A biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy and its preparation method

Abstract

This invention belongs to the fields of pharmaceutical formulation and nanomedicine technology, and discloses a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy and its preparation method. The core of this invention is reactive oxygen species (ROS) responsive / scavenging nanomicelles (TPTN), which are prepared into a biomimetic nanocomposite (MTPTN) by encapsulating the mitochondrial outer membrane with TPTN through co-extrusion. This biomimetic nanomedicine can effectively cross the damaged vascular endothelial barrier and target and accumulate at the site of cardiac lesions. It utilizes the cell / intracellular transport mechanism mediated by mitochondrial outer membrane surface proteins and homologous targeting to achieve targeted enrichment of mitochondria in damaged cardiomyocytes. The encapsulated ROS responsive / scavenging nanomicelles TPTN can effectively remove excess ROS at the lesion site, inhibit inflammatory infiltration, and thus prevent and treat the occurrence and development of dilated cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical formulation and nanomedicine technology, and relates to a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy, specifically to a mitochondrial membrane biomimetic modified reactive oxygen species responsive / scavenging nanomicelle and its preparation method. Background Technology

[0002] Dilated cardiomyopathy (DCM) is a major cause of heart failure and the most common indication for heart transplantation worldwide. 1,2 Currently, different treatment strategies are used in clinical practice based on the etiology, symptoms, and severity of DCM, including drug intervention, device implantation, and surgery. 3 These treatments can alleviate symptoms of heart failure and improve cardiac function in patients with dilated cardiomyopathy (DCM), but they cannot prevent the further progression of DCM. Furthermore, currently used DCM drugs typically have low accumulation and short residence time within the diseased heart, and due to non-specific drug distribution and dose-limiting toxicity, they cause significant side effects. Therefore, for the precision treatment of DCM, new treatment methods and site-specific drug delivery strategies still require further exploration.

[0003] As we all know, the heart is the organ that consumes the most energy. 4 Mitochondria produce adenosine triphosphate (ATP) through the electron transport chain and the oxidative phosphorylation (OXPHOS) system, which is crucial for maintaining normal physiological function of the myocardium. 5 However, mitochondrial dysfunction and OXPHOS abnormalities can lead to excessive production of reactive oxygen species (ROS), increased mitochondrial oxidative / nitrifying stress, and loss of mitochondrial membrane potential, thereby affecting electron transport chain (ETC) processes, hindering ATP synthesis, inducing cell death, and ultimately causing cardiac damage. 6 Therefore, mitochondrial dysfunction is associated with various cardiac diseases, including myocarditis, myocardial infarction, drug-induced cardiotoxicity, heart failure, myocardial hypertrophy, and deep vein thrombosis (DCM). Notably, a growing body of research indicates that mitochondrial dysfunction plays a crucial role in the pathogenesis of DCM. 7-9 Since mitochondria are also a major source of ROS in cardiomyocytes, mitochondrial dysfunction induces excessive ROS production, leading to the production and secretion of pro-inflammatory cytokines and triggering a pro-inflammatory response in cardiomyocytes. 10,11 Furthermore, mitochondrial dysfunction leads to metabolic imbalance, reduced ATP production, and decreased calcium levels. 2+ Disruptions in homeostasis, imbalances in mitochondrial fusion and division, and disruptions in mitophagy collectively contribute to the development of DCM. 12 Therefore, specifically regulating myocardial mitochondrial homeostasis and oxidative stress / inflammatory responses at lesion sites is a promising precision treatment strategy for DCM.

