Nanoparticle for treating myocardial ischemia-reperfusion injury, preparation method and application

Abstract

The invention relates to the field of biological medicine, in particular to a nanoparticle for treating myocardial ischemia reperfusion injury, a preparation method and application. According to the scheme, the nanoparticles comprise liposome containing ML351, a phase change material PFP is sealed in the liposome, and a TA-Ce metal polyphenol network coating is wrapped outside the liposome. The TA-Ce coating ensures that the system actively anchors myocardial cells through multiple acting forces, continuously removes ROS, and creates a primary protection environment for the myocardial cells. Under ultrasonic triggering, the PFP in the inner core is subjected to phase change blasting, local enrichment and accurate release of ML351 are realized, lipid peroxide is reduced, a ferroptosis pathway is blocked, and sequential treatment under space-time control is realized. A network structure of TA-Ce removes ROS, and TA-Ce and ML351 inhibit lipid peroxidation, and upstream and downstream synergy is formed on an anti-oxidation and anti-ferroptosis pathway, so that the overall treatment effect is greatly enhanced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nanoparticle for treating myocardial ischemia-reperfusion injury, its preparation method, and its application. Background Technology

[0002] Myocardial ischemia-reperfusion injury (MI / RI) is a complex pathological process in which, after reperfusion therapy (such as thrombolysis or percutaneous coronary intervention) in patients with acute myocardial infarction, restored blood flow leads to further aggravation of myocardial tissue damage. It not only fails to fully achieve the expected goal of salvaging ischemic myocardium but may also induce malignant arrhythmias, myocardial stunning, and microcirculatory disturbances, severely impacting patient prognosis. The core of this paradox lies in the fact that while restored blood flow brings oxygen and substrates, it also triggers active damage at multiple levels, including oxidative stress, calcium overload, inflammatory responses, and the activation of novel cell death mechanisms.

[0003] However, most traditional drugs used to treat myocardial ischemia-reperfusion injury have a "collateral effect," neglecting the interactions between oxidative stress and lipid metabolism. Furthermore, due to the periodic beating of the heart, these traditional drugs suffer from low bioavailability and insufficient retention. In addition, their poor resistance and lack of imaging capabilities prevent efficient drug delivery and systemic safety.

[0004] Recent studies have revealed that ferroptosis is the main type of cardiomyocyte death after myocardial ischemia-reperfusion. During ischemia-reperfusion, the burst of reactive oxygen species (ROS) and the impairment of the antioxidant system jointly lead to impaired clearance of phospholipid hydroperoxides. Simultaneously, the intracellular free iron pool increases, catalyzing lipid peroxidation through the Fenton reaction, ultimately resulting in systemic cell membrane rupture. Therefore, inhibiting the ferroptosis pathway has become an emerging therapeutic target. Summary of the Invention

[0005] The present invention aims to provide a nanoparticle for treating myocardial ischemia-reperfusion injury, its preparation method and application, in order to solve the problem that traditional drugs mainly rely on passive targeting with enhanced vascular permeability, resulting in low enrichment concentration and short residence time in organs with fast blood flow and strong perfusion, such as the heart.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a nanoparticle for treating myocardial ischemia-reperfusion injury, comprising a drug-loaded liposome, a phase change material PFP encapsulated within the liposome, and a TA-Ce metal polyphenol network coating coated outside the liposome; the drug loaded in the liposome is one or more of lipoxygenase inhibitors, anti-inflammatory drugs, and calcium overload inhibitors.

[0007] The principles and advantages of this scheme are as follows: (1) From passive enrichment to active anchoring: Traditional nanomedicines mainly rely on passive targeting with enhanced vascular permeability, which has the problems of low enrichment concentration and short residence time in organs with fast blood flow and strong perfusion, such as the heart. However, this approach uses a TA-Ce metal polyphenol network coating to coat the outside of liposomes. By utilizing the affinity and tissue specificity of tannic acid (TA) for collagen, the nanoparticles can be actively and firmly anchored in the heart, thereby significantly improving the initial enrichment concentration and residence time of nanoparticles at the lesion site. This overcomes the inherent difficulties of cardiac targeting and lays a spatial foundation for treatment.

[0008] (2) Constructing a spatiotemporally controlled sequential therapy: The TA-Ce coating contains cerium (Ce), which can be used in Ce 3+ (Reduction) and Ce 4+ The nanoparticles undergo valence state transitions between oxidized forms, exhibiting superoxide dismutase / catalase-like activity. This effectively scavenges ROS in the microenvironment of myocardial infarction (MI) / respiratory inflammatory response (RI), aiding in inflammation resolution, reducing oxidative stress, and creating a preliminary protective environment for cardiomyocytes. Simultaneously, upon reaching the lesion site, the nanoparticles, triggered by ultrasound, undergo a phase transition and burst of the core PFP, locally enriching and precisely releasing the drug loaded onto the liposomes. This allows for targeted treatment at levels such as calcium overload (when the loaded drug is a calcium overload inhibitor), inflammatory response (when the loaded drug is an anti-inflammatory drug), or activation of novel cell death mechanisms (when the loaded drug is a lipoxygenase inhibitor). Therefore, this nanoparticle approach achieves precise targeting and timed drug release. During treatment, the TA-Ce coating first exerts its effect, continuously scavenging ROS and reducing oxidative stress, followed by the release of the drug loaded onto the liposomes for enhanced therapy. This achieves spatiotemporally controlled sequential treatment, resulting in higher therapeutic efficiency than single-therapy approaches.

