Membrane organelle nano-vesicle for preventing and treating myocardial hypertrophy as well as preparation method and application of membrane organelle nano-vesicle

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a membranous organelle nano-vesicle for preventing and treating myocardial hypertrophy and a preparation method and application thereof, and the membranous organelle nano-vesicle comprises endoplasmic reticulum extracted from mammal liver tissue and nano-scale vesicles which are obtained after ultrasonication treatment and have a calcium ion buffering function. The liver-derived endoplasmic reticulum nano-vesicles and the derivatives thereof are extracted and are directly physically fused with the myocardial cell endoplasmic reticulum, so that the technical spanning from chemical signal regulation to organelle function reconstruction is fundamentally realized, the calcium pump activity of SERCA2a is accurately and efficiently enhanced, the endoplasmic reticulum calcium homeostasis is directly repaired, and the liver-derived endoplasmic reticulum nano-vesicles and the derivatives thereof are used for preparing the myocardial cell endoplasmic reticulum nano-vesicles. Meanwhile, energy metabolism is synergistically improved, and cell apoptosis is inhibited, so that the pathological process of myocardial hypertrophy is remarkably reversed in an animal model, and the compound has excellent biological safety and industrialization potential due to the biogenic characteristic of the compound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to membranous organelle nanovesicles for the prevention and treatment of myocardial hypertrophy, their preparation methods, and applications. Background Technology

[0002] Cardiac hypertrophy is a cardiac pathological state characterized by increased cardiomyocyte volume and thickening of the ventricular wall, typically an adaptive response to stress factors such as pressure overload, neurohormonal stimulation, or genetic mutations. During the occurrence and development of pathological cardiomyopathy, intracellular calcium ions (Ca²⁺) play a crucial role. + Homeostasis imbalance is one of the core components. The sarcoplasmic reticulum (SR), a specialized endoplasmic reticulum in cardiomyocytes, serves as the main intracellular calcium ion reservoir. It releases Ca²⁺ ions through ranitin receptors (RyR2) on its membrane. + To induce contraction, and through the sarcoplasmic reticulum / endoplasmic reticulum Ca² + -ATPase (SERCA2a) converts Ca²⁺ in the cytoplasm + Recovery is necessary to achieve relaxation. Decreased SERCA2a activity leads to cytoplasmic Ca²⁺... + Overload exacerbates endoplasmic reticulum stress (ERS), ultimately triggering cardiomyocyte apoptosis and pathological remodeling.

[0003] Currently, clinical strategies for preventing and treating myocardial hypertrophy mainly rely on drug intervention, such as angiotensin-converting enzyme inhibitors (ACEIs), beta-blockers, and calcium channel blockers. These treatments primarily slow disease progression by reducing cardiac load or indirectly regulating cell signaling pathways, but they cannot fundamentally repair the calcium processing capacity of damaged cardiomyocytes, especially by directly restoring SERCA2a function and endoplasmic reticulum calcium homeostasis.

[0004] In existing technologies, researchers have attempted to intervene in calcium homeostasis at different levels to treat myocardial hypertrophy. Patent document CN116615438A discloses a peptide and its composition capable of binding to L-type calcium channels (LTCC). This peptide partially inhibits Ca²⁺... + Influx of calcium indirectly reduces cytoplasmic calcium overload, thus having a certain therapeutic effect on myocardial hypertrophy. However, the target of this technology is limited to ion channels on the cell membrane surface, which is an indirect chemical regulation. It cannot directly act on the sarcoplasmic reticulum / endoplasmic reticulum, a core organelle of calcium circulation, and cannot directly enhance the activity of SERCA2a or repair its function. Furthermore, there is a risk that long-term inhibition of calcium influx may affect the normal contractile function of the myocardium.

[0005] Another patent document, CN109106715A, discloses the application of the small molecule compound 8-hydroxy-5-nitroquinoline in the preparation of drugs for the prevention and treatment of myocardial hypertrophy. This compound inhibits Ca²⁺... + The Calmodulin-dependent protein kinase II signaling pathway indirectly regulates SERCA2a expression levels. While this approach shows some effect at the gene and signaling pathway levels, its mechanism of action remains indirect regulation through upstream signaling, failing to directly enhance endoplasmic reticulum calcium pump function. Furthermore, as a small molecule chemical drug, it may have potential toxic side effects due to non-specific effects and lacks specific targeting ability for the endoplasmic reticulum in cardiomyocytes.

[0006] In summary, it is necessary to propose a membrane organelle nanovesicle that can directly target the endoplasmic reticulum, fundamentally and physically restore its calcium homeostasis, and is safe and effective for the prevention and treatment of myocardial hypertrophy, as well as its preparation method and application. Summary of the Invention

[0007] The purpose of this invention is to provide a membrane organelle nanovesicle for the prevention and treatment of myocardial hypertrophy, its preparation method and application. By extracting liver-derived endoplasmic reticulum nanovesicles (ENVs) and their derivatives, they are directly physically fused with the endoplasmic reticulum of cardiomyocytes, fundamentally realizing a technological leap from chemical signal regulation to organelle function reconstruction. This achieves precise and efficient enhancement of SERCA2a calcium pump activity, directly repairs endoplasmic reticulum calcium homeostasis, and synergistically improves energy metabolism and inhibits cell apoptosis, thereby significantly reversing the pathological process of myocardial hypertrophy in animal models. Furthermore, due to its biological origin, it possesses excellent biosafety and industrialization potential.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a membrane organelle nanovesicle for the prevention and treatment of myocardial hypertrophy, wherein the membrane organelle nanovesicle comprises endoplasmic reticulum extracted from mammalian liver tissue, which is obtained by ultrasonic disruption and has a calcium ion buffering function.

