Coenzyme i-glycyrrhizin supermolecular nanodrug for treating myocardial infarction, and preparation method and application thereof

Abstract

The present application relates to a kind of coenzyme I-gardenoside element supramolecular nanomedicine for treating myocardial infarction and its preparation method and application, belong to nanomedicine and cardiovascular disease treatment technical field.Solve the problem that there is no myocardial infarction treatment drug that can simultaneously realize acute phase mitochondrial protection and chronic period metabolic remodeling regulation in prior art.The nanomedicine of the present application is formed by coenzyme I-gardenoside element skeleton and free coenzyme I.The nanomedicine of the present application is supramolecular nanomedicine with lesion enrichment, acid response phase release and mitochondrial related sustained delivery capacity, and has important application in the space-time specific regulation myocardial infarction after metabolic reprogramming, reduce ventricular remodeling and delay the progress of heart failure.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine and cardiovascular disease treatment technology, specifically relating to a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction, its preparation method, and its application. Background Technology

[0002] Myocardial infarction (MI) is one of the leading cardiovascular diseases causing death and disability worldwide. It carries a high risk of acute death, and its long-term complications severely impact patients' quality of life and prognosis. Although treatment strategies such as reperfusion therapy, beta-blockers, and implantable cardioverter-defibrillators have significantly improved acute-phase survival rates in recent years, they still cannot effectively prevent the chronic progression of MI to heart failure (HF). Numerous studies have shown that more than half of patients die within five years of MI due to continued deterioration of cardiac function. Therefore, how to inhibit ventricular remodeling and maintain long-term metabolic homeostasis is a crucial scientific question in the field of cardiovascular treatment.

[0003] Traditionally, it has been believed that the primary pathological event following myocardial infarction stems from the massive necrosis of cardiomyocytes caused by acute ischemia. However, recent studies have revealed that the ongoing metabolic reprogramming of surviving cardiomyocytes after infarction is also a significant factor driving disease progression. Normal adult myocardium primarily relies on fatty acid oxidation (FAO) for energy, but under ischemic and hypoxic conditions, cardiomyocytes gradually shift to glycolysis to meet short-term energy needs. This metabolic shift offers some protection in the acute phase, but long-term, sustained glycolysis dependence leads to lactic acid buildup, intracellular acidosis, decreased mitochondrial oxidative phosphorylation, and insufficient ATP production, further inducing lipotoxicity, oxidative stress, inflammatory activation, and fibrosis.

[0004] At the mitochondrial level, decreased oxidative phosphorylation capacity, electron transport chain disruption, and membrane potential disturbances following myocardial infarction can trigger the generation of large amounts of reactive oxygen species (ROS). Excessive ROS not only leads to mitochondrial deoxyribonucleic acid (DNA) damage and membrane lipid oxidation, but can also induce the opening of mitochondrial permeability transition pores, promote cytochrome c release, and activate endogenous apoptosis pathways. Furthermore, mitochondrial DNA (mtDNA) leakage can continuously activate chronic inflammatory responses through innate immune signaling pathways, further promoting ventricular fibrosis and remodeling.

[0005] In recent years, uncoupling protein 2 (UCP2) has received widespread attention due to its involvement in mitochondrial membrane potential regulation, oxidative stress control, and metabolic regulation. Some studies suggest that UCP2 can reduce ROS load in the acute phase, thereby alleviating ischemic injury; however, other studies have found that persistently high expression of UCP2 leads to decreased mitochondrial oxidative phosphorylation efficiency and insufficient adenosine triphosphate (ATP), which is closely related to hypoxia-inducible factor 1α (HIF-1α)-mediated pathological glycolytic reprogramming. Therefore, the role of UCP2 in different time windows exhibits a clear phased effect.

[0006] On the other hand, coenzyme I is also known as nicotinamide adenine dinucleotide (NAD) + Coenzyme I (CoI) plays a crucial role in myocardial energy metabolism, serving as an essential coenzyme for maintaining cellular redox homeostasis and mitochondrial function. Studies have shown that CoI depletion is a significant metabolic imbalance following myocardial infarction, and supplementation with CoI or its precursors (such as nicotinamide mononucleotide and nicotinamide ribose) can improve mitochondrial function and alleviate oxidative stress. However, free CoI suffers from poor membrane permeability, low tissue selectivity, and a short in vivo half-life, hindering efficient delivery to lesion sites. Meanwhile, geniposide, one of the few known UCP2 inhibitors, has the potential to regulate glycolysis and mitochondrial metabolism, but its high chemical activity, poor in vivo stability, and lack of lesion accumulation limit its further application.

[0007] In summary, current treatment strategies primarily focus on controlling acute-phase damage, such as simple antioxidant or anti-inflammatory treatments, or promoting blood flow restoration. However, a treatment system that can simultaneously address both acute-phase mitochondrial protection and subsequent chronic metabolic remodeling regulation is lacking. Especially after myocardial infarction, treatment needs differ significantly at different stages: the acute phase requires rapid restoration of redox homeostasis and inhibition of mitochondrial damage, while the chronic remodeling phase requires restoring mitochondrial metabolic flexibility and rebuilding glucose and lipid metabolism balance. Therefore, developing novel nanomedicines capable of lesion enrichment, phased release, and sustained mitochondrial-related metabolic regulation is of great significance for improving the long-term prognosis of myocardial infarction. Summary of the Invention

[0008] The technical problem this invention aims to solve is the lack of existing drugs for treating myocardial infarction that can simultaneously achieve mitochondrial protection in the acute phase and regulation of metabolic remodeling in the chronic phase. This invention provides a supramolecular nanomedicine of coenzyme I-geniposide, characterized by lesion accumulation and spatiotemporally phased release, for the treatment of myocardial infarction, along with its preparation method and applications. This nanomedicine, possessing lesion accumulation, acid-responsive phased release, and mitochondrial-related sustained delivery capabilities, has important applications in spatiotemporally specific regulation of metabolic reprogramming after myocardial infarction, mitigating ventricular remodeling, and delaying the progression of heart failure.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A supramolecular nanomedicine of coenzyme I-geniposide for the treatment of myocardial infarction, wherein the nanomedicine is assembled from the coenzyme I-geniposide backbone and free coenzyme I; coenzyme I is nicotinamide adenine dinucleotide.

[0010] Preferably, the coenzyme I-geniposide backbone is formed by the Schiff base reaction between coenzyme I and geniposide; the free coenzyme I is assembled with the coenzyme I-geniposide backbone through hydrogen bond π-π stacking and intermolecular interactions to form a supramolecular nanostructure.

[0011] The nanomedicine is a spherical or near-spherical nanoparticle.

[0012] More preferably, the nanoparticle size is 140–170 nm.

[0013] Preferably, the nanomedicine exhibits a negatively charged surface at pH 7.4 and undergoes charge reversal under acidic conditions.

[0014] Preferably, the nanomedicine can achieve biphasic release of coenzyme I under pH 5.0 conditions.

[0015] A method for preparing a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction includes the following steps: Coenzyme I was dissolved in PBS buffer to obtain a coenzyme I solution; Gardenoside was dissolved in anhydrous ethanol to obtain an anhydrous ethanol solution containing gardenoside. Add the coenzyme I solution to the anhydrous ethanol solution containing geniposide, stir, and allow coenzyme I to undergo a Schiff base reaction with geniposide to form the coenzyme I-geniposide backbone precursor. Subsequently, the coenzyme I-geniposide backbone precursor and free coenzyme I were co-assembled in PBS buffer, and after dialysis and freeze-drying, a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction was obtained.

[0016] Preferably, the stirring is carried out at 42°C for 24 hours.

[0017] Application of a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction in the preparation of drugs for the treatment of myocardial infarction.

[0018] Furthermore, the coenzyme I-geniposide supramolecular nanomedicine for treating myocardial infarction is used in the preparation of drugs to improve ventricular remodeling and the progression of heart failure after myocardial infarction.

[0019] The beneficial effects of this invention are: The coenzyme I-geniposide supramolecular nanomedicine of the present invention is a bioadaptive nanomedicine with the ability to enrich lesions and release them in stages. It can not only control acute ischemic injury, but also further regulate chronic metabolic remodeling, thereby achieving staged intervention in the entire process of myocardial infarction.

[0020] This invention constructs nanomedicines through a supramolecular co-assembly strategy, avoiding the complex encapsulation process of traditional nanocarriers and improving the compositional uniformity and structural stability of nanomedicines.

[0021] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can rapidly accumulate in the myocardial infarction area and remain locally for a long time, and its fluorescence signal can persist in the infarction area for more than 3 days.

[0022] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can reverse the surface charge in response to the acidification environment of the endosome, thereby promoting the escape of the endosome and achieving effective intracellular delivery.

[0023] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can achieve a "burst + sustained" biphasic release of coenzyme I under acidic conditions, wherein about 50% of coenzyme I is rapidly released in the first 2 hours, followed by sustained release for more than 72 hours.

[0024] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can continuously release geniposide in subsequent stages, thereby inhibiting UCP2-related pathological glycolysis and improving mitochondrial metabolism.

[0025] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can significantly reduce ROS levels, restore the coenzyme I / reduced coenzyme I balance and improve mitochondrial membrane potential.