[0009] (3) Nanoparticles have high drug loading capacity and ultrasound imaging capability. In addition to the effects mentioned in (1) and (2) above, due to the cavitation effect of ultrasound, nanoparticles can be used as ultrasound contrast agents to provide visualization guidance during treatment.

[0010] (4) The TA-Ce metal polyphenol network coating on the nanoparticles wraps around the liposomes, thereby locking the liposomes and blocking them, preventing the nanoparticles from leaking out before reaching the lesion site, and ensuring precise drug release.

[0011] In summary, the TA-Ce coating in the nanoparticles for treating myocardial ischemia-reperfusion injury in this protocol ensures that the nanoparticles actively anchor to cardiomyocytes, continuously clearing ROS and creating an initial protective environment for cardiomyocytes. Under ultrasound triggering, the core PFP undergoes a phase transition explosion, locally enriching and precisely releasing the drug loaded on the liposomes, achieving spatiotemporally controlled sequential therapy and greatly enhancing the overall therapeutic effect. In conclusion, this application proposes a non-invasive, safe, spatiotemporally controlled sequential therapeutic nanoparticle, which can be monitored by ultrasound imaging and has certain clinical translational potential.

[0012] To achieve the above objectives, the present invention also adopts the following technical solution: a method for preparing nanoparticles for treating myocardial ischemia-reperfusion injury, comprising the following steps: S1. Preparation of liposome nanoparticles encapsulated with phase change material PFP: DPPC, DSPE-PEG-2000, CH and drugs are mixed in CHCl3. The drugs are one or more of lipoxygenase inhibitors, anti-inflammatory drugs and calcium overload inhibitors. Then, under water bath conditions of 45-55℃, a thin film is formed by vacuum rotary evaporation, and then hydrated with deionized water; Then, under ice bath conditions, PFP was added and emulsified using an acoustic vibratory analyzer; then, after low-temperature centrifugation, washing, and resuspension, liposome nanoparticles containing the phase change material PFP were obtained. S2. Liposome nanoparticles coated with TA-Ce metal polyphenol network: TA and (NH4)2Ce(NO3)6 were added to the liposome nanoparticles prepared in S1, which were encapsulated with phase change material PFP, and the solution pH was adjusted to 8.0 by adding NaOH. After washing and resuspending at low temperature, nanoparticles for treating myocardial ischemia-reperfusion injury were obtained.

[0013] Thus, the nanoparticles for treating myocardial ischemia-reperfusion injury described in this application were prepared using the above-described preparation method.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: the application of TA-Ce metal polyphenol network coating in the preparation of nanoparticles for treating myocardial ischemia-reperfusion injury, wherein the TA-Ce metal polyphenol network coating is wrapped around the outside of the nanoparticles.

[0015] Therefore, in preparing nanoparticles for treating myocardial ischemia-reperfusion injury (not limited to the nanoparticles provided in this application, but also other structural nanoparticles), by coating the nanoparticles with a TA-Ce metal polyphenol network coating, it is ensured that the nanoparticles can actively anchor to cardiomyocytes, significantly improving the initial enrichment concentration and retention time of the nanoparticles at the lesion site, overcoming the inherent difficulties of cardiac targeting, and laying a spatial foundation for treatment; at the same time, it can also clear ROS, creating a certain protective environment for cardiomyocytes.

[0016] To achieve the above objectives, the present invention adopts the following technical solution: the application of the nanoparticles for treating myocardial ischemia-reperfusion injury provided in this application in the preparation of drugs for treating myocardial ischemia-reperfusion injury, specifically in the preparation of sequential therapeutic drugs for treating myocardial ischemia-reperfusion injury under spatiotemporal control.

[0017] To achieve the above objectives, the present invention adopts the following technical solution: the application of the nanoparticles for treating myocardial ischemia-reperfusion injury provided in this application in the preparation of ultrasound contrast agents, and the ultrasound contrast agents are applied to myocardial ischemia-reperfusion injury.

[0018] Preferably, as an improvement, the lipoxygenase inhibitor is ML351.

[0019] Therefore, liposomes are loaded with ML351, with the phase change material PFP encapsulated within the liposomes, and coated with a TA-Ce metal polyphenol network. The TA-Ce coating ensures that the system actively anchors cardiomyocytes through multiple forces, including π-π stacking, cation-π interactions, and strong binding with amino and thiol groups, continuously clearing ROS and creating an initial protective environment for cardiomyocytes. Under ultrasound triggering, the core PFP undergoes a phase transition explosion, locally enriching and precisely releasing ML351, reducing lipid peroxidation, blocking the ferroptosis pathway, and achieving spatiotemporally controlled sequential therapy. The TA-Ce network structure clears ROS and, together with ML351, inhibits lipid peroxidation, forming an upstream and downstream synergistic effect in the antioxidant and anti-ferroptosis pathways, greatly enhancing the overall therapeutic effect.

[0020] Preferably, as an improvement, in S2, the acoustic vibration is carried out under ice bath conditions, the acoustic vibration height is 5mm from the bottom of the container, the acoustic vibration power is 40W, and the acoustic vibration is carried out for 6 minutes in a 5s on, 5s off sequence.