[0009] The basic principle of this technical solution lies in using endogenous membrane organelle nanovesicles to repair and enhance the function of diseased cardiomyocytes at the organelle level. Its core lies in introducing endoplasmic reticulum nanovesicles, extracted and prepared from mammalian liver, into cardiomyocytes via a delivery system. These vesicles, due to the similarity of their biological membrane structures, can undergo physical membrane fusion with the host cell's endoplasmic reticulum.

[0010] This fusion process is not a simple matter of material supplementation, but a functional structural integration. The functional membrane proteins carried by the exogenous vesicles, especially the SERCA2a calcium pump and its related regulatory proteins, are directly introduced and integrated into the endoplasmic reticulum membrane of the host cell, thereby physically enhancing the active transport capacity of calcium ions and directly correcting the core calcium circulation disorder in myocardial hypertrophy.

[0011] This principle transcends the traditional indirect regulatory model of drugs at the signaling molecule level, achieving a shift from chemical intervention to physical functional reconstruction. Through this direct repair at the organelle level, calcium homeostasis can be systematically restored, mitochondrial energy metabolism improved, and the apoptosis pathway triggered by endoplasmic reticulum stress inhibited, thereby fundamentally reversing the pathological process of myocardial hypertrophy.

[0012] Furthermore, the mammals are rats or mice; the membrane proteins on the membrane organelle nanovesicles include Calnexin, GRP78, and SERCA2a.

[0013] A method for preparing membranous organelle nanovesicles for the prevention and treatment of myocardial hypertrophy includes the following steps: (a) Take fresh animal liver tissue, wash it with PBS and cut it into small pieces, and centrifuge it at 500×g for 5 minutes; (b) Add cold reagent A at a ratio of 1 mg tissue: 10 μl and let stand on ice for 10 minutes. Use a homogenizer to homogenize the tissue thoroughly until there are no obvious solid clumps. (c) Centrifuge the homogenate at 4℃ and 500-1000×g for 5-10 minutes and collect the supernatant; (d) Centrifuge the supernatant obtained in step (c) at 4°C and 10,000-11,000 × g for 10 minutes, discard the precipitate, and collect the supernatant; (e) The supernatant obtained in step (d) is ultracentrifuged at 4°C and 50,000-100,000×g for 45 minutes, and the thin layer of precipitate at the bottom of the centrifuge tube is collected. (f) The thin-layer precipitate was resuspended and mixed with cold reagent B, and centrifuged at 4°C and 50,000-100,000×g for 45 minutes. The supernatant was discarded, and the precipitate was resuspended with preservation solution to obtain the membrane organelle sample. (g) The membrane organelle sample was treated with a cell disruptor under ice bath, 100W power, and intermittent sonication for 5 minutes to obtain the membrane organelle nanovesicles. This extraction method, through the synergistic effect of differential centrifugation and controlled ultrasound, can efficiently separate endoplasmic reticulum nanovesicles with intact membrane structure and functional activity from liver tissue. The series of centrifugation steps effectively removes impurities such as cell nuclei and mitochondria, achieving high-purity enrichment of endoplasmic reticulum components. Subsequent intermittent ice-bath ultrasound treatment, while maintaining biological activity, reconstructs endoplasmic reticulum fragments into uniformly sized nanovesicles that are easily taken up by cells. The final product not only preserves membrane proteins (such as SERCA2a) well, but also achieves high-purity, high-activity, and high-yield endoplasmic reticulum extraction without the use of chemical reagents. The obtained ENVs possess calcium ion buffering and membrane fusion potential suitable for their application in the treatment of myocardial hypertrophy.

[0014] A membrane-modified nanoparticle for the prevention and treatment of myocardial hypertrophy comprises membrane organelle nanovesicles as described in claim 1 or 2 and polymer nanoparticles, wherein the membrane organelle nanovesicles and the polymer nanoparticles are mixed at a polymer to nanovesicle membrane protein weight ratio of 1:1, and then obtained by ultrasonic fusion and centrifugation.

[0015] A hybrid membrane fusion vesicle for the prevention and treatment of myocardial hypertrophy is prepared by mixing the membrane organelle nanovesicles as described in claim 1 or 2 with a cell membrane at a weight ratio of 1:1, followed by ultrasonic fusion and ultra-high-speed centrifugation.

[0016] By precisely fusing endoplasmic reticulum nanovesicles with synthetic polymers and natural cell membranes, two derivative systems with complementary functions were successfully constructed: (1) membrane-modified nanoparticles encapsulate polymer nanonuclei (such as polylactic acid nanoparticles) with membrane organelle nanovesicles, which significantly enhances the structural stability and in vivo circulation time of the vesicles; (2) hybrid membrane fusion vesicles, through the integration of membrane organelle nanovesicles with cell membranes, have the dual functions of calcium homeostasis repair and inflammation targeting. Together, they expand the therapeutic application scenarios and efficacy of basic vesicles.

[0017] A pharmaceutical composition for the prevention and treatment of myocardial hypertrophy comprises a therapeutically effective amount of membrane organelle nanovesicles, membrane-modified nanoparticles or hybrid membrane fusion vesicles, and a pharmaceutically acceptable carrier or excipient.

[0018] Furthermore, the pharmaceutically acceptable carrier includes a buffered salt solution, and the dosage form of the pharmaceutical composition is an injection.