[0026] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can significantly reduce cardiomyocyte apoptosis and mtDNA leakage, and inhibit inflammatory activation.

[0027] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can improve fatty acid oxidation and oxidative phosphorylation capacity and restore the coordination of glucose and lipid metabolism.

[0028] The coenzyme I-geniposide supramolecular nanomedicine of the present invention can significantly reduce myocardial fibrosis, decrease ventricular dilation, and improve ejection fraction.

[0029] The coenzyme I-geniposide supramolecular nanomedicine of the present invention has good biocompatibility and safety, and no obvious organ toxicity was observed after multiple administrations. Attached Figure Description

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0031] Figure 1 This is a schematic diagram illustrating the synthesis process and structure of coenzyme I-geniposide supramolecular nanomedicine.

[0032] Figure 2 Transmission electron microscope image and particle size distribution of coenzyme I-geniposide supramolecular nanomedicine element.

[0033] Figure 3 This is a molecular weight distribution diagram of geniposide C in the coenzyme I-geniposide supramolecular nanomedicine.

[0034] Figure 4 Solid-state supramolecular nanomedicine for coenzyme I-geniposide 13 C10 NMR spectrum.

[0035] Figure 5 Infrared spectra of coenzyme I, geniposide, and coenzyme I-geniposide supramolecular nanomedicines.

[0036] Figure 6 XPS elemental analysis diagram of coenzyme I-geniposide supramolecular nanomedicine.

[0037] Figure 7 The ultraviolet spectra of coenzyme I, geniposide, and coenzyme I-geniposide supramolecular nanomedicines are shown.

[0038] Figure 8 This is a graph showing the release curves of coenzyme I from the supramolecular nanomedicine of coenzyme I-geniposide under different pH conditions.

[0039] Figure 9 This is a graph showing the release curves of geniposide from the coenzyme I-geniposide supramolecular nanomedicine under different pH conditions.

[0040] Figure 10 This is a graph evaluating the scavenging capabilities of coenzyme I-geniposide supramolecular nanomedicine against superoxide anions, hydroxyl radicals, and hydrogen peroxide.

[0041] Figure 11 This image shows the distribution and myocardial enrichment of coenzyme I-geniposide supramolecular nanomedicine in mice.

[0042] Figure 12 This is a diagram showing the long-term retention of coenzyme I-geniposide supramolecular nanomedicine in infarcted myocardium.

[0043] Figure 13 Map showing how coenzyme I-geniposide supramolecular nanomedicine promotes endosome escape and mitochondrial localization.

[0044] Figure 14 This is a graph showing the binding ability of coenzyme I-geniposide supramolecular nanomedicine to mitochondrial outer membrane proteins.

[0045] Figure 15 This is a schematic diagram of the molecular dynamics simulation and co-assembly of coenzyme I-geniposide supramolecular nanomedicine.

[0046] Figure 16 This image shows the effect of coenzyme I-geniposide supramolecular nanomedicine on improving cardiac function in mice with myocardial infarction. (AB) show the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) at 1, 3, 7, 14, 21, and 28 days post-surgery, respectively; (CD) show the quantitative analysis of LVFS and LVEF at day 28 post-surgery, respectively; and (E) shows representative B-mode and M-mode echocardiograms at day 28 post-surgery, respectively.

[0047] Figure 17 This diagram illustrates the therapeutic effect of coenzyme I-geniposide supramolecular nanomedicine on myocardial ischemia / reperfusion injury.

[0048] Figure 18 The image shows the results of coenzyme I-geniposide supramolecular nanomedicine in reducing myocardial fibrosis.

[0049] Figure 19 Ultrastructure diagram of mitochondria after myocardial infarction, as a supramolecular nanomedicine for coenzyme I-geniposide.

[0050] Figure 20 The diagram shows the results of balanced glycolysis and oxidative phosphorylation of the coenzyme I-geniposide supramolecular nanomedicine.

[0051] Figure 21 The figure shows the results of coenzyme I-geniposide supramolecular nanomedicine regulating fatty acid and glucose substrate preferences.

[0052] Figure 22Figure showing the results of improving coenzyme I / reduced coenzyme I homeostasis using coenzyme I-geniposide supramolecular nanomedicine.

[0053] Figure 23 This diagram illustrates the inhibitory effects of coenzyme I-geniposide supramolecular nanomedicine on ROS and mitochondrial ROS. (A) shows the ROS level detected by DHE fluorescence staining in myocardial tissue; (B) shows the mitochondrial ROS level detected by MitoSOX fluorescence staining in myocardial tissue; (C) shows the intracellular ROS level detected by DCFH-DA fluorescence staining in each group of myocardial cells; (D) shows the fluorescence intensity of DCFH-DA quantitatively analyzed by flow cytometry; (E) shows the mitochondrial ROS level detected by MitoSOX fluorescence staining in each group of myocardial cells; and (F) shows the fluorescence intensity of MitoSOX quantitatively analyzed by flow cytometry.

[0054] Figure 24 The image shows the results of coenzyme I-geniposide supramolecular nanomedicine inhibiting cell apoptosis.

[0055] Figure 25 This diagram illustrates the regulatory role of coenzyme I-geniposide supramolecular nanomedicine on the SIRT1-PGC-1α-PPARα pathway. Specifically, (A–J) represents Western blot analysis of UCP2, SIRT1, PGC-1α, and PPARα protein expression and grayscale quantitative analysis in myocardial tissues (A, C–F) and cardiomyocytes (B, G–J); (K–N) represents immunoprecipitation (IP) analysis of PGC-1α acetylation levels in myocardial tissues (K, M) and cardiomyocytes (L, N); and (O–X) represents Western blot analysis of fatty acid uptake and β-oxidation-related proteins CD36, CPT1A, ACADL, and ACADM in myocardial tissues (O, P–S) and cardiomyocytes (T, U–X), along with their respective grayscale quantitative analysis.

[0056] Figure 26 This is a graph showing the detection of mRNA expression levels of glycolysis-related genes. (A–E) represent the results of qRT-PCR detection of glucose transport and glycolysis-related genes in myocardial tissue, respectively. Slc2a1, Hk2, Ldha, Pdk4 and Pdk mRNA expression level of 1; (F–J) represent the mRNA expression levels of cardiomyocytes detected by qRT-PCR. Slc2a1, Hk2, Ldha, Pdk4 and Pdk1 The mRNA expression level diagram.

[0057] Figure 27This is a graph showing the expression levels of glycolysis-related proteins. (A–E) are Western blot graphs of the expression and gray-scale quantitative analysis of glucose transport and glycolysis-related proteins GLUT1, HKII, LDHA, and PDK4 in myocardial tissue; (F–J) are Western blot graphs of the expression and gray-scale quantitative analysis of GLUT1, HKII, LDHA, and PDK4 in cardiomyocytes.

[0058] Figure 28 To verify that coenzyme I-geniposide is transmitted via NAD+ + – Phenotypic replication experiment diagram of the mechanism of UCP2 in treating myocardial infarction. (A) shows the regulation of NAD by coenzyme I-geniposide. + – A schematic diagram illustrating the mechanism of action of UCP2 in treating cardiomyocyte metabolic reprogramming and cell damage; (B–K) are Western blot analysis of the expression of UCP2, cGAS, STING, Bax, Bcl-2, LDHA, ATP5A, SIRT1 and ACADL proteins in each group, and their respective gray-scale quantitative analysis; (L) is an immunofluorescence staining image of HIF-1α expression (red) and F-actin (green) in each group, with DAPI (blue) staining of cell nuclei; (M) is an immunofluorescence staining image of JC-1 (aggregates red, monomers green) to detect mitochondrial function, with Hoechst (blue) staining of cell nuclei; (N) is a PI / Calcein AM double staining image of cell viability in each group.

[0059] Figure 29 To verify that coenzyme I-geniposide is transmitted via NAD+ + –Salvage experiment on the mechanism of UCP2 in treating myocardial infarction. (A–J) show the expression of UCP2, cGAS, STING, Bax, Bcl-2, LDHA, ATP5A, SIRT1, and ACADL proteins in each group of cells, along with their respective grayscale quantitative analysis. (K) shows the expression of HIF-1α (red) and F-actin (green) in each group of cells detected by immunofluorescence staining, with cell nuclei stained using DAPI (blue). (L) shows the mitochondrial function detected by JC-1 (aggregates red, monomers green) fluorescence staining, with cell nuclei stained using Hoechst (blue). (M) shows the cell viability detection using PI / Calcein AM double staining.

[0060] Figure 30 The figure shows the effects of coenzyme I-geniposide supramolecular nanomedicine on HIF-1α / HRE nuclear translocation (A) and transcriptional activity (B).

[0061] Figure 31This image shows the results of the coenzyme I-geniposide supramolecular nanomedicine inhibiting inflammation activation.