[0004] Studies have shown that nanocarriers are ideal targeted delivery tools. Among them, commonly used mitochondrial-targeting modification units, such as rhodamine, triphenylphosphine, and Szeto-Schiller peptides (such as SS-31 and SS-20), have been investigated for mitochondrial-targeted delivery of antioxidants and protective drugs to the heart to repair mitochondrial damage and thus exert cardioprotective effects. 13,14 However, these conjugates formed by modifications to mitochondrial targeting units often exhibit off-target effects, being rapidly cleared by the kidneys and having a short residence time in the heart, thus leading to decreased efficacy. Furthermore, studies have found that synthetic nanoparticles and biomimetic nanoparticles (NPs) have proven to be promising targeted delivery carriers for the treatment of cardiac diseases. 15,16 In addition to passive targeting, NPs can also be functionalized through molecular targeting units or cell membranes to give them active targeting capabilities. 17-19 Despite these advances, achieving effective mitochondrial targeting in cardiomyocytes remains highly challenging. 14 Therefore, based on the existing technology, this invention synthesizes reactive oxygen species (ROS) responsive / scavenging nanomicelles and encapsulates them in the outer mitochondrial membrane. Utilizing the cell / intracellular transport mechanism mediated by mitochondrial outer membrane surface proteins and homologous targeting, it achieves targeted enrichment of damaged cardiomyocytes and mitochondria within cardiomyocytes. Furthermore, the encapsulated ROS responsive / scavenging nanomicelles can effectively remove excess ROS from the lesion site, inhibit inflammatory infiltration, and thus prevent and treat the occurrence and development of dilated cardiomyopathy. Summary of the Invention

[0005] The purpose of this invention is to provide a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy, based on the existing technology and current status. Specifically, it relates to a mitochondrial membrane biomimetic modified reactive oxygen species (ROS) responsive / scavenging nanomicelle and its preparation method. This biomimetic antioxidant nanomedicine can effectively target mitochondria, scavenge ROS at the lesion site, inhibit inflammatory responses, and further prevent and treat the occurrence and development of dilated cardiomyopathy.

[0006] To achieve the above objectives, the technical solution adopted by this invention is a method for preparing a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy. This method uses reactive oxygen species-responsive / scavenging nanomicelles (TPTN) as the core and encapsulates the TPTN with the outer mitochondrial outer membrane (OMM) as the shell, thus preparing a biomimetic nanomedicine encapsulated in the outer mitochondrial outer membrane. The specific steps include:

[0007] S1: Extraction of mitochondrial outer membrane: Extraction of mitochondrial outer membrane from animal heart tissue.

[0008] S2: Synthesis of reactive oxygen species responsive / scavenging nanomicelles TPTN: Using cyanuric chloride as the backbone, terminal hydroxyl polyethylene glycol, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxo radical and 4-hydroxymethylphenylboronic acid pinacol ester were gradually added to carry out an affinity substitution reaction in an ultra-dry organic solvent to synthesize an amphiphilic polymer TPT with reactive oxygen species responsive / scavenging ability. The TPT then self-assembled in aqueous solution to form reactive oxygen species responsive / scavenging nanomicelles TPTN.

[0009] S3: Preparation of biomimetic nanomedicine MTPTN: The mitochondrial outer membrane extracted in S1 was mixed with the TPTN nanomicelles prepared in S2, and after sonication, they were co-extruded into a polycarbonate membrane to obtain MTPTN.

[0010] The extraction of the mitochondrial outer membrane in step S1 above specifically includes the following steps: collecting mouse hearts, washing with PBS to remove blood, cutting, digesting, centrifuging, adding pre-cooled mitochondrial separation reagent, homogenizing, collecting mitochondria after gradient centrifugation; adding ultrapure water to the collected mitochondria and stirring, adding sucrose solution, homogenizing, collecting the mitochondrial outer membrane after gradient centrifugation.

[0011] Preferably, the hydroxyl-terminated polyethylene glycol has an average molecular weight of 2000 Da.

[0012] Preferably, the molar ratio of cyanuric chloride to hydroxyl-terminated polyethylene glycol is 1:1.

[0013] Preferably, the molar ratio of cyanuric chloride, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxygen radical, and pinacol 4-hydroxymethylphenylboronic acid is 1:1:1.

[0014] Preferably, the mass ratio of the mitochondrial outer membrane to TPTN nanomicelles is 1:1.

[0015] The present invention also provides a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy prepared using the above method.

[0016] The above-mentioned biomimetic nanomedicines for the prevention and treatment of dilated cardiomyopathy are used in the preparation of drugs for the prevention and treatment of dilated cardiomyopathy and diseases related to oxidative stress / inflammation or mitochondrial damage.