[0019] The application of a membrane organelle nanovesicle, membrane-modified nanoparticle, or hybrid membrane fusion vesicle in the preparation of a drug for the prevention and / or treatment of myocardial hypertrophy.

[0020] Furthermore, the myocardial hypertrophy is pathological myocardial hypertrophy, and the mechanism of action of the drug includes one or more of the following: enhancing the activity of SERCA2a in cardiomyocytes, restoring endoplasmic reticulum calcium homeostasis, and inhibiting endoplasmic reticulum stress and apoptosis.

[0021] Furthermore, the drug is an intravenously injectable drug, and the content of membrane organelle nanovesicles, membrane-modified nanoparticles, or hybrid membrane fusion vesicles in the drug is 0.1 mg / mL-100 mg / mL.

[0022] The beneficial effects of this technical solution are: (1) This technical solution surpasses the indirect regulation of existing drugs at the cell membrane or signaling pathway level and achieves direct intervention at the organelle level for the first time. Through the physical fusion of endogenous membranous organelle nanovesicles with the endoplasmic reticulum of cardiomyocytes, the function of the core calcium pump protein SERCA2a is directly supplemented and enhanced, and calcium homeostasis is repaired from the root, with a more precise and fundamental target.

[0023] (2) This approach not only effectively restores calcium circulation, but also achieves triple synergistic repair of "calcium homeostasis-energy metabolism-cell survival" by repairing the endoplasmic reticulum, a core hub. Experiments have shown that this technology can simultaneously increase ATP levels, restore mitochondrial membrane potential, reduce oxidative stress, and inhibit apoptosis, demonstrating a comprehensive therapeutic effect in reversing the pathological process of myocardial hypertrophy.

[0024] (3) Due to the use of a physical membrane fusion mechanism, endogenous functional proteins are structurally integrated into the host cell. Their functional enhancement effect does not depend on the continuous drug concentration, so the effect is more lasting and stable, avoiding the problems of traditional chemical drugs that require frequent administration due to rapid metabolism and large fluctuations in efficacy.

[0025] (4) The core active ingredient is derived from the endoplasmic reticulum of the organism itself, which is a natural biological material with low immunogenicity and good biocompatibility. Animal experiments have confirmed that no liver or kidney function damage or significant inflammatory response occurred in the treatment group, and serum myocardial injury markers were significantly reduced, demonstrating extremely high biocompatibility and suitability for long-term treatment. Attached Figure Description

[0026] Figure 1 This is the effect of ENVs on the survival rate of ISO-induced H9C2 cardiomyocytes in Experimental Example 1 of this invention.

[0027] Figure 2 This is the effect of ENVs on ISO-induced mitochondrial function (ATP level and membrane potential) of H9C2 cardiomyocytes in Experiment Example 2 of this invention.

[0028] Figure 3 This study describes the effects of ENVs and the traditional drug Nifedipine on the survival rate of H9C2 cardiomyocytes in Experimental Example 3 of this invention.

[0029] Figure 4 This is an example of the effect of ENVs and the traditional drug Nifedipine on the calcium ion content of H9C2 cardiomyocytes in Experiment 4 of this invention.

[0030] Figure 5 This is an example of the effects of ENVs and the traditional drug Nifedipine on the ATP levels of H9C2 cardiomyocytes in Experimental Example 5 of this invention.

[0031] Figure 6 This is an example of the uptake and localization of ENVs in H9C2 cardiomyocytes in Experiment 6 of this invention (20x).

[0032] Figure 7 This is an example of the uptake and localization of ENVs in H9C2 cardiomyocytes in Experiment 6 of this invention (40x).

[0033] Figure 8 This is an example of the uptake and localization of ENVs in H9C2 cardiomyocytes in Experiment 6 of this invention (60x).

[0034] Figure 9 This is the effect of ENVs on ISO-induced opening of the mitochondrial permeability transition pore (MPTP) in Experimental Example 7 of this invention.

[0035] Figure 10 This is a comparison of gross cardiac specimens of mice treated with different formulations in Experiment Example 8 of this invention.

[0036] Figure 11 This is the effect of different formulations on the cardiac weight index of hypertrophic mice in Experimental Example 8 of the present invention.

[0037] Figure 12 The effect of ENVs and their derivatives on cardiac function (echocardiography) in hypertrophic mice in Experimental Example 9 of this invention.

[0038] Figure 13 This is the effect of ENVs and their derivatives on the intracellular calcium ion concentration in myocardial hypertrophy mice in Experimental Example 9 of this invention.

[0039] Figure 14 The effects of ENVs and their derivatives on the levels of ATP, reactive oxygen species (ROS), and membrane potential in cardiomyocytes of hypertrophic mice, as shown in Experimental Example 9 of this invention.

[0040] Figure 15 This study describes the effect of ENVs and their derivatives on the serum myocardial injury marker CK-MB level in hypertrophic mice, as shown in Experimental Example 9 of this invention.

[0041] Figure 16This is the effect of ENVs and their derivatives on the serum myocardial injury marker cTnI level in hypertrophic mice, as shown in Experimental Example 9 of this invention.

[0042] Figure 17 These are photographs of the internal organs of mice after ENVs administration in Experiment Example 10 of this invention.

[0043] Figure 18 This is a line graph showing the change in mouse body weight after ENVs administration in Experiment Example 10 of this invention.

[0044] Figure 19 The changes in serum ALT and AST indices in mice after ENVs administration in Experiment Example 10 of this invention are shown.