[0062] Figure 32 This is a graph showing the safety evaluation results of coenzyme I-geniposide supramolecular nanomedicine. Detailed Implementation

[0063] The inventive concept of this invention is as follows: This invention addresses the continuous pathological processes following myocardial infarction, including acute oxidative stress, mitochondrial damage, energy metabolism disorders, amplified inflammation, and chronic ventricular remodeling. It proposes a staged therapeutic strategy based on coenzyme I-geniposide supramolecular nanomedicine. This invention does not simply mix coenzyme I with geniposide; instead, it constructs supramolecular nanostructures with acid-responsive release, lesion enrichment, and mitochondrial localization capabilities through Schiff base structures, hydrogen bonds, and π-π stacking interactions, achieving a transformation from molecular structure design to pharmacological function. In the acute phase of myocardial infarction, coenzyme I-geniposide supramolecular nanomedicine can rapidly accumulate in ischemic myocardial regions, scavenging various reactive oxygen species, replenishing coenzyme I, restoring redox homeostasis, protecting mitochondrial function, and inhibiting cardiomyocyte apoptosis. In the subacute and chronic phases, it can further regulate SIRT1-PGC-1α-PPARα, UCP2 / HIF-1α, and inflammation-related pathways, restoring oxidative phosphorylation and fatty acid oxidation, reducing abnormal glycolysis, lactic acid accumulation, and inflammatory responses, thereby alleviating fibrosis and ventricular remodeling. This invention enables continuous and systematic intervention in acute injury and chronic remodeling of myocardial infarction, and combines structural innovation, mechanistic innovation and treatment mode innovation, and has good prospects for preclinical translation.

[0064] A supramolecular nanomedicine of coenzyme I-geniposide for the treatment of myocardial infarction, wherein the nanomedicine is assembled from the coenzyme I-geniposide backbone and free coenzyme I; coenzyme I is nicotinamide adenine dinucleotide.

[0065] In this invention, the coenzyme I-geniposide backbone is formed by the Schiff base reaction between coenzyme I and geniposide; free coenzyme I is assembled with the coenzyme I-geniposide backbone through hydrogen bonding and π-π stacking to form a supramolecular nanostructure.

[0066] In this invention, the nanomedicine is a spherical or near-spherical nanoparticle with a particle size of 140–170 nm.

[0067] In this invention, the nanomedicine exhibits a negatively charged surface at pH 7.4 and undergoes charge reversal under acidic conditions.

[0068] In this invention, the nanomedicine is capable of acid-responsive phase release, such as biphasic release of coenzyme I at pH 5.0. The biphasic release includes an early burst release and a subsequent sustained release, that is, the acid-responsive phase release includes an early rapid release of coenzyme I and a subsequent sustained release of the coenzyme I-geniposide backbone.

[0069] In this invention, the nanomedicine has the ability to accumulate in ischemic myocardial lesions and the ability to persistently locate mitochondria-related lesions.

[0070] In this invention, the nanomedicine is able to interact with classic mitochondrial outer membrane proteins.

[0071] In this invention, the nanomedicine can be used to regulate metabolic reprogramming after myocardial infarction, which includes enhanced glycolysis, decreased fatty acid oxidation, lactic acid accumulation, and impaired oxidative phosphorylation.

[0072] In this invention, the nanomedicine can regulate the homeostasis of coenzyme I / reduced coenzyme I, oxidative phosphorylation function, and the balance of glycolysis and fatty acid oxidation metabolism.

[0073] In this invention, the nanomedicine can coordinate the regulation of the SIRT1-PGC-1α-PPARα signaling pathway and the UCP2 / HIF-1α signaling pathway.

[0074] A method for preparing a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction includes the following steps: Coenzyme I was dissolved in PBS buffer to obtain a coenzyme I solution; Gardenoside was dissolved in anhydrous ethanol to obtain an anhydrous ethanol solution containing gardenoside. Add the coenzyme I solution to an anhydrous ethanol solution containing geniposide and stir for 24 hours to allow coenzyme I to undergo a Schiff base reaction with geniposide to form the coenzyme I-geniposide backbone precursor. Subsequently, the coenzyme I-geniposide backbone precursor and free coenzyme I were co-assembled in PBS buffer, and after dialysis and freeze-drying, a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction was obtained.

[0075] Application of a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction in the preparation of drugs for the treatment of myocardial infarction.

[0076] Furthermore, the preparation of coenzyme I-geniposide supramolecular nanomedicines for the treatment of myocardial infarction enables the synergistic application of drugs for the treatment of myocardial infarction by restoring redox homeostasis in the acute phase and regulating metabolic remodeling in the chronic phase.

[0077] Furthermore, the application of the coenzyme I-geniposide supramolecular nanomedicine for treating myocardial infarction in the preparation of drugs to improve ventricular remodeling and heart failure progression after myocardial infarction.

[0078] In this invention, preferably, the nanomedicine is administered via intravenous injection at a dose of 1–10 mg / kg, and more preferably, the dose of the nanomedicine is 5 mg / kg.

[0079] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments and will not limit the scope of protection of the present invention.

[0080] Example 1: Synthesis of Coenzyme I-Gardinoside Supramolecular Nanomedicine Synthesis process as follows Figure 1 As shown, 13.269 g of coenzyme I was accurately weighed and dissolved thoroughly in 70 mL of PBS buffer (pH 6.0, concentration 50 mM) to obtain a coenzyme I solution. The obtained coenzyme I solution was placed in a 4°C automated dispensing system for later use. 2.700 g of geniposide was accurately weighed and dissolved thoroughly in 30 mL of anhydrous ethanol to obtain an anhydrous ethanol solution containing geniposide. This solution was transferred to a round-bottom flask. A magnetic rotor was added to the round-bottom flask, and the flask was placed in a 42°C water bath. The automated dispensing system was started, and the coenzyme I solution was added dropwise to the anhydrous ethanol solution containing geniposide at a constant rate, while the mixture was slowly stirred for 24 hours to allow the coenzyme I to undergo a Schiff base reaction with geniposide, forming the coenzyme I-geniposide backbone precursor.

[0081] After the reaction was complete, anhydrous ethanol was added to the reaction system to fully disperse the unreacted raw materials and products. The mixture was then centrifuged at 13,000 rpm, and the supernatant was discarded. The resulting precipitate was resuspended in 30% ethanol solution, centrifuged, and washed repeatedly eight times to remove unreacted coenzyme I, geniposide, and small molecule impurities. After washing, the precipitate was freeze-dried to obtain coenzyme I-geniposide backbone precursor powder. Next, 0.766 g of the coenzyme I-geniposide backbone precursor and 0.500 g of coenzyme I (i.e., free coenzyme I) were weighed and added to a round-bottom flask. 25 mL of PBS buffer (pH 7.4, 50 mM) was added. The mixture was slowly stirred at 37°C in the dark for 12 hours, allowing the coenzyme I-geniposide backbone precursor and free coenzyme I to assemble into a supramolecular nanostructure through hydrogen bonding, π-π stacking interactions, and intermolecular interactions. After the reaction, the resulting solution was placed in a dialysis bag and dialyzed with ultrapure water at 4°C for 12 hours to remove free small molecules and salts. After dialysis, the solution was freeze-dried to obtain coenzyme I-geniposide supramolecular nanoparticles, denoted as NGB. The obtained powder was stored at -20°C protected from light. In this example, the yield of the freeze-dried coenzyme I-geniposide backbone precursor was approximately 46.3 ± 1.9%; after further assembly, the recovery rate of the coenzyme I-geniposide supramolecular nanoparticles was approximately 76.6 ± 3.2%.

[0082] The following describes the structural and performance characteristics of the coenzyme I-geniposide supramolecular nanomedicine prepared in the examples. The characterization results are shown in the accompanying drawings of the specification. In the drawings, coenzyme I is represented using NAD+. + The terms "geinpin" or "GP" are used to represent geniposide, and "NGB" is used for coenzyme I-geniposide supramolecular nanomedicines. "Sham" represents the sham-operated group, and "MI" represents the model group.

[0083] Example 2: Morphology, particle size and molecular weight characterization of coenzyme I-geniposide supramolecular nanomedicine The coenzyme I-geniposide supramolecular nanoparticle powder obtained in Example 1 was added to ultrapure water to prepare a dispersion of a suitable concentration (24 μg / mL). After ultrasonic dispersion, it was dropped onto the surface of a copper mesh, allowed to stand for adsorption, and then excess liquid was removed and dried. Its morphology was observed using a transmission electron microscope.

[0084] like Figure 2As shown, the coenzyme I-geniposide supramolecular nanomedicine exhibits a uniform, near-spherical nanostructure with a particle size of approximately 140–160 nm, indicating its ability to form stable nanoscale supramolecular aggregates in an aqueous system. Further analysis of the aqueous dispersion of the coenzyme I-geniposide supramolecular nanomedicine was conducted, and its hydrated particle size and Zeta potential were measured using dynamic light scattering. Before testing, the sample was filtered through a 0.22 μm filter or centrifuged at low speed to remove large particulate impurities. The results showed that the peak hydrated particle size of the coenzyme I-geniposide supramolecular nanomedicine was approximately 169.5 nm, with a narrow particle size distribution, indicating good dispersibility in aqueous solution.

[0085] The molecular weight distribution of coenzyme I-geniposide supramolecular nanomedicines was analyzed by gel permeation chromatography. The sample was dissolved in the mobile phase, filtered, and then injected for analysis. The results showed a single elution peak and a polydispersity index of approximately 1.086, indicating that the obtained coenzyme I-geniposide supramolecular nanomedicines had a homogeneous composition and did not form obvious multi-peak impurities or large, heterogeneous aggregates. Figure 3 ).