[0021] The inventors encountered the following difficulties in coating nanoparticles with S2: PFP is prone to foaming and volatilization, so repeated preparations failed to produce coated nanoparticles. Through research, the inventors discovered that PFP has a low boiling point and volatilizes due to heat during acoustic vibration. Therefore, they performed acoustic vibration under ice bath conditions to reduce volatilization. However, ice bath conditions alone were insufficient to produce coated nanoparticles. Finally, by adjusting the acoustic vibration height and power, and setting these parameters under ice bath conditions, the inventors prevented PFP from foaming and volatilizing. Therefore, the acoustic vibration conditions and parameter settings are crucial for the successful preparation of nanoparticles.

[0022] Preferably, as an improvement, the pH adjustment rate in S2 is 400 μL / s.

[0023] During the preparation of S2, the inventors frequently encountered a problem where the coating failed to adhere to the liposomes. Research revealed that this failure was due to the slow pH adjustment rate in S2, leading to precipitation and hindering successful coating. In contrast, this new method requires very rapid pH adjustment, preventing precipitation and allowing for easy coating onto the liposomes. Therefore, the speed of pH adjustment is crucial for successful liposome coating. Attached Figure Description

[0024] Figure 1 Transmission electron microscopy (TEM) images of MP NPs.

[0025] Figure 2 The standard curve of ML351 was obtained by high performance liquid chromatography.

[0026] Figure 3 The particle size and potential of MP NPs and MP@T NPs are shown.

[0027] Figure 4 The ultraviolet spectrum, XPS spectrum, and Ce3d high-resolution XPS spectrum of MP@T NPs are shown.

[0028] Figure 5 The figure illustrates the dissolved oxygen levels in H2O2 solutions containing different concentrations of nanoparticles at the same concentration.

[0029] Figure 6 The dissolved oxygen levels in H2O2 solutions containing different concentrations of MP@T NPs are illustrated.

[0030] Figure 7 The H2O2 scavenging capacity of MP@T NPs and TA-Ce at different concentrations is illustrated.

[0031] Figure 8 The diagram illustrates the ability of different concentrations of MP@T NPs and TA-Ce to scavenge hydroxyl radicals using the TMB method.

[0032] Figure 9 This demonstrates the performance of EPR in detecting the scavenging of hydroxyl radicals by MP@T NPs.

[0033] Figure 10 The SOD-like activities of MP@T NPs and TA-Ce at different concentrations were illustrated.

[0034] Figure 11 This illustrates the SOD-like activity of MP@T NPs in scavenging superoxide anions as detected by EPR.

[0035] Figure 12This illustrates the total antioxidant capacity of MP@T NPs and TA-Ce at different concentrations as determined by the ABTS method.

[0036] Figure 13 The flow cytometry analysis illustrates the uptake of DiI-labeled MP NPs and MP@T NPs by H9c2 cells.

[0037] Figure 14 The image illustrates the fluorescence analysis of uptake of DiI-labeled MP NPs and MP@T NPs by H9c2 cells.

[0038] Figure 15 The image illustrates the fluorescence analysis of reactive oxygen species in H9c2 cells.

[0039] Figure 16 The image shows the intracellular oxygen fluorescence analysis of H9c2 cells.

[0040] Figure 17 The image shows the fluorescence analysis of superoxide anion in the mitochondria of H9c2 cells.

[0041] Figure 18 The image shows the fluorescence image of lipid peroxides detected by BODIPY.

[0042] Figure 19 The diagram illustrates the MDA content detection for each group.

[0043] Figure 20 This illustrates the protein blot analysis.

[0044] Figure 21 The changes in mitochondrial membrane potential in each group are illustrated.

[0045] Figure 22 Imaging in rats.

[0046] Figure 23 The images show the in vitro fluorescence images, quantitative in vitro fluorescence, and 3-hour heart sections of the rat heart.

[0047] Figure 24 The images show the cardiac biological transmission electron microscopy, enhanced Prussian blue staining, DHE staining, TNFα staining, and IL-10 staining.

[0048] Figure 25 The levels of IL-6, IL-1β, and IL-10 in the plasma of each group are shown.

[0049] Figure 26 The results show the staining patterns of HE, Masson, TUNEL, and cTnT.

[0050] Figure 27 Transmission electron microscopy images of MP@T NPs. Detailed Implementation

[0051] The following detailed description illustrates the specific implementation method: This embodiment discloses a nanoparticle for treating myocardial ischemia-reperfusion injury, comprising a drug-loaded liposome, an in vivo encapsulation of the phase change material perfluoropentane (PFP), and an external coating of the liposome with a TA-Ce metal polyphenol network. The drug loaded in the liposome is one or more of lipoxygenase inhibitors, anti-inflammatory drugs, and calcium overload inhibitors. In this embodiment, the drug loaded in the liposome is specifically a lipoxygenase inhibitor, more specifically ML351.