[0017] This invention uses dilated cardiomyopathy as a model to verify the application of the drug of this invention in the prevention and treatment of diseases related to oxidative stress / inflammation and / or mitochondrial damage. The administration methods of this invention include oral, intravenous, subcutaneous, and intramuscular injection, as well as any combination of the above methods.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The size of the biomimetic nanomedicine for preventing and treating dilated cardiomyopathy described in this invention can be controlled by the preparation process parameters. The preparation method is simple, the cycle is short, the cost is low, and it is easy to realize the industrialization of the nanomedicine.

[0020] (2) The biomimetic nanomedicine for preventing and treating dilated cardiomyopathy described in this invention has good biocompatibility and low immunogenicity. It can be degraded in vivo, and the degradation products have no toxic side effects on the body.

[0021] (3) The biomimetic nanomedicine for preventing and treating dilated cardiomyopathy described in this invention can be targeted and enriched at the site of cardiac lesions through active and passive targeting. After being internalized by cardiomyocytes, it is located in the mitochondria of cardiomyocytes, thereby achieving a three-level targeting effect on the diseased heart, cardiomyocytes and mitochondria.

[0022] (4) The biomimetic nanomedicine for preventing and treating dilated cardiomyopathy described in this invention can be triggered by a microenvironment with high levels of reactive oxygen species to release reactive oxygen species scavenging units (Tempol and PB), thereby achieving synergistic antioxidant stress and anti-inflammatory effects, reducing cardiac damage and ventricular remodeling, and thus preventing and treating the occurrence and development of dilated cardiomyopathy.

[0023] (5) The biomimetic nanomedicine for preventing and treating dilated cardiomyopathy described in this invention, through modification of the mitochondrial outer membrane, utilizes the cell / intracellular transport mechanism mediated by mitochondrial outer membrane surface proteins and homologous targeting, which can significantly improve the targeting effect and therapeutic efficacy of nanomedicine compared with simple reactive oxygen species responsive / scavenging nanomicelles TPTN. Attached Figure Description

[0024] Figure 1 Transmission electron microscopy, confocal images, particle size and surface potential maps of biomimetic nanomedicine (MTPTN) for the prevention and treatment of dilated cardiomyopathy;

[0025] Figure 2 The images show the in vitro fluorescence (A) and quantitative statistical graph (B) of the cardiac distribution of TPTN-Cy5 and MTPTN-Cy5 in dilated cardiomyopathy model mice 1 hour after tail vein injection.

[0026] Figure 3 The colocalization of TPTN-Cy5 and MTPTN-Cy5 with cardiomyocytes in the heart of mice with dilated cardiomyopathy after tail vein injection;

[0027] Figure 4 The images (A) and (B) show the fluorescence images and quantitative statistics of doxorubicin-induced uptake of TPTN-Cy5 and MTPTN-Cy5 by cardiomyocytes.

[0028] Figure 5The images show laser confocal microscopy (A) and quantitative statistical graph (B) of TPTN-Cy5 and MTPTN-Cy5 colocalizing with mitochondria in doxorubicin-induced cardiomyocytes.

[0029] Figure 6 The typical echocardiograms (A), left ventricular ejection fraction (LVEF) (B), left ventricular fractional shortening (LVFS) (C), end-diastolic left ventricular diameter (LVIDd) (D), and end-systolic left ventricular diameter (LVIDs) (E) of mice in each group after TPTN and MTPTN treatment.

[0030] Figure 7 The effects of TPTN and MTPTN treatment on serum cardiac troponin IcTnI (A) and brain natriuretic peptide BNP (B) in mice with dilated cardiomyopathy;

[0031] Figure 8 The antioxidant and anti-inflammatory effects of TPTN and MTPTN treatment on mice with dilated cardiomyopathy;

[0032] Figure 9 This refers to the protective effect of TPTN and MTPTN treatment on myocardial mitochondria in mice with dilated cardiomyopathy. Detailed Implementation

[0033] The invention will now be described in further detail with reference to specific embodiments. It should be understood that the embodiments of the present invention are for illustrative purposes only and are not intended to limit the invention. Various substitutions and modifications made based on ordinary technical knowledge and common practices in the art without departing from the technical concept of the present invention should be included within the scope of the present invention.