[0086] Example 3: Chemical structural characterization of coenzyme I-geniposide supramolecular nanomedicine The coenzyme I-geniposide supramolecular nanoparticle powder obtained in Example 1 was subjected to solid-state processing. 13 Structural analysis was performed using C NMR, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In solid-state... 13 In C NMR detection, dried coenzyme I-geniposide supramolecular nanoparticle powder is placed in an NMR sample tube to detect changes in the chemical shift of carbon atoms. Figure 4 The results showed that, compared with coenzyme I alone and geniposide alone, a new characteristic chemical shift signal appeared in coenzyme I-geniposide, suggesting that a new chemical linkage structure was formed between coenzyme I and geniposide. In Fourier transform infrared spectroscopy, the sample powder was thoroughly dried before scanning, and data were recorded from 4000 to 4000 cm⁻¹. -1 Absorption peaks within the range. Figure 5 The results showed that characteristic absorption peaks related to the Schiff base structure appeared in coenzyme I-geniposide, while some characteristic peaks in the raw material shifted or changed in intensity, indicating that a reaction occurred between coenzyme I and geniposide.

[0087] In X-ray photoelectron spectroscopy, dry powder is fixed on the surface of the sample stage, and the changes in the binding energy of elements such as C, N, O, and P are detected. Figure 6 The results showed that the chemical environment of nitrogen and carbon elements in coenzyme I-geniposide was altered, further proving that coenzyme I and geniposide successfully formed a Schiff base structure.

[0088] Example 4: Detection of UV absorption characteristics of coenzyme I-geniposide supramolecular nanomedicine Take coenzyme I (NAD) + Geniposide, geinpin, and coenzyme I-geniposide supramolecular nanomedicine (NGB) were dissolved in PBS buffer or ultrapure water to prepare test solutions with the same mass concentration (24 μg / mL). Absorption spectra were scanned in the wavelength range of 200–800 nm using a UV-Vis spectrophotometer.

[0089] Figure 7 The results showed that, compared with coenzyme I and geniposide, coenzyme I-geniposide exhibited a new absorption peak or absorption peak shift at a specific wavelength, indicating that coenzyme I and geniposide underwent an intermolecular reaction and assembly. This ultraviolet absorption characteristic can serve as an auxiliary criterion for determining the formation of coenzyme I-geniposide.

[0090] Example 5: Acid-responsive release behavior of coenzyme I-geniposide supramolecular nanomedicine A certain amount of coenzyme I-geniposide supramolecular nanoparticle powder was weighed and added to both pH 7.4 and pH 5.0 PBS buffers to prepare sample solutions of the same concentration (24 μg / mL). The pH 7.4 condition was used to simulate the physiological environment, and the pH 5.0 condition was used to simulate the acidic environment of endosomes or lysosomes. The samples were incubated at 37℃ in the dark, and samples were taken at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours. After each sampling, the released free coenzyme I and geniposide were separated by centrifugation or ultrafiltration, and their content was detected by HPLC-MS.

[0091] Figures 8-9 The results showed that at pH 5.0, coenzyme I was rapidly released at approximately 50.21% within 0–2 hours, followed by a gradual release of approximately 77.13% over 72 hours, exhibiting a biphasic release pattern of "burst release + sustained release." Geniposide was released cumulatively at approximately 75.42% over 72 hours, showing a sustained release pattern. In contrast, at pH 7.4, the release of both coenzyme I and geniposide was significantly reduced, indicating that coenzyme I-geniposide possesses acid-responsive release capabilities.

[0092] Example 6: Evaluation of the scavenging ability of coenzyme I-geniposide supramolecular nanomedicine for superoxide anions The NBT method was used to evaluate the scavenging ability of coenzyme I-geniposide supramolecular nanomedicine against superoxide anions. 390 μL of methionine solution, 23 μL of NBT solution, 7 μL of riboflavin solution, 15 μL of different concentrations of coenzyme I-geniposide supramolecular nanomedicine solution, and 1.065 mL of ultrapure water were added sequentially to 1.5 mL of PBS buffer. After thorough mixing, the mixture was transferred to a quartz cuvette. The final concentrations of the coenzyme I-geniposide supramolecular nanomedicine were 0, 3, 6, 12, and 24 μg / mL. The cuvette was irradiated under UV light for 5 minutes to generate superoxide anions in the riboflavin-methionine system. The absorbance at 560 nm was measured immediately after the reaction. NBT can be reduced by superoxide anions to formazan; if the coenzyme I-geniposide supramolecular nanomedicine has the ability to scavenge superoxide anions, the absorbance at 560 nm will decrease.

[0093] Figure 10 The results showed that as the concentration of coenzyme I-geniposide supramolecular nanomedicine increased, the absorbance at 560 nm gradually decreased, indicating that coenzyme I-geniposide supramolecular nanomedicine can scavenge superoxide anions in a concentration-dependent manner.

[0094] Example 7: Evaluation of the scavenging ability of coenzyme I-geniposide supramolecular nanomedicine against hydroxyl radicals The Fenton reaction system was used to evaluate the scavenging ability of coenzyme I-geniposide supramolecular nanomedicine against hydroxyl radicals. Reaction systems containing 0.05 mM ferrous sulfate, 0.1 mM 3,3',5,5'-tetramethylbenzidine, 1 mM hydrogen peroxide, and different concentrations of coenzyme I-geniposide supramolecular nanomedicine were prepared, and ultrapure water was added to a final volume of 3 mL. The final concentrations of the coenzyme I-geniposide supramolecular nanomedicine were 0, 3, 6, 12, and 24 μg / mL. After thorough mixing, the reaction system was transferred to a quartz cuvette and irradiated under UV light for 5 minutes. The absorbance at 652 nm was measured immediately after the reaction. The hydroxyl radicals generated by the Fenton reaction can oxidize TMB and produce a blue product with a maximum absorption peak at 652 nm. If the coenzyme I-geniposide supramolecular nanomedicine can scavenge hydroxyl radicals, the oxidation degree of TMB decreases, and the absorbance at 652 nm decreases.

[0095] Figure 10 The results showed that the coenzyme I-geniposide supramolecular nanomedicine could reduce the absorbance at 652 nm in a concentration-dependent manner, indicating that it has hydroxyl radical scavenging ability.

[0096] Example 8: Evaluation of the hydrogen peroxide scavenging ability of coenzyme I-geniposide supramolecular nanomedicine. The scavenging ability of coenzyme I-geniposide supramolecular nanomedicine for hydrogen peroxide was detected using a hydrogen peroxide detection kit. Coenzyme I-geniposide supramolecular nanomedicine solutions with final concentrations of 0, 3, 6, 12, and 24 μg / mL were prepared and mixed with hydrogen peroxide solution, and reacted at room temperature for 30 minutes. After the reaction, the detection reagent was added according to the hydrogen peroxide detection kit instructions, and the residual hydrogen peroxide content in the reaction system was measured.

[0097] Figure 10 The results showed that the residual hydrogen peroxide content in the system decreased after treatment with coenzyme I-geniposide supramolecular nanomedicine, and the decrease increased with the increase of coenzyme I-geniposide supramolecular nanomedicine concentration, indicating that coenzyme I-geniposide supramolecular nanomedicine has hydrogen peroxide scavenging ability.

[0098] Example 9: Preparation of Coenzyme I-Gardinoside-FITC Weigh 50 mg of coenzyme I-geniposide supramolecular nanomedicine and disperse it thoroughly in 8 mL of ultrapure water. Separately, dissolve 2 mg of fluorescein isothiocyanate (FITC) in 2 mL of DMSO to prepare a 1 mg / mL FITC solution. Add the FITC solution to the aqueous dispersion of coenzyme I-geniposide supramolecular nanomedicine and react at 25°C in the dark for 12 hours to label the coenzyme I-geniposide supramolecular nanomedicine with FITC. After the reaction, place the reaction solution in a dialysis bag and dialyze with ultrapure water for 12 hours to remove free FITC and DMSO. After dialysis, collect the product by centrifugation to obtain coenzyme I-geniposide-FITC. The obtained coenzyme I-geniposide-FITC was used for subsequent in vivo distribution, cellular uptake, and mitochondrial localization experiments. The entire preparation and storage process was carried out under light-protected conditions.

[0099] Example 10: In vivo distribution and lesion enrichment of coenzyme I-geniposide supramolecular nanomedicine A mouse model of myocardial infarction was established by permanent ligation of the left anterior descending artery. Mice with myocardial infarction were randomly divided into three groups: a coenzyme I-geniposide-FITC group, a free FITC group, and a free FITC + coenzyme I + geniposide group. Each group received the corresponding formulation via tail vein injection. Mice were sacrificed at 0 min, 10 min, 30 min, 1 hour, 3 hours, 6 hours, 1 day, 3 days, and 7 days after administration, and the heart, liver, spleen, lung, and kidneys were completely removed. Immediately after collection, the tissues were preserved in pre-cooled PBS at 4°C, surface fascia and blood vessels were removed, and the tissues were rinsed three times with PBS to remove residual blood.