[0052] This embodiment discloses a method for preparing nanoparticles for treating myocardial ischemia-reperfusion injury, comprising the following steps: S1. Preparation of liposome nanoparticles encapsulated with phase change material PFP (PFP@ML351 / lip NPs (MP NPs)): 6 mg of dipalmitoylphosphatidylcholine (DPPC), 2 mg of phospholipid-polyethylene glycol (DSPE-PEG-2000), 2 mg of cholesterol (CH) and 1 mg of ML351 were mixed in 4 ml of chloroform (CHCl3); Then, under water bath conditions of 45-55℃ (specifically 50℃), a thin film was formed by vacuum rotary evaporation (100 rpm) for 1 hour, and then hydrated with 10 ml of deionized water. Then, 100 μL of PFP (ice bath conditions) was added, and emulsification was performed using a vibratory emulsifier (40 W, (5 s on, 5 s off) for 6 min); then, the mixture was centrifuged at 4 °C (8000 rpm, 5 min), washed, and resuspended in 2 ml of ultrapure water to obtain MP NPs. S2. Preparation of PFP@ML351 / lip@TA-Ce NPs (MP@T NPs) nanoparticles, which is the TA-Ce metal polyphenol network coating on the outside of MP NPs in S1: 40 mg / mL tannic acid (TA) and 13.2 mg / mL cerium ammonium nitrate ((NH4)2Ce(NO3)6) were added to 0.5 mg / mL MP NPs and subjected to acoustic vibration (40 W, 30 s), and 0.1 M NaOH was added to adjust the pH of the solution to 8.0; then centrifuged at low temperature of 4℃ (8000 rpm, 5 min), washed, and resuspended in 2 ml of ultrapure water to obtain MP@T NPs.

[0053] The nanoparticles prepared in this embodiment for treating myocardial ischemia-reperfusion injury comprise liposomes containing ML351, encapsulated with a phase change material (PFP), and coated with a TA-Ce metal polyphenol network. The TA-Ce coating ensures that the nanoparticles actively anchor to cardiomyocytes through multiple forces, continuously clearing ROS and creating an initial protective environment for cardiomyocytes. Under ultrasound triggering, the core PFP undergoes a phase change explosion, locally enriching and precisely releasing ML351, reducing lipid peroxidation, blocking the ferroptosis pathway, and achieving spatiotemporally controlled sequential therapy. Thus, the TA-Ce network structure clears ROS, and together with ML351 inhibits lipid peroxidation, forming an upstream-downstream synergy in the antioxidant and anti-ferroptosis pathways, greatly enhancing the overall therapeutic effect. In summary, these nanoparticles can achieve non-invasive, safe, spatiotemporally controlled sequential therapy, alleviating MI / RI injury through multiple pathways, and can be monitored by ultrasound imaging, demonstrating certain clinical translational potential.

[0054] The following experiments demonstrate the properties and effects of the nanoparticles used in this embodiment for treating myocardial ischemia-reperfusion injury. In these experiments, the preparation method of DiI / DiR-labeled MP NPs or MP@T NPs differs from the method described above in that only the addition of DiI / DiR during lipid membrane synthesis is required.

[0055] I. Characterization of Nanoparticle Properties Combination Figure 1 As shown, transmission electron microscopy results indicate that the MP NPs nanoparticles are uniformly sized, nearly spherical, with a diameter of 147.27 ± 1.35 nm. Figure 2 As shown, the ML351 standard curve was determined by high performance liquid chromatography, and the encapsulation efficiency of MP NPs was 99.51±0.01%, and the drug loading rate was 9.05%.

[0056] Transmission electron microscope images ( Figure 27 As can be seen, the synthesized MP@T NPs are nearly spherical with a diameter of 176.60±4.57 nm. Inductively coupled plasma atomic emission spectrometry (ICP-AES) determined the Ce encapsulation efficiency in the MP@T NPs to be 18.19%. These results indicate that after encapsulation with the TA-Ce coating, the binding... Figure 3 As shown, the diameter of MP@T NPs is slightly increased compared to MP NPs. Additionally, combined with... Figure 3 As shown, the zeta potentials of MP NPs and MP@T NPs were measured to be -22.8 mV and -32.2 mV, respectively, indicating that the coating was successfully applied to MP@T NPs.

[0057] Combination Figure 4As shown, the UV spectroscopy indicates that TA-Ce (prepared by adding 40 μL of 40 mg / mL TA and 160 μL of 13.2 mg / mL cerium ammonium nitrate to 2 mL of ultrapure water, adjusting the pH to 8.0 with 200 μL of NaOH (0.1 M), centrifuging at room temperature (8000 rpm, 5 min), discarding the supernatant, and resuspending the precipitate in 4 mL of ultrapure water to obtain the TA-Ce solution) and MP@TNPs absorbed light in a broad spectral range from 400 nm to 800 nm. This demonstrates that MP@T NPs successfully carried a tannic acid-cerium coating, inheriting its characteristic absorption peaks, further illustrating the successful preparation of metal-polyphenol network hybrid liposomes.

[0058] Combination Figure 4 As shown, the surface chemical composition and valence state of MP@T NPs were evaluated by X-ray photoelectron spectroscopy (XPS). The results showed a clear Ce3d peak in the MP@T NPs, and the calculated ratio of trivalent to tetravalent cerium (Ce3d to tetravalent cerium) was obtained. 3+ / Ce 4+ The ratio of 1:4 indicates that the MP@T NPs mainly contain tetravalent cerium, which has antioxidant properties.

[0059] II. Study on the enzyme-like activity of TA-Ce metal polyphenol network coating TA-Ce possesses enzyme-like activity and is a highly efficient reactive oxygen species scavenger. To evaluate the enzyme-like activity of TA-Ce metal polyphenol network coatings, we evaluated the performance of MP@T NPs based on catalase-like H2O2 decomposition reaction, superoxide dismutase (SOD)-like superoxide dismutase reaction, hydroxyl radical scavenging ability, and total antioxidant activity.