[0034] Example 1: Preparation of reactive oxygen species responsive / scavenging nanomicelles TPTN

[0035] 462 mg of cyanuric chloride (TCT) was dissolved in 20 mL of anhydrous dichloromethane, and 2 g of hydroxyl-terminated polyethylene glycol (PEG) and 0.5 mL of N,N-diisopropylethylamine (DIPEA) were added. The mixture was stirred at 0 °C for 24 h. After the reaction was completed, the product was separated and dried to obtain 1.5 g of white solid monosubstituted product (TCT-PEG).

[0036] The above-mentioned TCT-PEG and 120 mg of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxo radical (TP) were dissolved in 10 mL of anhydrous 1,4-dioxane, and 0.5 mL of DIPEA were added. The mixture was reacted at 40 °C for 12 h under nitrogen protection. After the reaction was completed, 230 mg of 4-hydroxymethylphenylboronic acid pinacol ester (PB) was added to the mixture, and the mixture was reacted at 80 °C for 12 h under nitrogen protection. After the reaction was completed, the mixture was separated and dried to obtain the amphiphilic polymer TPT with reactive oxygen species responsive / scavenging ability. This polymer dissolved in water self-assembled to form reactive oxygen species responsive / scavenging nanomicelles TPTN.

[0037] Example 2: Extraction of mouse myocardial mitochondria

[0038] After perfusion of the mouse heart, the heart was removed, washed with PBS to remove blood, weighed, and the heart tissue was minced. 10 volumes of pre-chilled PBS were added, and the tissue was incubated on ice for 3 min, centrifuged at 600g for 20 s, and the supernatant was discarded. 8 volumes of pre-chilled trypsin digestion solution were added, and the tissue was incubated on ice for 20 min, centrifuged at 600g for 20 s, and the supernatant was discarded. 2 volumes of pre-chilled mitochondrial separation reagent were added, and the tissue was resuspended to wash away residual trypsin. The tissue was centrifuged at 600g for 20 s, and the supernatant was discarded. 8 volumes of pre-chilled mitochondrial separation reagent were added, and the tissue was homogenized 20-30 times on ice. The tissue was centrifuged at 600g at 4℃ for 5 min, and the supernatant was collected. The supernatant was then centrifuged at 7000g at 4℃ for 10 min, and the precipitate was the isolated mitochondria. The mitochondrial protein concentration was determined using the BCA method.

[0039] Example 3: Extraction of the outer mitochondrial membrane of mouse myocardium

[0040] Ultrapure water was added to the above mitochondrial precipitate to adjust the concentration to 5 mg / mL. The mixture was stirred on ice for 20 min, then an equal volume of 1.4 M sucrose solution was added, and the mixture was stirred on ice for 5 min. The mixture was then homogenized on ice 30–40 times. The homogenate was centrifuged at 12000 g and 4 °C for 10 min. The supernatant was collected and centrifuged at 100000 g and 4 °C for 30 min; the precipitate was the mouse cardiac mitochondrial outer membrane (OMM). The protein content in the OMM was determined using the BCA method, and the sample was stored at -80 °C for further studies. Approximately 5.9 ± 0.76 mg of OMM was extracted from 100 mg of purified mitochondria.

[0041] Example 4: Preparation of mitochondrial outer membrane biomimetic nanomedicine MTPT

[0042] The OMM obtained above was sonicated at 42 kHz for 5 min, then extruded through a 400 nm polycarbonate membrane 10 times, and then extruded through a 200 nm polycarbonate membrane 10 times to obtain mitochondrial outer membrane vesicles (OMMVs). An equal volume and concentration of TPTN were added and mixed, sonicated at 42 kHz for 5 min, and then extruded through a 200 nm polycarbonate membrane 10 times to obtain the biomimetic nanomedicine MTPTN with a TPTN core and an outer mitochondrial outer membrane.

[0043] Example 5: Investigation of MTPTN uptake by cardiomyocytes

[0044] H9C2 cardiomyocytes were used at a rate of 2 × 10⁻⁶ per well. 5 Cells were seeded at a density in 12-well plates and incubated overnight. H9C2 cells were pretreated with 5 μM doxorubicin (DOX) for 24 h, then incubated with 50 μg / mL Cy5-labeled TPTN or MTPTN for 1 h. Cells were fixed with 4% paraformaldehyde, stained with DAPI, and observed under laser confocal microscopy.