[0045] Figure 20 The change in blood urea nitrogen content in mice after administration of ENVs in Experiment Example 10 of this invention. Detailed Implementation

[0046] The specific implementation method is described below with reference to the accompanying drawings.

[0047] Example 1 This embodiment provides a method for preparing endoplasmic reticulum nanovesicles, the extraction and preparation method including the following steps: (1) Take 50mg-100mg of fresh animal tissue samples and wash them with PBS. (2) Cut the mixture into small pieces with scissors and wash twice with cold PBS. Centrifuge at 500×g for 5 minutes. (3) Add 500 μl-1 ml of cold reagent A and place on ice for 10 minutes. (4) Homogenize thoroughly with a Dounce homogenizer for 30-40 cycles until no obvious solid lumps remain. Then centrifuge at 4°C and 1000×g for 5 minutes. (5) Aspirate the supernatant into another pre-cooled clean centrifuge tube. (6) Centrifuge at 4℃ and 11000×g for 10 minutes, discard the precipitate and collect the supernatant. (7) Centrifuge the supernatant at 4℃ and 50,000×g for 45 minutes. Discard the supernatant and collect the precipitate. [Note]: 1) If conditions permit, the centrifugation force can be increased to 100,000×g, which is beneficial to improve the recovery rate of smooth endoplasmic reticulum vesicles. 2) If a large centrifuge rotor is used and the liquid volume is too small for centrifugation, PBS can be added to increase the liquid volume. 3) If high-speed centrifugation is not available, a low-speed centrifugation endoplasmic reticulum extraction kit (EX2690) can be selected. 4) The precipitate is a thin layer at the bottom of the centrifuge tube. It is not an obvious clump. (8) Add 400 μl of cold reagent B to the precipitate and mix well. (9) Centrifuge at 4℃ and 50,000×g for 45 minutes. [Note]: 1) If a large centrifuge rotor is used and the liquid volume is too small for centrifugation, PBS can be added to increase the liquid volume. 2) If high-speed centrifugation is not available, a low-speed centrifugation endoplasmic reticulum extraction kit (EX2690) can be selected. 3) If it is desired to recover all smooth endoplasmic reticulum vesicles, the centrifugation force needs to be increased to 100,000×g. (10) Discard the supernatant and resuspend the precipitate in endoplasmic reticulum preservation solution. [Note]: 1) Resuspension with the endoplasmic reticulum preservation solution in the kit is optional. Choose a suitable buffer solution to resuspend the endoplasmic reticulum according to the needs of downstream experiments or use it directly for downstream experiments. 2) When it is necessary to extract endoplasmic reticulum proteins, an appropriate amount of protein lysis buffer can be added directly to the precipitate for lysis. 3) The precipitate is a thin layer at the bottom of the centrifuge tube. It is not a significant clump. (11) The endoplasmic reticulum sample was obtained and subjected to endoplasmic reticulum ENV nanovesicles by sonication in an ice bath for 5 minutes using a cell disruptor with a power of 100W, on for 2 seconds and off for 3 seconds. [Note]: 1) The EX1370 endoplasmic reticulum extraction kit used in this preparation process was purchased from Solarbio Science & Technology Co., Ltd. Reagents A, B and C were all provided by the EX1370 endoplasmic reticulum extraction kit.

[0048] Example 2 This embodiment provides a method for preparing membrane-modified nanoparticles ENVs-NPs, which are composed of endoplasmic reticulum ENVs nanovesicles prepared in Example 1 and polylactic acid nanoparticles.

[0049] (1) Weigh 7.5 mg of PLA and 10 mg of PVA, add 1 mL of THF to PLA, and add 5 mL of ultrapure water to PVA, and dissolve by sonication. Add the PVA solution dropwise to the PLA solution under magnetic stirring until all the PLA solution has been added. Sonicate the mixture at 300W for 5 min, turning it on for 2 s and off for 3 s. Finally, vacuum rotary evaporate the obtained nanosolution for 30 min to remove most of the THF, and measure the particle size by DLS. Number and record the prepared polylactic acid nanoparticles.

[0050] (2) The endoplasmic reticulum ENVs nanovesicles obtained from the extraction and preparation in Example 1 were mixed with PLA nanoparticles at a polymer to membrane protein weight ratio of 1:1. The mixture was sonicated in an ice bath at 100W for 5 minutes, with the cycle on for 1 second and off for 2 seconds. To remove the uncoated membrane, the mixture was centrifuged at 12000g for 10 minutes, and the precipitate was ENVs-NPs.

[0051] Example 3 This embodiment provides a method for preparing hybrid membrane fusion NEM-ENVs, which consists of endoplasmic reticulum ENVs nanovesicles prepared in Example 1 and neutrophil membranes.

[0052] Extraction of neutrophil membranes: (1) First, prepare two clean petri dishes and add 2 mL of RPIM1640 complete culture medium to each petri dish for later use.

[0053] (2) Anesthetize the mice with ether, remove the cervical vertebrae to kill the mice, immediately cut off the two hind legs of the mice, and quickly separate and remove all the tissues, leaving only the tibia and femur.

[0054] (3) Next, separate the tibia and femur at the knee joint, cut off both ends of the joint of the tibia and femur, and use a 1mL syringe to repeatedly blow RPIM1640 complete culture into the tibia and femur until the bone turns white, to obtain about 2mL of bone marrow cell suspension, and then add 2mL of RPIM1640 complete culture medium to 4mL for later use.