[0060] First, the dissolved O2 concentration in H2O2 solutions containing the same concentration (1 mg / mL) of MP NPs, TA-Ce, and MP@T NPs was monitored using a dissolved oxygen meter. Combined with... Figure 5 As shown, the evaluation revealed that compared to the MP NPs group, the TA-Ce coating and MP@T NPs exhibited significantly greater decomposition ability for H2O2. Further analysis of the dissolved O2 concentration in H2O2 solutions containing different concentrations (0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL) of MP@T NPs was conducted, combined with... Figure 6 As shown, preliminary verification indicates that MP@T NPs can effectively catalyze the decomposition of H2O2, and the decomposition effect is positively correlated with concentration. Combined with... Figure 7 As shown, when MP@T NPs is 500 μg / ml, the decomposition of H2O2 can reach 80%, which indicates that MP@T NPs have good CAT-like activity, which is mainly related to the TA-Ce coating present in MP@T NPs.

[0061] Hydroxyl radicals are also one of the main sources of intracellular oxidative stress. The inventors then used a TMB colorimetric method to detect the ability of MP@T NPs to scavenge hydroxyl radicals. The specific procedure was as follows: 160 μL of 8 mg / mL ferrous sulfate (FeSO4) solution and 20 μL of 1.5 mM H2O2 solution were mixed and reacted for 20 min. Then, 200 μL of MP@T NPs at different concentrations (31.25, 62.5, 125, 250, 500 μg / mL) were added and reacted for 30 min. After the reaction, 20 μL of TMB (12 mg / mL) was added for color development. After 10 min, the mixture was centrifuged (3000 rpm, 1 min), and the supernatant was used to detect the OD value at 652 nm. Figure 8 As shown, by measuring the absorbance at 652 nm, it was found that the light absorption intensity decreased with increasing MP@T NPs concentration. When the MP@T NPs concentration was 500 μg / mL, the scavenging rate of hydroxyl radicals reached 69%, indicating that MP@T NPs can effectively scavenge hydroxyl radicals. Furthermore, the hydroxyl radical scavenging performance of MP@T NPs was further investigated using electron paramagnetic resonance (EPR) spectroscopy. The specific procedure was as follows: the reaction system contained 500 μL of ferrous sulfate solution (5 mg / mL), 10 μL of 5,5-dimethyl-1-pyrrolidone-N-oxide (98% DMPO), 10 μL of 30% H2O2 solution, and 80 μL of MP@T NPs at different concentrations (250, 500 μg / mL), with an aqueous solution used as a control group. Figure 9 As shown, Fe 2+ The addition of H2O2 led to the appearance of characteristic spectral signals of hydroxyl radicals. The amplitude decreased slightly after adding 250 μg / ml of MP@T NPs, but decreased significantly when the concentration of MP@T NPs increased to 500 μg / ml. This verifies the efficient scavenging performance of MP@T NPs on hydroxyl radicals.

[0062] The superoxide scavenging ability of MP@T NPs at different concentrations was determined using a commercially available superoxide detection kit (Beyotime) following the instructions. In the superoxide dismutation reaction experiment, [the following was also considered]: Figure 10As shown, the SOD-like activity of MP@T NPs increased with increasing concentration. When the concentration of MP@T NPs was 500 μg / mL, the SOD-like activity of MP@T NPs was 42%. Furthermore, the superoxide anion scavenging ability was detected using electron paramagnetic resonance (EPR) spectroscopy. The specific procedure was as follows: the reaction system contained 20 μL xanthine (10 mM), 20 μL xanthine oxidase (1 U / mL), 10 μL DMPO (200 mM), and 50 μL of MP@T nanoparticles at different concentrations (250, 500 μg / mL), with phosphate-buffered saline (PBS) as the control group. Figure 11 As shown, after co-incubation of xanthine and xanthine oxidase, a characteristic peak of superoxide anion appears. The addition of 500 μg / ml MP@T NPs can effectively weaken the spectral signal, confirming the high SOD activity of MP@T NPs.

[0063] The total antioxidant capacity of MP@T NPs at different concentrations was determined using a commercially available ABTS method total antioxidant capacity kit (Beyotime) according to the instructions. The total antioxidant level was detected using 2,2'-adiazonium bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radicals, combined with... Figure 12 As shown, at 500 μg / ml, MP@T NPs achieved a scavenging rate of 83% against ABTS free radicals, indicating that MP@T NPs have a strong free radical scavenging ability, can eliminate oxidative stress, and achieve the purpose of antioxidation of cells and tissues.

[0064] III. Safety Study of MP@T NPs The CCK-8 assay showed that after incubation of 100 μg / ml MP@T NPs with H9c2 cells and HUVEC cells for 24 h, the cell survival rate was over 80%, indicating that MP@T NPs have high biosafety.