[0100] Images of the distribution of FITC fluorescence signals in various organs were acquired using a fluorescence microscope. Figure 11The results showed that in the coenzyme I-geniposide-FITC group, a significant fluorescent signal was observed in the ischemic myocardium within 10 minutes after administration, reaching its peak at approximately 1 hour; the fluorescent signal could persist for more than 3 days. In contrast, free FITC was mainly rapidly distributed to the kidneys and cleared.

[0101] Further TTC staining was performed on the cardiac tissue. Figure 12 The results showed that the coenzyme I-geniposide-FITC fluorescence signal was mainly distributed in the ischemic or infarct-related areas indicated by TTC staining, indicating that the coenzyme I-geniposide supramolecular nanomedicine has the ability to enrich myocardial ischemic lesions.

[0102] Example 11: Intracellular lysosomal escape and mitochondrial localization of coenzyme I-geniposide supramolecular nanomedicine H9c2 cells were seeded at 1×10^5 cells / well in 12-well plates and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody at 37°C in a 5% CO2 incubator until cell adhesion occurred. Coenzyme I-geniposide-FITC was added to the cell culture medium and co-incubated with the H9c2 cells. MitoTracker Red-labeled mitochondria or LysoTracker Red-labeled lysosomes were added at different time points. After staining, the cells were gently washed with PBS to remove unbound probes, and the colocalization of FITC green fluorescence and mitochondrial or lysosomal red fluorescence was observed under a fluorescence microscope.

[0103] Figure 13 The results showed that the coenzyme I-geniposide supramolecular nanomedicine could colocalize with lysosomal signals in the early stage of incubation, suggesting that it entered the cell via endocytosis. As the incubation time increased, the colocalization of coenzyme I-geniposide-FITC with lysosomes decreased, while the colocalization with mitochondria increased, indicating that the coenzyme I-geniposide supramolecular nanomedicine can escape from lysosomes and further localize to mitochondria.

[0104] Example 12: Molecular docking of coenzyme I-geniposide supramolecular nanomedicine with mitochondrial outer membrane proteins The binding affinity of coenzyme I-geniposide supramolecular nanomedicines to mitochondrial outer membrane-associated proteins was evaluated using molecular docking. TOM7, TOM20, TOM34, TOM40, TOM70, VDAC1, and VDAC2 were selected as receptor proteins, and docking analysis was performed using MOE2022 software. After importing the receptor protein structures into the software, hydrogenation, water molecule removal, energy minimization, and active pocket settings were performed. Coenzyme I-geniposide supramolecular nanomedicines were used as ligand molecules, and after conformational optimization, they were docked with the aforementioned receptor proteins. Binding energies, binding sites, and binding modes such as hydrogen bonding, hydrophobic interactions, and π-π interactions were recorded.

[0105] Figure 14 The results showed that the coenzyme I-geniposide supramolecular nanomedicine had a strong binding ability to mitochondrial outer membrane proteins such as TOM20, TOM40, VDAC1 and VDAC2, suggesting that the mitochondrial localization ability of the coenzyme I-geniposide supramolecular nanomedicine may be related to its affinity for mitochondrial outer membrane proteins.

[0106] Example 13: Molecular dynamics simulation of coenzyme I-geniposide supramolecular nanomedicine To verify the feasibility of co-assembling the co-enzyme I-geniposide backbone precursor and free coenzyme I to form the supramolecular nanostructure NGB, molecular dynamics simulations were used for analysis. First, the molecular force field parameters of the co-enzyme I-geniposide backbone precursor and coenzyme I were established using a GAFF force field. The molecular geometry was quantum chemically optimized, and single-point energy calculations were performed to obtain the electrostatic potential distribution. Subsequently, the RESP method was used to fit the atomic partial charges. In the initial model, 20 co-enzyme I-geniposide backbone precursor molecules and 20 coenzyme I molecules were randomly placed in an 8 nm × 8 nm × 8 nm cubic box, and TIP3P water molecules were added for solvation. The simulation was performed using GROMACS 2022.5 software. Before the formal simulation, the steepest descent method was used to minimize energy and remove unreasonable atomic contacts. Then, molecular dynamics simulations were performed in the NPT ensemble with a time step of 2 fs, a temperature maintained at 300 K, and a pressure maintained at 1 bar. During the simulation, the cutoff radius for short-range van der Waals forces and electrostatic interactions was set to 1.0 nm, while long-range electrostatic interactions were calculated using the PME method. After the simulation, GROMACS and VMD software were used to analyze molecular aggregation, the number of hydrogen bonds, π-π packing, and changes in intermolecular distances.

[0107] Figure 15 The results showed that the coenzyme I-geniposide precursor and coenzyme I could gradually aggregate in the aqueous phase to form stable aggregates, indicating that the formation of its supramolecular nanostructure is based on molecular interactions.

[0108] Example 14: The effect of coenzyme I-geniposide supramolecular nanomedicine on improving cardiac function in mice with myocardial infarction Male C57BL / 6J mice aged 8–12 weeks and weighing 20–25 g were selected. After isoflurane anesthesia, the mice underwent endotracheal intubation and mechanical ventilation. A 1 cm incision was made in the left third and fourth intercostal spaces to expose the heart. The left anterior descending coronary artery was ligated with 10-0 sutures approximately 2 mm below the left atrial appendage to establish a permanent myocardial infarction model. In the sham surgery group, only thoracotomy and suture threading were performed; the left anterior descending coronary artery was not ligated.

[0109] Postoperative analgesia and anti-infection treatment were administered. The experiment was divided into four groups: sham surgery group, myocardial infarction model group, myocardial infarction + coenzyme I-geniposide group, myocardial infarction + coenzyme I group, myocardial infarction + geniposide group, and myocardial infarction + nicotinamide mononucleotide group. Coenzyme I-geniposide, coenzyme I, and geniposide were administered via tail vein injection at a dose of 5 mg / kg; nicotinamide mononucleotide was administered via intraperitoneal injection at a dose of 500 mg / kg. Administration was performed on postoperative days 0, 3, and 6. Cardiac function was assessed using a small animal ultrasound imaging system on postoperative days 1, 3, 7, 14, 21, and 28. Left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular end-diastolic diameter, and left ventricular end-systolic diameter were recorded, and left ventricular ejection fraction and left ventricular shortening fraction were calculated.

[0110] Figure 16 The AE results showed that, compared with the sham surgery group, the left ventricular ejection fraction and left ventricular shortening fraction continued to decrease in the myocardial infarction model group, while the left ventricular end-diastolic and end-systolic volumes and diameters increased, indicating significant cardiac function impairment and ventricular dilation after myocardial infarction. Compared with the model group (MI), the NGB group significantly improved left ventricular ejection fraction and left ventricular shortening fraction, reduced left ventricular dilation, and improved ventricular systolic function, with overall recovery effects superior to the coenzyme I alone group, the geniposide alone group, and the nicotinamide mononucleotide group. Among these, coenzyme I alone and geniposide alone showed limited improvement, and nicotinamide mononucleotide had a certain protective effect but was lower than that of NGB, indicating that NGB can more effectively improve the long-term decline in cardiac function after myocardial infarction through lesion enrichment and staged delivery.

[0111] Example 15: Therapeutic effect of coenzyme I-geniposide supramolecular nanomedicine on myocardial ischemia / reperfusion injury Male C57BL / 6J mice aged 8–12 weeks were selected. After isoflurane anesthesia, endotracheal intubation, and mechanical ventilation, the heart was exposed via thoracotomy. The left anterior descending coronary artery was temporarily ligated with 10-0 sutures approximately 2 mm below the left atrial appendage to induce myocardial ischemia. After 1 hour of ischemia, the ligation was released to restore coronary blood flow, and reperfusion was continued for 24 hours to establish a myocardial ischemia / reperfusion injury model.

[0112] The experimental groups included a sham surgery group, a myocardial ischemia / reperfusion injury model group, a myocardial ischemia / reperfusion injury + coenzyme I-geniposide group, a myocardial ischemia / reperfusion injury + coenzyme I group, a myocardial ischemia / reperfusion injury + geniposide group, and a myocardial ischemia / reperfusion injury + nicotinamide mononucleotide group. Administered drugs 5 minutes before reperfusion: coenzyme I-geniposide, coenzyme I, and geniposide were administered via tail vein injection at a dose of 5 mg / kg; nicotinamide mononucleotide was administered via intraperitoneal injection at a dose of 500 mg / kg.

[0113] Figure 17The results showed that the myocardial ischemia / reperfusion injury model group exhibited significant expansion of myocardial infarction area, tissue structure destruction, and cell damage. After NGB treatment, the myocardial infarction area decreased, the degree of tissue damage lessened, and myocardial cell apoptosis-related damage was inhibited, suggesting a protective effect against acute ischemia / reperfusion injury. Compared with coenzyme I and geniposide alone, NGB showed a more stable acute myocardial protective effect; compared with nicotinamide mononucleotide, NGB had a better advantage in limiting infarction expansion and reducing cell damage, but its inhibitory advantage on mitochondrial DNA leakage-related damage in the early reperfusion phase was not absolute, suggesting that the overall efficacy of NGB is not only due to acute-phase protection but also related to its subsequent continuous metabolic regulatory effects.