[0055] (4) Next, take a new 15 mL centrifuge tube with a pointed tip, add 4 mL of separation reagent A from the mouse bone marrow neutrophil extraction kit, and then slowly add separation reagent C along the wall to make the liquid surface clearly separated. Finally, slowly add 4 mL of bone marrow cell suspension along the wall, being careful not to disrupt the solution interface.

[0056] (5) Then slowly and steadily place the centrifuge tube into a horizontal rotor centrifuge. Centrifuge for 30 minutes at room temperature and 1000g.

[0057] (6) After centrifugation, slowly and steadily remove the centrifuge tube and carefully aspirate the neutrophils between the separation reagent C solution and the separation reagent A solution, as well as the neutrophils in the separation reagent A solution, into a new 15mL centrifuge tube with a pointed tip using a pipette with a long tip.

[0058] (7) Add 10 ml of cell washing buffer to wash the cells and centrifuge at 450 g for 10 min at room temperature.

[0059] (8) Remove the supernatant, then add an appropriate amount of red blood cell lysis buffer, lyse on ice for 10 min, and centrifuge at room temperature and 250g for 10 min. (9) Continue to remove the supernatant, resuspend the cells in 5 mL of cell washing buffer, and centrifuge at 250 g for 10 min at room temperature. Wash three times. (10) Finally, add an appropriate amount of 1640 drug-containing culture medium containing 0.1% LPS to resuspend the cells, then transfer the cell suspension to a cell culture flask, place it in a cell culture incubator, and incubate at 37°C for 2 hours to activate the neutrophils.

[0060] (11) Transfer the neutrophils in the cell culture flask to a new 15 mL centrifuge tube, centrifuge at room temperature and 250 g for 10 min using a horizontal rotor centrifuge, remove the supernatant to obtain the activated neutrophil precipitate.

[0061] (12) Wash the cells twice with cell washing solution and suspend the activated neutrophils in pre-cooled separation buffer (add PMSED before use).

[0062] (13) Then, the cells were destroyed by ultrasound on ice using a probe ultrasound device with a power of 200w for 5min, with the power on for 2s and the power off for 3s.

[0063] (14) The suspension after sonication was centrifuged at 4°C and 10000g for 10 min to remove mitochondria and cell debris.

[0064] (15) Collect the supernatant, determine its protein concentration using the BCA method, and calculate the protein content. (16) After appropriate amount dispensing, freeze-dry the activated neutrophil membranes and store them at -80℃ for later use.

[0065] Hybrid membrane fusion: The neutrophil membrane prepared in the above steps and the endoplasmic reticulum ENVs nanovesicles extracted and prepared in Example 1 were added together in a 1:1 ratio, and then sonicated at 100W for 5 minutes, turned on for 2 seconds and off for 3 seconds, and then centrifuged at 100,000g for 20 minutes to collect the precipitate.

[0066] Experimental Example 1 Verification of the effect of endoplasmic reticulum ENVs nanovesicles prepared in Example 1 on cell survival rate in the H9C2 myocardial hypertrophy model.

[0067] Make 6 sub-wells with 5,000 cells per well, and add the cell suspension to the 96-well plate at an appropriate density, 100 μL per well; Complete culture medium was added to the control group for culture. 150 μmol / L isoproterenol hydrochloride was added to the model and treatment groups. After 24 hours, the culture medium was removed, and the samples were washed once with PBS. Complete culture medium was then added to the control and model groups for culture, while different concentrations of ENVs were added to the treatment groups. After 24 hours, 10 μL of CCK-8 solution was added to each well for 1 hour. The absorbance was measured at 450 nm.

[0068] Experimental results are as follows Figure 1As shown, after 24 hours of intervention with different concentrations of endoplasmic reticulum nanovesicles (ENVs), the survival rate of model cells was significantly improved, showing a dose-dependent increase within the range of 10 μg / ml-30 μg / ml. Compared with the model group, the cell survival rate of the medium and high concentration ENVs treatment groups recovered to levels close to those of the normal control group.

[0069] The endoplasmic reticulum nanovesicles prepared in this invention can effectively reverse ISO-induced cardiomyocyte damage, significantly improve the survival rate of mast cardiomyocytes, and have a clear cell protection effect.

[0070] Experiment Example 2 Verification of the effects of mitochondria (MITO) and endoplasmic reticulum ENV nanovesicles prepared in Example 1 on mitochondrial functional ATP and JC-1.

[0071] The cell suspension was spread in a 6-well plate with 80,000 cells per well, and 6 auxiliary wells were made with 2 mL per well. After 12 hours of cell culture, the control group was treated with complete culture medium, while the other groups were treated with 150 μmol / L ISO HCl for 24 hours (completely protected from light). After treatment, the cell culture medium was discarded. The control group was treated with complete culture medium, while the treatment groups were treated with different concentrations of ENVs for 24 hours. Cells were then washed twice with PBS. ATP JC-1 assays were performed according to the prescribed procedure.

[0072] Experimental results are as follows Figure 2 As shown, intervention with different concentrations of endoplasmic reticulum nanovesicles (ENVs) significantly improved mitochondrial function in cardiomyocytes. Compared with the model group, the ATP level in the ENVs treatment group increased in a dose-dependent manner. Simultaneously, JC-1 assay results showed that mitochondrial membrane potential was restored, indicating that mitochondrial structural integrity and function were effectively protected.

[0073] The results confirm that the endoplasmic reticulum nanovesicles prepared in this invention can effectively reverse ISO-induced mitochondrial functional damage, provide energy support for cardiomyocytes and inhibit apoptosis pathways by enhancing ATP synthesis capacity and stabilizing mitochondrial membrane potential.