[0045] Example 6: Detection of MTPTN co-localization with cardiomyocyte mitochondria

[0046] H9C2 cardiomyocytes were used at a rate of 2 × 10⁻⁶ per well. 5 Cells were seeded at a density in 12-well plates and incubated overnight. H9C2 cells were pretreated with 5 μM doxorubicin (DOX) for 24 h, then incubated for 1 h with 1 mL of medium containing 50 μg Cy5-labeled TPTN or MTPTN. The old medium was then discarded, and 200 nM MitoTracker Green was added and incubated at 37 °C for 30 min to label cell mitochondria. After washing with PBS, the cells were fixed with 4% paraformaldehyde, stained with DAPI, and observed under laser confocal microscopy.

[0047] Example 7: In vivo distribution assay of MTPTN in the heart of a mouse model of dilated cardiomyopathy

[0048] Male C57BL / 6 mice aged 8-12 weeks were used to establish a dilated cardiomyopathy (DCM) model by intraperitoneal injection of 5 mg / kg doxorubicin (DOX) once a week for three consecutive weeks (on days 0, 7, and 14). On day 15, DCM mice were injected via the tail vein with 5 mg / kg of Cy5-labeled TPTN or MTPTN. The mice were sacrificed 1 hour later, and their hearts were harvested and placed in an in vivo imaging system to measure the fluorescence intensity of the heart tissue.

[0049] Example 8: In vivo pharmacodynamics of MTPTN in the prevention and treatment of dilated cardiomyopathy

[0050] Male C57BL / 6 mice aged 8-12 weeks were used to establish a dilated cardiomyopathy (DCM) model by intraperitoneal injection of 5 mg / kg doxorubicin (DOX) once a week for three consecutive weeks (on days 0, 7, and 14). On the same day as the first DOX injection, mice in the treatment group were injected with 0.5 mg / kg TPTN or MTPTN via the tail vein, while mice in the model group were injected with an equal volume of physiological saline via the tail vein, every 4 days for 6 weeks. Echocardiography was performed on the day before sacrifice to assess cardiac function and ventricular diameters. Mice were sacrificed on day 42, and blood was collected from the eyeballs. The supernatant was used to detect serum levels of cardiac troponin I (cTnI) and brain natriuretic peptide (BNP). Heart tissue was collected, homogenized with pre-cooled PBS, and the supernatant was used to detect levels of oxidative stress markers (H2O2 and MDA) and inflammatory markers (TNF-α and IL-1β). A portion of the heart tissue was prepared and examined under a transmission electron microscope for mitochondria in the myocardium.

[0051] The biomimetic nanomedicines prepared above for the prevention and treatment of dilated cardiomyopathy are used through... Figures 1 to 9 Verification experiments were conducted:

[0052] Figure 1 MTPTN, a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy, was prepared using the above method. Its outer shell is the mitochondrial outer membrane, and its core is reactive oxygen species (ROS) responsive / scavenging nanomicelles (TPTN). TPTN uses cyanuric chloride (TCT) as a backbone, and is formed by the sequential attachment of terminal hydroxyl polyethylene glycol, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxo radical, and 4-hydroxymethylphenylboronic acid pinacol ester through affinity substitution reactions to form an amphiphilic polymer, which then self-assembles in water to form nanomicelles. Transmission electron microscopy (TEM) shows that MTPTN is a well-defined spherical shape with a uniform particle size distribution. Confocal microscopy results show that the TPTN is encapsulated within the OMM (oxygenated membrane), exhibiting a typical yolk-shell structure. Malvern particle size analyzer results show that the average particle size of MTPTN is 212±4 nm, and the surface potential is -12.6±0.2 mV.

[0053] Figure 2 These are in vivo imaging results of the cardiac distribution of Cy5-labeled TPTN and MTPTN in a mouse model of dilated cardiomyopathy after tail vein injection. The results show that both TPTN-Cy5 and MTPTN-Cy5 significantly accumulate in the heart of the mouse model of dilated cardiomyopathy. Notably, compared with TPTN-Cy5, MTPTN-Cy5 showed a significantly increased accumulation in the heart of the mouse model of dilated cardiomyopathy. This indicates that MTPTN has a stronger targeting effect on the diseased heart of mice with dilated cardiomyopathy.

