further illustration of the present invention and is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. Based on the embodiments of the present invention, any non-essential improvements and adjustments made by those skilled in the art according to the above description of the present invention are within the scope of protection of the present invention.

Claims

1. A Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers, characterized in that, include: The self-assembling peptide RADA16-I, with the sequence AcN-RADARADARADARADA-CONH2, serves as the basic unit of the nanofiber scaffold and forms a three-dimensional nanofiber network through self-assembly. The functionalized modified peptide is attached to the C-terminus of the self-assembled peptide RADA16-I as a specific binding domain for Serelaxin. And the Serelaxin drug molecule, which is loaded into a three-dimensional nanofiber network by binding to the functionalized modified peptide.

2. The Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to claim 1, characterized in that, The functionalized modified peptide has the sequence GGCWIPIFVVKF, which is covalently linked to the C-terminus of the self-assembled peptide RADA16-I via an amino acid linker chain -GG-, forming the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I.

3. The Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to claim 2, characterized in that, The functionalized self-assembly peptide GGCWIPIFVVKF-RADA16-I is mixed with the self-assembly peptide RADA16-I in a 1:1 ratio under physiological conditions to form a functionalized self-assembly peptide nanofiber hydrogel.

4. A Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to any one of claims 3, characterized in that, The functionalized self-assembled peptide nanofiber hydrogel has nanofibers with a diameter of 10–15 nm and a length of 100–300 nm.

5. A method for preparing a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to any one of claims 1-4, characterized in that, Includes the following steps: (1) The functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I was prepared by solid-phase synthesis and purified by high performance liquid chromatography; (2) The self-assembled peptide RADA16-I and the functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I were mixed in a 1:1 ratio, PBS buffer was added, and the self-assembly was triggered by sonication for 30 min to form a functionalized self-assembled peptide nanofiber hydrogel. (3) Mix Serelaxin with the precursor solution of functionalized self-assembled peptide nanofiber hydrogel and let stand for 30 min to allow Serelaxin to fully combine with functionalized self-assembled peptide GGCWIPIFVVKF-RADA16-I and load it into the three-dimensional nanofiber network.

6. The use of a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to any one of claims 1-4 in the preparation of a drug for myocardial repair.

7. The application of a Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to claim 6 in the preparation of a drug for myocardial repair, characterized in that, The formulation reduces the differentiation of cardiac fibroblasts into myofibroblasts by inhibiting the TGF-β1 / Smad2 signaling pathway, and / or promotes the transdifferentiation of cardiac fibroblasts into endothelial cells and cardiomyocytes by activating the PI3K / Akt pathway and upregulating Hoxa-9 and VEGF factors.

8. The application of the Serelaxin sustained-release formulation based on functionalized self-assembled peptide nanofibers according to claim 7 in the preparation of a drug for myocardial repair, characterized in that, The formulation continuously releases Serelaxin in the body for more than 28 days.

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