[0065] IV. Study on the targeting ability of MP@T NPs MP NPs and MP@T NPs (50 μg / mL) were prepared in high-glucose DMEM medium and incubated with H9c2 cells for different time periods. Figure 13 As shown, flow cytometry was used to observe the uptake of DiI-labeled MP NPs and MP@T NPs by H9c2 cells. It was found that cell uptake increased with prolonged incubation time with MP NPs and MP@T NPs, indicating a time-dependent uptake of DiI-labeled MP NPs and MP@T NPs by H9c2 cells. After 4 hours of co-incubation, the phagocytosis of MP@T NPs by H9c2 cells was significantly stronger than that of MP NPs. Figure 14As shown, fluorescence analysis further evaluated the endocytosis of MP@T NPs by H9C2 cells. Compared with MP NPs, MP@T NPs also showed better uptake after 4 hours, indicating that MP@T NPs can efficiently target cardiomyocytes.

[0066] V. Study on how MP@T NPs reduce excessive ROS and decrease oxidative stress levels in the myocardial ischemia-reperfusion microenvironment. Oxidative oxidative stress (MI / RI) leads to excessive reactive oxygen species (ROS) production in the myocardium, damaging cardiomyocytes and causing cell death. Therefore, studying the protective function of MP@TNPs on cells under oxidative stress is crucial. This experiment established an oxygen-glucose deprivation / reperfusion (OGD / R) model of H9c2 cells to induce free radical damage. Specifically, H9c2 cells were seeded in 12-well plates and incubated for 24 hours in a cell incubator (37°C, 5% CO2). The old culture medium was then replaced with sugar-free DMEM, and the plates were sealed in hypoxic bags. After 4 hours of incubation, the culture medium was replaced with high-glucose DMEM containing nanoparticles, and incubation continued for another 4 hours. After establishing the model, the ROS scavenging was detected using a DCFH-DA probe that emits green fluorescence in the presence of free radicals. Figure 15 As shown, the results indicate that OGD / R treatment significantly increases the level of free radicals in cardiomyocytes, while the TA-Ce and MP@T NPs groups significantly reduce free radical levels compared to the MP NPs group. The MP NPs group also reduced free radical levels because ML351 reduced cellular lipid oxidation by inhibiting LOX synthase, thereby mitigating free radical levels. This demonstrates that the TA-Ce metal polyphenol network coating possesses excellent antioxidant and free radical scavenging capabilities.

[0067] Subsequently, the intracellular O2 production in each group was assessed using a tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (RDPP) probe, the red fluorescence of which was quenched by O2 oxidation. Figure 16 As shown, the results indicate that the red fluorescence of the TA-Ce and MP@T NPs treatment groups was significantly quenched compared to the OGD / R group, suggesting that the TA-Ce coating also exhibits good CAT-like activity at the cellular level.

[0068] Since mitochondria are the primary source of reactive oxygen species in mammals, and literature reports a strong binding affinity of superoxide anions (TA) to mitochondrial outer membrane proteins, we used the MitoSOX probe to investigate the distribution of superoxide anions within mitochondria under different conditions. Fluorescence results showed that the binding... Figure 17 As shown, H9c2 cells treated with OGD / R exhibited obvious red fluorescence, while the red fluorescence of the MP NPs group was slightly weakened, and the cells in the TA-Ce and MP@T NPs groups showed almost no such phenomenon.

[0069] All of the above results indicate that treatment with MP@T NPs can effectively reduce excessive ROS in the myocardial ischemia-reperfusion microenvironment and lower oxidative stress levels.

[0070] VI. MP@T NPs reduce lipid peroxidation and block ferroptosis pathways, enabling spatiotemporally controlled sequential therapy research. The aim of this study is to demonstrate that, under conditions where MP@T NPs are taken up by cardiomyocytes through the TA-Ce coating and cellular oxidative stress is reduced, the spatiotemporal control of US allows cells to take up more ML351, further alleviating ferroptosis damage, thereby achieving sequential therapeutic enhancement and thus realizing the protective effect of MP@T NPs on cardiomyocytes.

[0071] First, the inventors confirmed the biocompatibility of the US. The CCK8 method showed different power levels (0, 0.2, 0.4, 0.6, 0.8, 1, 1.2 W / cm²). 2 After 24 hours of incubation, the survival rate of H9c2 cells after irradiation was over 90%, indicating that US has high biosafety and that US also has high biosafety for H9c2 cells treated with OGD / R.

[0072] In myocardial ischemia-reperfusion injury, lipid peroxidation levels in H9c2 cells increase. The overall lipid peroxidation level in H9c2 cells was specifically detected using the BODIPY 581 / 591 C11 probe, combined with... Figure 18 As shown ( Figure 18 In each group shown, the Control group was in high-glucose medium, and the OGD / R group was in oxygen-glucose deprivation mode as before, using ML351 (32 μM), MP NPs (50 μg / mL), MP@T NPs (50 μg / mL), and MP NPs+US (50 μg / mL, 50% duty cycle, 0.5 W / cm²). 2 , 10s), MP@T NPs+US (50 μg / mL, 50% duty cycle, 0.5 W / cm 2 The fluorescence results showed that OGD / R treatment resulted in significant green fluorescence, indicating that the cells were in a state of intense lipid peroxidation. The ratio of red to green in the ML351 group, MP NPs and MP NPs+US group was increased, indicating that the degree of cell oxidation was relatively mild. The degree of oxidation in the MP@T NPs and MP@T NPs+US groups was basically inhibited, indicating that the sequential treatment of TA-Ce and ML351 in MP@T NPs greatly reduced the level of lipid peroxidation.