[0054] Figure 3This study describes the colocalization of TPTN-Cy5 and MTPTN-Cy5 with cardiomyocytes in the hearts of mice with dilated cardiomyopathy after tail vein injection. The results showed that both TPTN-Cy5 and MTPTN-Cy5 exhibited significant colocalization with cardiomyocytes in the heart tissue, with MTPTN-Cy5 showing significantly higher colocalization than TPTN-Cy5, consistent with the aforementioned in vivo imaging results.

[0055] Figure 4 This study investigated the uptake of TPTN-Cy5 and MTPTN-Cy5 in doxorubicin-induced H9C2 cardiomyocytes. The results showed that the red fluorescence of the MTPTN-Cy5 group was significantly stronger than that of the TPTN-Cy5 group, indicating that MTPTN-Cy5 can be internalized by cardiomyocytes more effectively compared to TPTN-Cy5.

[0056] Figure 5 This study compared the mitochondrial targeting effects of TPTN and MTPTN in cardiomyocytes. The results showed that although both TPTN-Cy5 and MTPTN-Cy5 exhibited mitochondrial targeting ability in cardiomyocytes, fluorescence observation and quantitative analysis indicated that the co-localization efficiency of MTPTN-Cy5 with mitochondria was significantly higher than that of TPTN-Cy5.

[0057] Figures 2-5 The results collectively demonstrate that the mitochondrial biomimetic nanomedicine MTPTN, prepared by encapsulating TPTN in OMMV, exhibits stronger tertiary targeting capabilities against the heart, cardiomyocytes, and myocardial mitochondria than TPTN.

[0058] Figure 6 The efficacy of TPTN and MTPTN in treating dilated cardiomyopathy mice was evaluated using echocardiography. Results showed that both TPTN and MTPTN improved cardiac function and reduced ventricular remodeling in mice with dilated cardiomyopathy. Furthermore, MTPTN showed superior therapeutic effects compared to TPTN.

[0059] Figure 7 The results showed that both TPTN and MTPTN could reduce the levels of cTnI and BNP in the serum of mice with dilated cardiomyopathy, and MTPTN was more effective than TPTN, indicating that MTPTN was more effective in reducing myocardial damage and alleviating heart failure in mice with dilated cardiomyopathy than TPTN.

[0060] Figure 8 The results showed that both TPTN and MTPTN could significantly reduce the levels of cardiac oxidative stress-related markers H2O2, MDA, and inflammatory factors TNF-α and IL-1β in mice with dilated cardiomyopathy, and MTPTN showed better therapeutic effects.

[0061] Figure 9Transmission electron microscopy results of mouse myocardial tissue showed that both TPTN and MTPTN could alleviate mitochondrial damage in a mouse model of dilated cardiomyopathy, with MTPTN being more effective than TPTN. Figure 7-9 The results together demonstrate that TPTN and MTPTN can treat dilated cardiomyopathy in mice through antioxidant, anti-inflammatory, and mitochondrial damage reduction effects, with MTPTN showing significantly better efficacy than TPTN.

[0062] In summary, the biomimetic nanomedicine for preventing and treating dilated cardiomyopathy described in this invention enhances the tertiary targeting ability of TPTN to the heart, cardiomyocytes, and myocardial mitochondria through the outer mitochondrial membrane. Simultaneously, this biomimetic nanomedicine exerts its therapeutic effect through the synergistic action of scavenging local reactive oxygen species, anti-inflammation, and reducing mitochondrial damage, thereby preventing and treating dilated cardiomyopathy and cardiovascular diseases related to oxidative stress / inflammation or mitochondrial damage.

[0063] References

[0064] 1. Weintraub RG, Semsarian C and Macdonald P. Dilatedcardiomyopathy. Lancet. 2017; 390:400-414.

[0065] 2. Marrow BA, Cook SA, Prasad SK and McCann GP. Emerging Techniques for RiskStratification in Nonischemic Dilated Cardiomyopathy: JACC Review Topic of the Week. J AmColl Cardiol. 2020;75:1196-1207.

[0066] 3. Fatkin D, Huttner IG, Kovacic JC, Seidman JG and Seidman CE. PrecisionMedicine in the Management of Dilated Cardiomyopathy: JACC State-of-the-ArtReview. J Am Coll Cardiol. 2019;74:2921-2938.