[0074] Experimental Example 3 The effects of ENVs prepared in Example 1 and the conventional drug Nifedipine on cell survival in the H9C2 myocardial hypertrophy model were investigated.

[0075] With 5,000 cells per well and 6 accessory wells, the cell suspension was added to each well of a 96-well plate at an appropriate density, 100 μL per well. The control group was cultured in complete medium, while the model and treatment groups were cultured in 150 μmol / L isoproterenol hydrochloride. After 24 hours, the medium was removed, and the cells were washed once with PBS. The control and model groups were then cultured in complete medium, while the treatment groups were cultured in 30 μg / mL ENVs and 5 μM Nifedipine, respectively. After 24 hours of treatment, 10 μL of CCK-8 solution was added to each well for 1 hour. The absorbance was measured at 450 nm.

[0076] Experimental results are as follows Figure 3 As shown, in the ISO-induced H9C2 myocardial hypertrophy model, treatment with 30 μg / mL ENVs significantly improved cell survival, with a significantly better effect than the 5 μM Nifedipine treatment group, indicating that ENVs are superior to traditional calcium channel blockers in terms of cell protection.

[0077] Experiment Example 4 Effects of ENVs and the conventional drug Nifedipine prepared in Example 1 on cellular calcium ion content in an H9C2 myocardial hypertrophy model. Cell suspension was seeded in 6-well plates, with 80,000 cells per well and 6 accessory wells. 2 mL of medium was added to each well. After 12 h of cell culture and adherence, the control group received complete culture medium, while the other groups were treated with 150 μmol / L ISO·HCl for 24 h (completely protected from light). After treatment, the cell culture medium was discarded. The control group received complete culture medium, while the treatment groups received 30 μg / mL ENVs and 5 μM Nifedipine, respectively. After 24 h of treatment, the cells were washed twice with PBS. Calcium ion content was measured according to the prescribed procedure.

[0078] Experimental results are as follows Figure 4 As shown, ENVs treatment can significantly reduce intracellular calcium overload induced by ISO, and its ability to restore calcium homeostasis is superior to that of Nifedipine, suggesting that ENVs can correct pathological calcium disorders at their source.

[0079] Experimental Example 5 Example 1: Detection of the effects of ENVs prepared in Example 1 and the conventional drug Nifedipine on mitochondrial function ATP in H9C2 myocardial hypertrophy model Cell suspension was seeded in 6-well plates, with 80,000 cells per well and 6 accessory wells. 2 mL of medium was added to each well. After 12 h of cell culture and adherence, the control group received complete culture medium, while the other groups were treated with 150 μmol / L ISO·HCl for 24 h (completely protected from light). After treatment, the cell culture medium was discarded. The control group received complete culture medium, while the treatment groups received 30 μg / mL ENVs and 5 μM Nifedipine, respectively. After 24 h of treatment, the cells were washed twice with PBS. ATP assays were performed according to the prescribed procedure.

[0080] Experimental results are as follows Figure 5 As shown, ENVs can significantly restore the decrease in ATP levels caused by ISO, and their effect on improving mitochondrial energy metabolism is better than that of Nifedipine, further confirming the multiple repair advantages of ENVs at the metabolic level.

[0081] Experimental Example 6 Cellular uptake verification experiment of endoplasmic reticulum ENVs nanovesicles prepared in Example 1 Grouping: ①Control; ②ISO·HCl+30μg / mL ENVs.

[0082] Each group should be prepared in one well, with 60,000 cells per well, seeded into a 6-well plate. A climbing slide should be placed in each well, with 2 mL per well.

[0083] After culturing cells for 4 hours and allowing them to adhere, the control group was treated with complete culture medium, while the other groups were treated with 200 μmol / L ISO·HCl for 24 hours (in the dark). After treatment, the cell culture medium was discarded, the control group was treated with complete culture medium, and the treatment groups were treated with 30 μg / mL ENVs for 24 hours. Cells were then washed twice with PBS. ER-Tracker Green working solution (green) at a 1:1000 ratio was added to fully cover the cells, 300-500 μL per well. Cells were incubated at 37°C for 30 minutes to prepare for fluorescent probe loading. Cells were fixed with 4% formaldehyde at 37°C for 2 minutes. After fixation, cells were washed 2-3 times with appropriate washing buffer, 5 minutes each time. Then, an appropriate amount of Hoechst staining solution (blue) (1:1000 concentration) was added evenly to the culture medium, gently mixed, 300-500 μL per well, and incubated at 37°C for 20 minutes. The staining solution was discarded, and cells were washed 2-3 times with PBS or culture medium before fluorescence detection. Place the slide onto a glass slide and observe cell uptake under laser confocal microscopy, ensuring that the exposure parameters of all images are consistent.

[0084] Experimental results are as follows Figure 6 , Figure 7 and Figure 8As shown, these are 20x, 40x and 60x magnification photographs, respectively. Laser confocal microscopy revealed that after being labeled with ER-Tracker Green (endoplasmic reticulum green fluorescent probe), the endoplasmic reticulum region of the treated group cells showed strong green fluorescence, and the fluorescence signal was clearly located and typically distributed with the cell nucleus (blue).

[0085] The results directly demonstrate that the endoplasmic reticulum nanovesicles (ENVs) prepared in this invention can be effectively taken up by cardiomyocytes and specifically enriched in the endoplasmic reticulum region of the cells, laying the material basis for their direct functional fusion with the endoplasmic reticulum of the host cells.