[0114] Example 16: The effect of coenzyme I-geniposide supramolecular nanomedicine on improving myocardial tissue pathological damage and fibrosis. Heart tissues from mice in each group were harvested on day 28 post-myocardial infarction surgery and fixed in 4% paraformaldehyde for 24 hours. After fixation, the tissues were dehydrated, embedded in paraffin, and cut into 4 μm thick sections. For HE staining, the sections were dewaxed and rehydrated sequentially in xylene, graded ethanol, and distilled water, then stained with Harris hematoxylin for 3 minutes, rinsed with tap water, rapidly differentiated in 1% hydrochloric acid alcohol, then blued with 0.6% ammonia, and finally stained with eosin for 1 minute. After dehydration in graded ethanol and clearing in xylene, the sections were mounted with neutral resin. For Masson staining, the Masson trichrome staining kit was used, followed by tissue processing, hematoxylin staining, acid fuchsin staining, phosphomolybdic acid treatment, aniline blue staining, dehydration and clearing, and mounting. The myocardial tissue structure and collagen deposition were observed under a microscope, and the proportion of fibrosis area was calculated using image analysis software.

[0115] Figure 18 The results showed that the myocardial tissue in the myocardial infarction model group was disordered, with thinning of the myocardial wall in the infarct and marginal zones, and a significant increase in inflammatory cell infiltration and collagen deposition, indicating persistent ventricular remodeling and fibrosis. After NGB treatment, the structural integrity of the myocardial tissue was significantly improved, ventricular wall thickness was better preserved, the area of ​​collagen deposition and fibrosis decreased, and inflammation-related tissue damage was alleviated. In contrast, coenzyme I and geniposide alone had a weaker effect on improving chronic remodeling, and nicotinamide mononucleotide could partially alleviate fibrosis, but its inhibitory effect on ventricular structural recovery and collagen deposition was lower than that of NGB, indicating that NGB can more effectively block the pathological process of inflammation progressing to fibrosis after myocardial infarction.

[0116] Example 17: Protective effect of coenzyme I-geniposide supramolecular nanomedicine on the ultrastructure of myocardial mitochondria Myocardial tissue from each group of mice was collected, and tissue from the infarct margin or corresponding region was rapidly excised and immediately placed in pre-cooled electron microscopy fixative and fixed overnight at 4°C. The tissue was washed three times with PBS for 5 minutes each time the following day. Subsequently, it was fixed with 1% osmium tetroxide solution at room temperature for 2 hours, and then washed three more times with PBS. The tissue was dehydrated using a graded series of ethanol solutions and then embedded in epoxy resin. Ultrathin sections of 60–80 nm thickness were prepared using an ultramicrotome and double-stained with 3% uranium acetate and lead citrate. Finally, the ultrastructure of cardiomyocyte mitochondria was observed using transmission electron microscopy.

[0117] Figure 19 The results showed that in the myocardial infarction model group, cardiomyocytes exhibited significant mitochondrial swelling, cristae breakage, vacuolation, and membrane structure damage, indicating that ischemic injury led to severe ultrastructural disorder of mitochondria. After NGB treatment, the overall morphology of mitochondria was relatively intact, the cristae structure was well preserved, and the degree of swelling and vacuolation was reduced, indicating that NGB can protect the mitochondrial structure of post-infarction cardiomyocytes. Compared with coenzyme I alone, geniposide alone, and nicotinamide mononucleotide alone, NGB showed more comprehensive recovery of mitochondrial morphology and functional markers; among them, coenzyme I and nicotinamide mononucleotide mainly showed certain metabolic support effects, geniposide mainly showed some mitochondrial stress regulation effects, while NGB could simultaneously exert mitochondrial structural protection and metabolic recovery effects.

[0118] Example 18: Regulatory effect of coenzyme I-geniposide supramolecular nanomedicine on oxidative phosphorylation and glycolysis in cardiomyocytes H9c2 cells were seeded in specialized microplates. After cell adhesion, all groups except the normal control group were treated with 400 μM cobalt chloride for 24 hours to establish a hypoxic injury model. The treatment groups were given coenzyme I-geniposide supramolecular nanomedicine, coenzyme I, geniposide, or nicotinamide mononucleotide, respectively, at a final concentration of 20 μg / mL. Oxygen consumption rate was measured using a mitochondrial stress assay kit, and extracellular acidification rate was measured using a glycolysis stress assay kit. Cells were equilibrated according to the kit instructions before testing, and mitochondrial function regulators or glycolysis regulators were added sequentially to assess basal respiration, maximal respiration, ATP-related respiration, and glycolysis capacity.

[0119] Figure 20The results showed that hypoxia-induced damage decreased oxygen consumption rate, ATP-related respiration, and maximal respiration capacity in H9c2 cells, while increasing extracellular acidification rate, indicating impaired oxidative phosphorylation accompanied by enhanced pathological glycolysis. After NGB treatment, cellular oxygen consumption rate, basal respiration, maximal respiration, and ATP-related respiration were restored, while glycolysis levels and glycolytic reserves decreased, indicating that NGB can re-coordinate glycolysis and mitochondrial oxidative phosphorylation. Compared with coenzyme I and nicotinamide mononucleotide alone, NGB not only restored mitochondrial respiration but also more effectively inhibited abnormal glycolysis; compared with geniposide alone, NGB restored oxidative phosphorylation capacity while inhibiting glycolysis, demonstrating a more complete metabolic rebalancing effect.

[0120] Example 19: Regulatory Effects of Coenzyme I-Geniposide Supramolecular Nanomedicine on Fatty Acid Oxidation, Glucose Uptake, and Lactic Acid Accumulation Cardiac tissue or H9c2 cell samples were collected, homogenized or lysed with physiological saline or PBS, and centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was collected. The fatty acid oxidation capacity of the samples was detected using a fatty acid oxidation capacity assay kit, and the total protein concentration was determined using the BCA method for normalization analysis. Separately, treated H9c2 cells were washed three times with PBS and starved in serum-free, glucose-free DMEM medium for 2 hours. Then, fluorescently labeled fatty acid probe BODIPY 558 / 568 C12 and fluorescently labeled glucose probe 2-NBDG were added, and incubation continued for 6 hours. After incubation, the cells were washed with PBS, and the uptake of fatty acids and glucose by the cells was observed under a fluorescence microscope. Simultaneously, myocardial tissue or cell lysates were collected, and lactate content was detected using a lactate assay kit.

[0121] Figure 21 The results showed that under hypoxic or myocardial infarction conditions, myocardial cells exhibited decreased fatty acid oxidation capacity, increased glucose uptake, and elevated lactate accumulation, suggesting a shift in energy substrate utilization from fatty acid oxidation to glycolysis dependence. After NGB treatment, fatty acid oxidation capacity was enhanced, fatty acid uptake and utilization were restored, and abnormal glucose uptake and lactate production decreased, indicating that it could promote the recovery of myocardial energy substrate utilization from pathological glycolysis to mitochondrial oxidative metabolism. In contrast, coenzyme I and nicotinamide mononucleotide alone could partially support fatty acid oxidation or mitochondrial metabolism, but were insufficient in inhibiting excessive glucose uptake and lactate accumulation; geniposide alone could reduce glycolysis and lactate production to some extent, but had limited effect on restoring fatty acid oxidation. NGB has both effects, therefore it is superior to other drug groups in restoring glucose and lipid metabolism balance.

[0122] Example 20: Regulation of the intracellular coenzyme I / reduced coenzyme I ratio by coenzyme I-geniposide supramolecular nanomedicine The levels of coenzyme I and reduced coenzyme I in cells, myocardial tissue, and serum samples were detected using a coenzyme I / reduced coenzyme I assay kit based on the WST-8 method, and the coenzyme I / reduced coenzyme I ratio was calculated. In cell experiments, H9c2 cells were treated with 400 μM cobalt chloride to establish a hypoxia model, followed by treatment with coenzyme I-geniposide. Cells were collected at 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, and 24 hours, lysed, and the levels and ratio of coenzyme I and reduced coenzyme I were measured. In animal experiments, coenzyme I-geniposide or nicotinamide mononucleotide were administered 2 hours after myocardial infarction. Distal myocardial tissue, infarct / marginal myocardial tissue, and serum samples were collected at 0, 1, 2, 3, 6, 12, 24, 48, and 72 hours after myocardial infarction to detect the levels of coenzyme I and reduced coenzyme I.

[0123] Figure 22 The results showed that myocardial ischemia or hypoxia led to a decrease in coenzyme I levels in infarcted / marginal zone myocardium and hypoxic cells, and an disordered coenzyme I / reduced coenzyme I ratio, indicating impaired redox homeostasis. After NGB treatment, coenzyme I levels in infarcted / marginal zone myocardium and hypoxic cells recovered rapidly and maintained a relatively stable coenzyme I / reduced coenzyme I ratio in subsequent stages, indicating that NGB can achieve redox regulation combining early supplementation and sustained maintenance. Compared with free coenzyme I, NGB, due to its ability to accumulate in lesions and release intracellularly in stages, can more effectively correct local coenzyme I deficiency; compared with nicotinamide mononucleotide, NGB has a more localized and targeted effect on the recovery of infarcted / marginal zones; geniposide alone does not have a coenzyme I supplementing effect, and therefore cannot improve coenzyme I / reduced coenzyme I homeostasis on its own.