[0067] 4.Li Q,Li C,Elnwasany A,Sharma G,An YA,Zhang G,Elhelaly WM,Lin J,GongY,Chen G,

[0068] Wang M,Zhao S,Dai C,Smart CD,Liu J,Luo X,Deng Y,Tan L,Lv SJ,DavidsonSM,Locasale JW,Lorenzi PL,Malloy CR,Gillette TG,Vander Heiden MG,Scherer PE,Szweda LI,Fu GPKM and Wang ZVM Exerts in Critical Cardiac Roles. RemodelingUnder PressureOverload in the Heart.Circulation.2021:144:712-727.

[0069] 5.Bayona-Bafaluy MP,Iglesias E,Lopez-Gallardo E,Emperador S,Pacheu-Grau D,Labarta L,Montoya J and Ruiz-Pesini E.Genetic aspects of the oxidativephosphorylation dysfunction indilated cardiomyopathy.Mutat Res Rev. MutatRes.2020:786:108334.

[0070] 6.Ji W,Tang X,Du W,Lu Y,Wang N,Wu Q,Wei W,Liu J,Yu H,Ma B,Li L andHuang W.

[0071] Optical / electrochemical methods for detecting mitochondrial energymetabolism.Chem SocRev.2022;51:71-127.

[0072] 7.Enomoto H,Mittal N,Inomata T,Arimura T,Izumi T,Kimura A,Fukuda Kand Makino S.

[0073] Dilated cardiomyopathy-linked heat shock protein family D member1mutations causeup-regulation of reactive oxygen species and autophagythrough mitochondrial dysfunction.

[0074] Cardiovasc Res.2021;117:1118-1131.

[0075] 8.Sharma S,Bhattarai S,Ara H,Sun G,St Clair DK,Bhuiyan MS,Kevil C,Watts MN,DominicP,Shimizu T,McCarthy KJ,Sun H,Panchatcharam M and MiriyalaS.SOD2 deficiency incardiomyocytes defines defective mitochondrialbioenergetics as a cause of lethal dilatedcardiomyopathy.Redox Biol.2020;37:101740.

[0076] 9.Hou T,Zhang R,Jian C,Ding W,Wang Y,Ling S,Ma Q,Hu X,Cheng H andWang X.

[0077] NDUFAB1 confers cardio-protection by enhancing mitochondrialbioenergetics throughcoordination of respiratory complex and supercomplexassembly.Cell Res.2019;29:754-766.10.Elliott EI,Miller AN,Banoth B,Iyer SS,Stotland A,Weiss JP,Gottlieb RA,Sutterwala FS andCassel SL.Cutting Edge:Mitochondrial Assembly of the NLRP3 Inflammasome Complex IsInitiated atPriming.J Immunol.2018;200:3047-3052.

[0078] 11.Wang Y,Liu X,Shi H,Yu Y,Yu Y,Li M and Chen R.NLRP3 inflammasome,animmune-inflammatory target in pathogenesis and treatment of cardiovasculardiseases.ClinTransl Med.2020;10:91-106.

[0079] 12.Ramaccini D,Montoya-Uribe V,Aan FJ,Modesti L,Potes Y,WieckowskiMR,Krga I,GlibeticM,Pinton P,Giorgi C and Matter ML.Mitochondrial Functionand Dysfunction in DilatedCardiomyopathy.Front Cell Dev Biol.2020;8:624216.

[0080] 13.Silva FS,Simoes RF,Couto R and Oliveira PJ.Targeting Mitochondriain CardiovascularDiseases.Curr Pharm Des.2016;22:5698-5717.

[0081] 14.Murphy MP and Hartley RC.Mitochondria as a therapeutic target forcommon pathologies.NatRev Drug Discov.2018;17:865-886.

[0082] 15.Lozano O,Torres-Quintanilla A and Garcia-Rivas G.Nanomedicine forthe cardiac myocyte:

[0083] Where are we?J Control Release.2018;271:149-165.

[0084] 16.Sahoo S,Kariya T and Ishikawa K.Targeted delivery of therapeuticagents to the heart.NatureReviews Cardiology.2021;18:389-399.