[0086] Experimental Example 7 Mitochondrial permeability transition pore (MPTP) detection after treatment with endoplasmic reticulum ENVs nanovesicles prepared in Example 1 Whether the mitochondrial permeability transition pore (MPTP) is open can be observed using laser confocal microscopy. Grouping: ①Control; ②ISO·HCl; ③ISO·HCl +MITO 25μg / mL; ④ISO·HCl +ENVs 30μg / mL.

[0087] The Mitochondrial Permeability Transition Pore Assay Kit (MPTP Assay Kit) from Beyotime is used. It is a kit that uses the fluorescent probe Calcein AM, which is permeability-sensitive, to detect the degree of opening of mitochondrial permeability transition pores. It is also commonly used to study cell death such as apoptosis and necrosis, and can more directly detect changes in the degree of opening of mitochondrial permeability transition pores.

[0088] Experimental results are as follows Figure 9 As shown, the degree of mitochondrial MPTP opening is determined by the intensity of Calcein green fluorescence in mitochondria; stronger green fluorescence indicates a lower degree of opening, and weaker green fluorescence indicates a higher degree of opening. Compared with the normal control group, the intensity of Calcein green fluorescence in the mitochondria of cardiomyocytes in the model group was significantly reduced, indicating that the mitochondrial permeability transition pore (MPTP) was open in large quantities. After treatment with endoplasmic reticulum nanovesicles (ENVs), the intensity of green fluorescence in the cells was significantly restored, and the effect was comparable to that of the positive control drug MITO group.

[0089] This result directly confirms that ENVs can effectively inhibit the abnormal opening of mitochondrial MPTP in cardiomyocytes, thereby inhibiting the subsequent apoptosis pathway. This is also an important mechanism by which they protect cardiomyocytes and treat myocardial hypertrophy.

[0090] Experimental Example 8 Efficacy of endoplasmic reticulum (ENV) nanovesicles and their derivatives prepared in Examples 1-3 in the treatment of animal models (1) Animal modeling After one week of acclimatization, C57BL / 6J mice were randomly divided into two groups (n=3 or more per group): a control group and a model group. The model was induced by subcutaneous injection of ISO solution at a dose of 10 mg / kg. The control group received an equal volume of 0.1% VC solution. The treatment continued for 14 days. On day 15, mice were euthanized by cervical dislocation. The thoracic cavity was opened with scissors, the heart was removed, weighed, and photographed. The tibia length was measured using calipers. (The successful establishment of the myocardial hypertrophy model was determined by detecting the cardiac organ indices of C57 mice: the ratio of heart weight to mouse body weight (HW / BW) and the ratio of heart length to tibia length (HW / TL).) Compared with the control group, the heart weight / body weight ratio (HW / BW) and heart weight / tibia length ratio (HW / TL) of mice in the model group were significantly increased, indicating that the subcutaneous injection of ISO successfully established a mouse model of myocardial hypertrophy.

[0091] (2) Animal therapy Within the successfully modeled group, treatment groups were divided into: ① ISO.HCl 10 mg / kg + NEM-ENVs 4 mg / kg; ② ENVs 4 mg / kg; ③ ENVs-NPs 4 mg / kg; ④ Istarixime The model and treatment groups were induced by subcutaneous injection of ISO solution at a dose of 10 mg / kg. The control group was injected with an equal volume of 0.1% VC solution. Treatment continued for 14 days. On day 15, NEM-ENVs 4 mg / kg, ENVs 4 mg / kg, ENVs-NPs 4 mg / kg, and Istarixime were injected via tail vein. The control and model groups were injected with an equal volume of physiological saline via tail vein. Treatment was administered every two days. After two weeks of treatment, mice were euthanized by cervical dislocation. The chest cavity was opened with scissors, the heart was removed, weighed, and photographed. The tibia length was measured using calipers.

[0092] Treatment results as Figure 10 and Figure 11 As shown, after two weeks of treatment with endoplasmic reticulum nanovesicles (ENVs) and their derivatives (ENVs-NPs, NEM-ENVs), cardiac hypertrophy indices (HW / BW and HW / TL) in all treatment groups decreased significantly.

[0093] The results confirmed at the animal level that the endoplasmic reticulum nanovesicles and their derivatives prepared in this invention can effectively reverse ISO-induced myocardial hypertrophy.

[0094] Experimental Example 9 Echocardiography and Ca2+ measurements of endoplasmic reticulum (ENV) nanovesicles and their derivatives prepared in Examples 1-3 after treatment of model animals 2+ Physiological markers including concentration, ATP, ROS, JC-1, CK-MB, and serum cTnl were detected. Echocardiography includes left ventricular ejection fraction (LVEF): reflecting overall cardiac systolic function; a decrease in LVEF suggests impaired cardiac function; fractional shortening (FS): assessing the ability of the left ventricle to shorten during systole, with higher sensitivity than LVEF; and left ventricular end-diastolic diameter (LViDD): a marker of ventricular dilation, indicating myocardial remodeling and decreased compliance (a chronic phase indicator).

[0095] Other physiological indicators were tested using commonly used clinical methods, and the results are as follows: Figures 12 to 16 As shown, compared with the model group, mice treated with endoplasmic reticulum nanovesicles (ENVs) and their derivatives showed significantly improved cardiac function (manifested as increased LVEF and FS values), while intracellular calcium levels in cardiomyocytes were also reduced. 2+ Concentration, ATP levels, and mitochondrial membrane potential tended to normalize, while oxidative stress levels (ROS) and serum myocardial injury markers (CK-MB, cTnI) were significantly reduced.