[0073] Following OGD / R treatment, oxidative stress was induced by various pathological factors. Glutathione peroxidase (GPR) levels in different groups were measured using a commercially available glutathione peroxidase assay kit (Beyotime) according to the instructions. This was combined with... Figure 19 As shown, glutathione peroxidase (GPx) was consumed in large quantities, resulting in a significant decrease in GPx level (28.6 mU / mg). Treatment with MP@T NPs and MP@T NPs+US restored GPx activity (63.9 mU / mg and 67.2 mU / mg, respectively).

[0074] Studies have shown that if GPX4 activity is inhibited or expression is absent, the abundant polyunsaturated fatty acid phospholipid substrates provided by ACSL4 will lead to the uncontrolled accumulation of lipid peroxides, ultimately triggering ferroptosis. Western blot (WB) experiments were used to further investigate the levels of ACSL4 and GPX4 after different treatments to study the mechanism of ferroptosis inhibition. Figure 20 As shown, the results indicated that ACSL4 protein expression was decreased and GPX4 protein expression was upregulated in the MP@T NPs and MP@T NPs+US groups, suggesting that sequential therapy enhances the ability to resist ferroptosis by downregulating ACSL4 and upregulating GPX4.

[0075] To further evaluate mitochondrial activity during ferroptosis, the mitochondrial membrane potential levels in different groups were measured using a commercially available mitochondrial membrane potential assay kit (Solepro) according to the instructions. The depolarization effect of the cardiomyocyte mitochondrial membrane was investigated using JC-1 dual fluorescent dye. The results showed that... Figure 21 OGD / R treatment reduced the ratio of red fluorescence signal (aggregated state) to green fluorescence signal (monomeric dye), indicating a decrease in mitochondrial membrane potential after OGD / R damage. Conversely, incubation with MP@T NPs prevented this trend, suggesting that MP@T NPs can maintain mitochondrial integrity in cardiomyocytes after exposure to free radical damage, and their protective effect is greater than that of MP NPs.

[0076] VII. In vivo studies in rats A myocardial ischemia / reperfusion (MI / RI) model was established in SD rats by ligating the left anterior descending artery (LAD) to induce acute myocardial ischemia. The specific procedure was as follows: After anesthetizing rats with 1% sodium pentobarbital (40 mg / kg, intraperitoneal injection), the LAD was ligated using 6 / 0 sutures. Successful LAD occlusion was confirmed by ST-segment elevation and a change in ventricular tissue color from bright red to pale on electrocardiogram (ECG). After successful modeling, the muscle and skin were sutured layer by layer, and the incision was disinfected. This experiment used the SD rat MI / RI model to verify the ultrasound imaging function of MP@T NPs and MP NPs. Before modeling, images were acquired in grayscale and contrast modes. Then, 1 mL (2 mg / mL) of MP@T NPs and MP NPs nanoparticles of equal concentration were injected via the tail vein. Images were acquired 30 min later, and images were compared after irradiation of the precordial region with ultrasound (US) for 3 min. Figure 22 As shown, the results revealed almost no contrast signal in the heart before modeling and 30 minutes after injection. Following ultrasound irradiation, MP@T NPs showed significantly stronger contrast signals in the cardiac region than MP NPs; this enhancement is attributed to the targeting of cardiac tissue demonstrated by TA's affinity for collagen. These findings indicate that MP@T NPs effectively aggregate in the cardiac region and can function as an ultrasound contrast agent.

[0077] Based on the in vitro therapeutic effects of MP@T NPs, we further investigated their in vivo therapeutic effects. First, we observed the retention effect of DiR-labeled MP@T NPs in MI / RI model SD rats. Combined with... Figure 23 As shown, after reperfusion, MP@T NPs were injected into the tail vein of rats. In vitro fluorescence results showed that MP@T NPs, due to the affinity of their TA coating for collagen, retained more in the heart than the MP NPs group, reaching peak retention 3 hours after injection. The same results were confirmed by heart sections obtained 3 hours after injection of DiI-labeled MP@T NPs.