[0085] 17.Shin M,Lee HA,Lee M,Shin Y,Song JJ,Kang SW,Nam DH,Jeon EJ,Cho M,DoM,Park S,Lee MS,Jang JH,Cho SW,Kim KS and Lee H.Targeting protein and peptidetherapeutics tothe heart via tannic acid modification.Nat Biomed Eng.2018;2:304-317.

[0086] 18.Zhao X,Luo W,Hu J,Zuo L,Wang J,Hu R,Wang B,Xu L,Li J,Wu M,Li P andLiu L.

[0087] Cardiomyocyte-targeted and 17beta-estradiol-loaded acousticnanoprobes as a theranosticplatform for cardiac hypertrophy.JNanobiotechnology.2018;16:36.

[0088] 19.Hu S,Wang X,Li Z,Zhu D,Cores J,Wang Z,Li J,Mei X,Cheng X,Su T andCheng K.

[0089] Platelet membrane and stem cell exosome hybrids enhance cellularuptake and targeting toheart injury.Nano Today.2021;39。

Claims

1. A method for preparing a biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy, characterized in that: Using reactive oxygen species-responsive / scavenging nanomicelles TPTN as the core and encapsulating TPTN with the outer mitochondrial outer membrane OMM as the shell, a biomimetic nanomedicine encapsulated in the outer mitochondrial outer membrane is prepared, specifically including the following steps: S1: Extraction of mitochondrial outer membrane: Extraction of mitochondrial outer membrane from animal heart tissue; S2: Synthesis of reactive oxygen species responsive / scavenging nanomicelles TPTN: Using cyanuric chloride as the backbone, hydroxyl-terminated polyethylene glycol, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxo radical and 4-hydroxymethylphenylboronic acid pinacol ester were gradually added to carry out an affinity substitution reaction in an ultra-dry organic solvent to synthesize an amphiphilic polymer TPT with reactive oxygen species responsive / scavenging ability. The TPT was then self-assembled in an aqueous solution to form reactive oxygen species responsive / scavenging nanomicelles TPTN. S3: Preparation of biomimetic nanomedicine MTPTN: The mitochondrial outer membrane extracted in S1 was mixed with the TPTN nanomicelles prepared in S2, and after sonication, they were co-extruded into a polycarbonate membrane to obtain MTPTN.

2. The method for preparing a biomimetic nanomedicine for preventing and treating dilated cardiomyopathy according to claim 1, characterized in that: Step S1, which involves extracting the outer mitochondrial membrane, includes the following steps: collecting mouse hearts, washing with PBS to remove blood, cutting, digesting, centrifuging, adding pre-cooled mitochondrial separation reagent, homogenizing, and collecting mitochondria after gradient centrifugation; adding ultrapure water to the collected mitochondria and stirring, adding sucrose solution, homogenizing, and collecting the outer mitochondrial membrane after gradient centrifugation.

3. The method for preparing a biomimetic nanomedicine for preventing and treating dilated cardiomyopathy according to claim 1, characterized in that: The average molecular weight of the hydroxyl-terminated polyethylene glycol in step S2 is 2000 Da.

4. The method for preparing a biomimetic nanomedicine for preventing and treating dilated cardiomyopathy according to claim 1, characterized in that: The molar ratio of cyanuric chloride to hydroxyl-terminated polyethylene glycol in step S2 is 1:

1.

5. The method for preparing a biomimetic nanomedicine for preventing and treating dilated cardiomyopathy according to claim 1, characterized in that: The molar ratio of cyanuric chloride, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxy radical, and pinacol 4-hydroxymethylphenylboronic acid in step S2 is 1:1:

1.

6. The method for preparing a biomimetic nanomedicine for preventing and treating dilated cardiomyopathy according to claim 1, characterized in that: In step S3, the mass ratio of the mitochondrial outer membrane to TPTN nanomicelles is 1:

1.

7. A biomimetic nanomedicine for the prevention and treatment of dilated cardiomyopathy, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 6.

8. The use of the biomimetic nanomedicine for preventing and treating dilated cardiomyopathy as described in claim 7 in the preparation of drugs for the prevention and treatment of dilated cardiomyopathy.

9. The application according to claim 8, characterized in that: The administration methods include oral, intravenous, subcutaneous, and intramuscular injection, as well as any combination of the above methods.

Images