[0124] Example 21: Inhibitory effect of coenzyme I-geniposide supramolecular nanomedicine on ROS and mitochondrial ROS In animal experiments, fresh myocardial tissue from mice in each group was used to prepare frozen sections. Total ROS levels were detected using DHE working solution, and cell nuclei were stained with DAPI. Adjacent sections were also stained with MitoSOX working solution to detect mitochondrial ROS levels, and cell nuclei were counterstained with DAPI. Images were acquired under a fluorescence microscope after staining. In cell experiments, H9c2 cells were subjected to hypoxia injury and different drug treatments, then washed twice with PBS, and the DCFH-DA fluorescent probe was added. The cells were incubated at 37°C in the dark for 30 minutes. After incubation, the cells were washed three times, and total ROS levels were detected using a fluorescence microscope and flow cytometry. For mitochondrial ROS detection, cells were washed twice with HBSS, and the MitoSOX Red probe was added. The cells were incubated at 37°C in the dark for 15 minutes. After washing, Hoechst staining solution was added and incubated for 10 minutes. After washing again, fluorescence imaging and flow cytometry were performed.

[0125] Figure 23 The results from the AF study showed that myocardial infarction or hypoxia significantly increased the levels of total ROS and mitochondrial ROS in myocardial tissue and H9c2 cells, indicating severe oxidative stress. After NGB treatment, the fluorescence signals of DHE, DCFH-DA, and MitoSOX were significantly reduced, indicating that NGB can simultaneously inhibit cytoplasmic and mitochondrial ROS. Compared with other drugs, coenzyme I and nicotinamide mononucleotide alone had a certain reducing effect on total ROS, and geniposide alone had a certain inhibitory effect on mitochondrial ROS, but none of the three could simultaneously cover both total and mitochondrial ROS. NGB, integrating the effects of coenzyme I supplementation and mitochondrial regulation by geniposide, exhibited a more comprehensive anti-oxidative stress effect.

[0126] Example 22: Inhibitory effect of coenzyme I-geniposide supramolecular nanomedicine on cardiomyocyte apoptosis In animal experiments, paraffin sections of mouse hearts from different treatment groups were dewaxed and rehydrated sequentially. Staining was performed according to the TUNEL apoptosis detection kit instructions, and cell nuclei were stained with DAPI. The proportion of TUNEL-positive cells was observed under a fluorescence microscope after staining.

[0127] In cell experiments, H9c2 cells from each group were collected and stained according to the Annexin V-FITC / PI apoptosis detection kit instructions. After staining, the apoptosis rate was detected using flow cytometry. During flow cytometry analysis, cell debris was first removed using FSC-A and SSC-A, then single cells were screened using FSC-A and FSC-H, and finally, live cells, early apoptotic cells, late apoptotic cells, and necrotic cells were distinguished based on Annexin V and PI signals.

[0128] Figure 24 The results showed that myocardial infarction or hypoxic injury significantly increased cardiomyocyte apoptosis, manifested as an increase in TUNEL-positive cells and an elevated proportion of apoptotic cells in Annexin V-FITC / PI assays, accompanied by lipid peroxidation, cytochrome c release, and activation of the Bax / caspase-3 apoptosis pathway. After NGB treatment, TUNEL-positive cells and the proportion of apoptotic cells detected by flow cytometry were significantly reduced. Cytochrome c leakage, Bax expression, and caspase-3 activation were inhibited, and Bcl-2 levels were restored, indicating that NGB can alleviate mitochondrial stress-mediated endogenous apoptosis. In contrast, nicotinamide mononucleotide (NMN) only showed partial anti-apoptotic effects, and coenzyme I and geniposide alone had limited protective effects in vivo, suggesting that the anti-apoptotic effect of NGB depends on its synergistic delivery and mitochondrial protective capabilities.

[0129] Example 23: Regulatory effect of coenzyme I-geniposide supramolecular nanomedicine on the SIRT1-PGC-1α-PPARα pathway Myocardial tissue or H9c2 cells from each group were collected, lysed thoroughly with RIPA lysis buffer, and centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was collected. Protein concentration was determined using the BCA method, and 30 μg of protein sample was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The PVDF membrane was washed with TBST and blocked with 5% skim milk powder at room temperature for 2 hours. Primary antibodies against SIRT1, PGC-1α, PPARα, CD36, CPT1A, ACADL, and ACADM were then added, and the membrane was incubated overnight at 4°C. The next day, after washing, the corresponding HRP-labeled secondary antibodies were added, and the membrane was incubated at room temperature for 1 hour. Finally, chemiluminescence immunoassay was used for imaging, and grayscale quantification was performed using image analysis software. Simultaneously, immunoprecipitation was used to detect PGC-1α acetylation levels. Tissue or cell lysates were incubated overnight at 4°C with Protein A / G magnetic beads pre-conjugated with PGC-1α antibody via rotation. After washing, the immune complexes were eluted, and the acetylation level of PGC-1α was detected using an anti-acetylated lysine antibody. At the same time, the total PGC-1α was detected as a basis for normalization.

[0130] Figure 25 The results from the AX study showed that myocardial infarction or hypoxic injury inhibited the SIRT1-PGC-1α-PPARα pathway, increased PGC-1α acetylation levels, and decreased the expression of fatty acid oxidation-related proteins such as CD36, CPT1A, ACADL, and ACADM. After NGB treatment, the expression of SIRT1, PGC-1α, and PPARα was restored, PGC-1α acetylation levels decreased, and fatty acid transport and β-oxidation-related proteins were upregulated, suggesting that NGB can activate the coenzyme I-dependent SIRT1-PGC-1α-PPARα metabolic regulatory axis. Compared with nicotinamide mononucleotide, NGB more fully restored this pathway and fatty acid oxidation proteins; compared with coenzyme I alone, NGB had better lesion delivery and sustained regulatory effects; compared with geniposide alone, NGB not only inhibited abnormal UCP2-related responses but also restored coenzyme I-dependent oxidative metabolic programs.

[0131] Example 24: Regulatory effect of coenzyme I-geniposide supramolecular nanomedicine on pathological glycolysis signaling H9c2 cell or myocardial tissue samples were collected from each group, and the expression levels of proteins such as HKII, LDHA, PDK4, and GLUT1 were detected by Western blot. q-PCR was also used for further detection. Slc2a1, Hk2, Ldha, Pdk4 and Pdk1 The expression levels of glycolysis-related genes were determined, with β-actin used as an internal reference gene. For q-PCR detection, total RNA was extracted using the TRIzol method, and cDNA was obtained after reverse transcription. Real-time quantitative PCR was then performed using the TB Green fluorescence method. The relative expression levels of each target gene were calculated based on the Ct values.

[0132] Figure 26 AJ and Figure 27 The AJ results showed that UCP2 / HIF-1α signaling was activated in mouse models of myocardial infarction or hypoxic cell models, and the expression of HIF-1α-related glycolysis genes and proteins was increased, manifesting as Slc2a1, Hk2, Ldha, Pdk4 and Pdk NGB treatment upregulated genes such as 1, and increased proteins such as GLUT1, HKII, LDHA, and PDK4. After NGB treatment, UCP2 / HIF-1α signaling was inhibited, downstream glycolytic transcriptional activity of HIF-1α decreased, and the expression of key glycolytic genes and proteins was downregulated, indicating that NGB can inhibit pathological glycolysis enhancement and improve the coupling between glycolysis and mitochondrial oxidative metabolism. Compared with geniposide alone, NGB showed more complete inhibition of UCP2 / HIF-1α and glycolytic signaling; compared with coenzyme I and nicotinamide mononucleotide alone, NGB showed greater inhibition of... Pdk4, Pdk1 It exhibits stronger inhibition of lactate production-related pathways, demonstrating a synergistic regulatory advantage.

[0133] Example 25: UCP2 knockdown and overexpression to verify the mechanism of action of coenzyme I-geniposide supramolecular nanomedicine To verify the role of UCP2 in the regulation of cardiomyocyte energy metabolism by coenzyme I-geniposide supramolecular nanomedicine, siRNA-mediated UCP2 knockdown and plasmid-mediated UCP2 overexpression experiments were conducted.

[0134] In the UCP2 knockdown assay, the siRNA targeting UCP2 was designated si-UCP2, and the negative control siRNA was designated si-NC. Following the instructions for the Lipofectamine RNAiMAX transfection reagent, si-UCP2 or si-NC was transfected into H9c2 cells. After overnight transfection, Western blot was used to detect UCP2 protein expression levels to confirm the knockdown efficiency.

[0135] In the UCP2 overexpression experiment, the UCP2 overexpression plasmid was designated oe-UCP2, and the empty vector control was designated oe-NC. Plasmid transfection was performed according to the Lipofectamine 3000 transfection reagent instructions. UCP2 protein expression was measured overnight after transfection, and only cells with a transfection efficiency of 70% or higher were used for subsequent experiments.

[0136] The UCP2 knockdown experiment included Control, Hypoxia, Coenzyme I-geniposide, si-UCP2, si-UCP2 + Coenzyme I, si-NC, and si-NC + Coenzyme I groups. The UCP2 overexpression experiment included Control, Hypoxia, Coenzyme I-geniposide, Coenzyme I-geniposide + oe-UCP2, Coenzyme I-geniposide + oe-NC, Coenzyme I-geniposide + oe-UCP2 + EX-527, and Coenzyme I-geniposide + oe-NC + EX-527 groups. Except for the Control group, all other groups used 400 μM cobalt chloride stimulation to establish a hypoxic injury model.