[0096] These results comprehensively confirm, from physiological function to molecular level, that the product of this invention can systematically reverse the core pathological process of myocardial hypertrophy, effectively improve cardiac function, and has good biosafety.

[0097] Experimental Example 10 Animal safety evaluation experiment Groups: ①Control; ②ISO·ENVs 4 mg / kg; ③ENVs 4 mg / kg; ④ENVs 8 mg / kg; ⑤ENVs 16 mg / kg; ⑥ISO·ENVs 4 mg / kg. The ISO·ENVs model was first established by subcutaneous injection of ISO solution at a dose of 10 mg / kg, administered continuously for 14 days. On day 15, ENVs 4 mg / kg were injected via the tail vein, once every two days, for 7 consecutive days. Body weight was recorded. The Control group received an equal volume of physiological saline via the tail vein. The ENVs 4 mg / kg, ENVs 8 mg / kg, and ENVs 16 mg / kg groups received ENVs via tail vein injection at different concentrations, administered only once, with body weight recorded for 7 consecutive days. Blood was collected and dissected after 7 days.

[0098] The experimental results are as follows Figure 17 , Figure 18 , Figure 19 and Figure 20As shown, single or multiple administrations of different doses of ENVs (4-16 mg / kg) did not cause significant abnormal changes in body weight, organ morphology, and liver and kidney function indicators (ALT, AST, blood urea nitrogen) in normal and ISO model mice, demonstrating that ENVs have good in vivo safety within the therapeutic dose range.

Claims

1. A membranous organelle nanovesicle for use in the prevention and treatment of cardiac hypertrophy, characterized in that, The membrane organelle nanovesicle comprises endoplasmic reticulum extracted from mammalian liver tissue, and is a nanoscale vesicle with calcium ion buffering function obtained after ultrasonic crushing treatment.

2. The membrane organelle nanovesicle of claim 1, wherein, The mammal is a rat or a mouse; and the membrane protein on the membrane organelle nanovesicle comprises Calnexin, GRP78 and SERCA2a.

3. A method for preparing the membrane organelle nanovesicle according to claim 1 or 2, characterized by, The method comprises the following steps: (a) taking fresh animal liver tissue, washing with PBS and cutting, and centrifuging at 500xg for 5 minutes; (b) adding cold reagent A at a ratio of 1 mg of tissue: 10 μl, and standing on ice for 10 minutes, and using a homogenizer to homogenize the tissue sufficiently until no obvious solid mass is present; (c) centrifuging the homogenate at 4°C, 500-1000xg for 5-10 minutes, and collecting the supernatant; (d) centrifuging the supernatant obtained in step (c) at 4°C, 10000-11000xg for 10 minutes, discarding the precipitate, and collecting the supernatant; (e) ultracentrifuging the supernatant obtained in step (d) at 4°C, 50000-100000xg for 45 minutes, and collecting the thin layer of precipitate at the bottom of the centrifuge tube; (f) resuspending and mixing the thin layer of precipitate with cold reagent B, ultracentrifuging at 4°C, 50000-100000xg for 45 minutes, discarding the supernatant, and resuspending the precipitate with a storage solution, to obtain the membrane organelle sample; (g) treating the membrane organelle sample using a cell crusher under the conditions of ice bath, power 100W and intermittent ultrasonic for 5 minutes, to obtain the membrane organelle nanovesicle.

4. A membrane-modified nanoparticle for use in the prevention or treatment of cardiac hypertrophy, characterized in that, The membrane organelle nanovesicle as claimed in claim 1 or 2 and polymer nanoparticles are mixed at a weight ratio of polymer to nanovesicle membrane protein of 1:1, ultrasonic fusion and centrifugation are performed, and the membrane modified nanoparticles are prepared.

5. A hybrid membrane fusion vesicle for use in the prevention of cardiac hypertrophy, characterized in that, The membrane organelle nanovesicle as claimed in claim 1 or 2 and cell membranes are mixed at a weight ratio of 1:1, ultrasonic fusion and ultracentrifugation are performed, and the hybrid membrane fusion vesicle is prepared.

6. A pharmaceutical composition for preventing and treating cardiac hypertrophy, characterized by comprising the compound of claim 1 as an active ingredient. The drug composition comprises a therapeutically effective amount of the membrane organelle nanovesicle as claimed in claim 1 or 2, or the membrane modified nanoparticle as claimed in claim 4, or the hybrid membrane fusion vesicle as claimed in claim 5, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition of claim 6, wherein The pharmaceutically acceptable carrier comprises a buffer salt solution, and the dosage form of the drug composition is an injection.

8. The membrane organelle nanovesicle as claimed in claim 1 or 2, or the membrane modified nanoparticle as claimed in claim 4, or the hybrid membrane fusion vesicle as claimed in claim 5, for use in the preparation of a drug for preventing and / or treating cardiac hypertrophy.

9. Use according to claim 8, wherein the compound is ###0002### The cardiac hypertrophy is pathological cardiac hypertrophy, and the mechanism of action of the drug comprises one or more of the following: enhancing the activity of cardiac cell SERCA2a, restoring endoplasmic reticulum calcium homeostasis, and inhibiting endoplasmic reticulum stress and cell apoptosis.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The drug is an intravenous injection drug, and the content of the membrane organelle nanovesicle, or the membrane modified nanoparticle, or the hybrid membrane fusion vesicle in the drug is 0.1 mg / mL-100 mg / mL.

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