[0137] Figure 28 AN and Figure 29 The AM results showed that changes in UCP2 expression could affect the protective effect of NGB on hypoxic cardiomyocyte metabolism and mitochondrial function. UCP2 knockdown partially mimicked the effect of NGB, manifested as a decrease in HIF-1α-related glycolysis signaling, restoration of mitochondrial membrane potential, improved cell viability, and a reduction in inflammation and apoptosis-related markers. Supplementation with coenzyme I on top of UCP2 knockdown further enhanced these protective effects, more closely resembling the results of NGB treatment. Conversely, UCP2 overexpression weakened the ameliorative effect of NGB on mitochondrial membrane potential, cell viability, and glycolysis reprogramming, with the reversal being more pronounced after combined SIRT1 inhibition. These results indicate that the effect of NGB is not solely derived from coenzyme I supplementation or simple UCP2 inhibition, but rather depends on the synergistic effect between the restoration of coenzyme I-SIRT1 signaling and UCP2 regulation.

[0138] Example 26: Effects of Coenzyme I-Geniposide Supramolecular Nanomedicine on HIF-1α / HRE Transcriptional Activity The effect of coenzyme I-geniposide supramolecular nanomedicine on HIF-1α-mediated transcriptional activity was evaluated using the HRE luciferase reporter system. H9c2 cells were co-transfected with the pHRE-TA-Luc reporter plasmid and the pRL-TK internal control plasmid. The pHRE-TA-Luc plasmid contains a hypoxia-responsive element upstream of the minimal TA promoter, which drives downstream firefly luciferase expression and reflects HRE transcriptional activity; the pRL-TK plasmid expresses Renida luciferase and was used to correct for transfection efficiency and cell number differences. After transfection, except for the normal control group, all other groups underwent hypoxia stimulation and were treated with coenzyme I-geniposide supramolecular nanomedicine. After treatment, firefly luciferase activity and Renida luciferase activity were measured sequentially according to the instructions of the dual luciferase reporter gene assay kit. Relative HRE transcriptional activity was expressed as firefly luciferase activity / Renida luciferase activity.

[0139] Figure 30The AB results showed that hypoxia stimulation significantly enhanced HIF-1α nuclear translocation and HRE-mediated transcriptional activity, suggesting that hypoxia-related pathological transcriptional responses were activated. After NGB treatment, HIF-1α nuclear enrichment decreased and HRE transcriptional activity decreased, indicating that NGB can inhibit the HIF-1α / HRE-mediated hypoxic transcriptional response. Compared with coenzyme I alone, geniposide alone, and nicotinamide mononucleotide alone, NGB showed more significant inhibition of HIF-1α nuclear translocation and HRE activity.

[0140] Example 27: Effects of Coenzyme I-Geniposide Supramolecular Nanomedicine on Inflammatory Pathways Related to Inflammatory Factors and Mitochondrial DNA Leakage Myocardial tissue from each group was collected, thoroughly rinsed with PBS to remove surface blood, and a 10% myocardial tissue homogenate was prepared. The homogenate was centrifuged at 15,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The levels of inflammatory factors such as IFN-γ, IFN-β, TGF-β1, and IL-1β were detected using an ELISA kit.

[0141] Conditioned culture media of H9c2 cells under different treatment conditions were collected, and the levels of IFN-β, TGF-β1 and IL-1β were detected using an ELISA kit.

[0142] Immunofluorescence was used to detect the distribution of dsDNA outside the mitochondria, and Western blot was used to detect the expression of inflammatory pathway proteins such as cGAS, STING, p65, p-p65, IRF3, and p-IRF3. After fixation and blocking, tissue or cell samples were incubated with dsDNA, TOM20, and other primary antibodies at 4°C for 16 hours; the next day, fluorescent secondary antibody was added, and the samples were incubated at 37°C for 1 hour, and then mounted with mounting medium containing DAPI to quench fluorescence.

[0143] Figure 31 The results showed that myocardial infarction or hypoxic injury promotes mitochondrial DNA leakage, manifested as abnormal distribution of dsDNA outside the mitochondria, accompanied by enhanced phosphorylation signals of the cGAS-STING pathway and its downstream IRF3 and NF-κB p65, and increased release of inflammatory and fibrosis-related factors such as IFN-β, IFN-γ, IL-1β, and TGF-β1. NGB treatment improved mitochondrial structural integrity, reduced abnormal cytoplasmic dsDNA distribution, inhibited cGAS-STING and downstream inflammatory signals, decreased inflammatory factor release, and reduced acute inflammatory cell infiltration and chronic fibrosis-related cell activation. Compared with nicotinamide mononucleotide, NGB more effectively inhibited mtDNA leakage, inflammatory pathway activation, and inflammation-fibrosis progression; coenzyme I alone and geniposide alone had limited inhibitory effects on the above inflammatory pathways in vivo, indicating that NGB can more effectively block the amplification of chronic inflammation and myocardial fibrosis driven by mitochondrial damage.

[0144] Example 28: In vivo safety evaluation of coenzyme I-geniposide supramolecular nanomedicine C57BL / 6J mice were randomly divided into a blank control group, an acute toxicity evaluation group for coenzyme I-geniposide, and a long-term toxicity evaluation group for coenzyme I-geniposide, with three mice in each group. Coenzyme I-geniposide was administered via tail vein injection at twice the therapeutic dose, once every 7 days.

[0145] Samples were collected from mice in the acute toxicity evaluation group one day after administration and from mice in the long-term toxicity evaluation group 28 days after administration. Blood samples were collected from mice to measure blood routine, alanine aminotransferase, aspartate aminotransferase, urea and creatinine, etc., to evaluate changes in hematology, liver function and kidney function.

[0146] Simultaneously, tissues from the heart, liver, spleen, lungs, kidneys, and bladder were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and prepared into tissue sections, which were then stained with hematoxylin and eosin (HE). The structures of the major organs were examined under a microscope to check for inflammatory infiltration, necrosis, hemorrhage, fibrosis, or other significant pathological changes.

[0147] Figure 32 The results showed that coenzyme I-geniposide did not cause significant weight loss, behavioral abnormalities, or death; no significant abnormalities were observed in hematological, liver, and kidney function indicators; and no significant tissue damage was observed in the major organs by HE staining, indicating that the supramolecular nanomedicine of coenzyme I-geniposide has good in vivo safety.

[0148] In summary, the coenzyme I-geniposide supramolecular nanomedicine provided by this invention is a bioadaptive nanomedicine with the ability to enrich lesions, release in stages in response to acid, and continuously regulate mitochondria. It can rapidly restore coenzyme I homeostasis and reduce mitochondrial damage in the acute phase, while regulating UCP2-related metabolic remodeling in subsequent stages, thereby achieving staged treatment of the entire process of myocardial infarction. It has important application value in improving long-term cardiac function and delaying the progression of heart failure.

[0149] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction, characterized in that, The nanomedicine is assembled from a coenzyme I-geniposide backbone and free coenzyme I; coenzyme I is nicotinamide adenine dinucleotide.

2. The nanomedicine according to claim 1, characterized in that, The coenzyme I-geniposide skeleton is formed by the Schiff base reaction between coenzyme I and geniposide; the free coenzyme I is assembled with the coenzyme I-geniposide skeleton through hydrogen bond π-π stacking and intermolecular interactions to form a supramolecular nanostructure.

3. The nanomedicine according to claim 1, characterized in that, The nanomedicine is a spherical or near-spherical nanoparticle.

4. The nanomedicine according to claim 3, characterized in that, The nanomedicine has a particle size of 140–170 nm.

5. The nanomedicine according to claim 1, characterized in that, The nanomedicine exhibits a negatively charged surface at pH 7.4, and undergoes charge reversal under acidic conditions.

6. The nanomedicine according to claim 1, characterized in that, The nanomedicine can achieve biphasic release of coenzyme I at pH 5.

0.

7. A method for preparing a nanomedicine according to any one of claims 1-6, characterized in that, Includes the following steps: Coenzyme I was dissolved in PBS buffer to obtain a coenzyme I solution; Gardenoside was dissolved in anhydrous ethanol to obtain an anhydrous ethanol solution containing gardenoside. Add the coenzyme I solution to the anhydrous ethanol solution containing geniposide, stir, and allow coenzyme I to undergo a Schiff base reaction with geniposide to form the coenzyme I-geniposide backbone precursor. Subsequently, the coenzyme I-geniposide backbone precursor and free coenzyme I were co-assembled in PBS buffer, and after dialysis and freeze-drying, a coenzyme I-geniposide supramolecular nanomedicine for the treatment of myocardial infarction was obtained.

8. The preparation method according to claim 7, characterized in that, The stirring process involved reacting at 42°C for 24 hours.

9. The use of the nanomedicine according to any one of claims 1-6 in the preparation of a medicament for treating myocardial infarction.

10. The application according to claim 9, characterized in that, The use of the nanomedicine according to any one of claims 1-6 in the preparation of drugs to improve ventricular remodeling and the progression of heart failure after myocardial infarction.

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