[0078] Literature reports that ferroptosis is the main type of cardiomyocyte death 24 h after myocardial ischemia-reperfusion. Based on in vitro fluorescence results, we established a sham-operated group. At 21 h, mice with a myocardial ischemia-reperfusion model were injected with 1 mL of saline, MP NPs (2 mg / mL), and MP@T NPs (2 mg / mL), respectively. After a 3-h retention period, the MP NPs+US group and the MP@T NPs+US group underwent US irradiation (50% duty cycle, 0.8 W / cm², 3 min) on the precordial region of the rats to increase cardiac uptake of nanoparticles. For the acute (3-day) myocardial ischemia-reperfusion model, tissue was obtained on day 3 for analysis. Figure 24As shown, transmission electron microscopy revealed that, compared with the sham-operated group, the mitochondrial volume was reduced, the mitochondrial double membrane density increased, and the mitochondrial cristae decreased or disappeared in the myocardial ischemia / reperfusion injury (MI / RI) group. These results indicate that MI / RI injury leads to significant damage to myocardial mitochondria. However, after intervention with MP@T NPs and US irradiation, both mitochondrial volume and cristae density recovered. This suggests that MP@T NPs improve cardiac function in rats with myocardial ischemia / reperfusion injury by repairing the mitochondria of ischemic cardiomyocytes. In addition to transmission electron microscopy, we also used enhanced Prussian blue staining to detect the therapeutic effect of ferroptosis. Prussian blue staining is mainly used to visualize iron ions in cells and tissues, especially non-heme iron. This staining method involves the reaction of potassium ferrocyanide with ferric iron to form an insoluble blue compound, thereby revealing iron deposition. Enhanced Prussian blue staining showed significant iron deposition in the MI / RI group, and the iron deposition was significantly reduced after intervention with MP@T NPs and US irradiation. After staining heart sections with the red fluorescent probe DHE, the myocardial ischemia-reperfusion area in the MI / RI group showed a strong red fluorescent signal, while the fluorescence signal in the MP@T NPs+US group was significantly weakened, indicating that MP@T NPs+US treatment has a significant ROS clearance capacity. Furthermore, we used anti-TNF-α and anti-IL-10 antibodies to detect the expression of inflammatory factors in the myocardial ischemia-reperfusion area. Compared with other groups, the MP@T NPs+US group showed an inhibitory effect on the pro-inflammatory cytokine TNF-α and an upregulation of the anti-inflammatory cytokine IL-10, thereby inhibiting inflammation and promoting the recovery of myocardial ischemia-reperfusion injury. In addition, we used IL-6, IL-10, and IL-1β ELISA kits to detect the expression of related inflammatory cytokines in plasma. Figure 25 As shown, compared with other groups, MP@T NPs+US also showed downregulation of pro-inflammatory cytokines IL-6 and IL-1β and upregulation of anti-inflammatory cytokine IL-10, thereby inhibiting inflammation and promoting the clearance of lipid peroxides.

[0079] For a chronic (14-day) myocardial ischemia-reperfusion model, treatment was administered twice weekly with both injection and ultrasound (US irradiation) (procedures as before). Ultrasound monitoring was performed on days 1, 7, and 14, and tissue samples were obtained for analysis on day 14. Pathological sections were stained with hematoxylin and Masson staining (HE staining: after dewaxing, hematoxylin staining of cell nuclei, eosin staining of cytoplasm, and finally dehydration and mounting, resulting in blue nuclei and pink cytoplasm; Masson staining: after dewaxing, cell nuclei were stained first, then muscle fibers were stained red with a compound red staining solution, and finally collagen fibers were stained blue). Figure 26As shown, the Sham group exhibited clear structural layers, with the cells arranged parallel to the direction of cardiac contraction without breakage or twisting, and very low collagen fiber content. After ischemia-reperfusion, the cardiac interstitial region significantly widened, with abundant blue collagen fiber proliferation. After MP@T NPs+US treatment, cardiomyocyte morphology tended to normalize, and collagen fiber content significantly decreased. Furthermore, TUNEL staining using the Roche in situ cell death assay kit showed that ischemia-reperfusion led to significant apoptosis of ischemic cardiomyocytes. In the myocardial ischemia-reperfusion injury model, MP@T NPs effectively protected ischemic cardiomyocytes from apoptosis. In addition, immunofluorescence staining of cTnT (cardiac troponin T) is an indicator for assessing cardiomyocyte survival, damage, and structural integrity. Staining results showed that the Sham group exhibited a uniform and continuous banded distribution with high fluorescence intensity; the fluorescence intensity in the damaged area of ​​the MI / RI group decreased significantly, and cTnT protein expression was significantly reduced. After MP@T NPs+US treatment, enhanced fluorescence intensity was observed in the damaged area, and structural integrity was partially restored. The above results demonstrate that MP@T NPs play a positive role in protecting cardiomyocyte structure and reducing cell death.

[0080] Furthermore, M-mode ultrasound monitoring during treatment showed that the motion waveform weakened or disappeared after ischemia-reperfusion. Compared with the sham-operated group, the ejection fraction and left ventricular short-axis shortening rate were significantly reduced in the myocardial ischemia-reperfusion model group, indicating that the rat myocardial ischemia-reperfusion model was successfully established. Statistical results showed that both MP NPs and MP@T NPs improved ejection fraction and left ventricular short-axis shortening rate in rats with myocardial ischemia-reperfusion injury. In comparison, MP@T NPs+US further improved ejection fraction and left ventricular short-axis shortening rate, indicating that MP@T NPs+US has a significant ameliorative effect on cardiac function in rats with myocardial ischemia-reperfusion injury.

[0081] Finally, we investigated the systemic toxicity of MP@T NPs in healthy SD rats. Complete blood counts and biochemical analyses of all rats administered 1 mL (2 mg / mL) of MP@T NPs showed that, compared to the control group, HE staining of major organs at all time points in SD rats did not reveal any abnormal inflammatory responses or tissue morphological damage.

[0082] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A nanoparticle for treating myocardial ischemia-reperfusion injury, characterized in that: The invention includes liposomes loaded with drugs, wherein the liposomes are internally encapsulated with a phase change material PFP and externally coated with a TA-Ce metal polyphenol network coating; the drugs loaded in the liposomes are one or more of lipoxygenase inhibitors, anti-inflammatory drugs, and calcium overload inhibitors.

2. The nanoparticle for treating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The lipoxygenase inhibitor is ML351.

3. A method for preparing nanoparticles for treating myocardial ischemia-reperfusion injury, characterized in that: Includes the following steps: S1. Preparation of liposome nanoparticles encapsulated with phase change material PFP: DPPC, DSPE-PEG-2000, CH and a drug are mixed in CHCl3, wherein the drug is one or more of lipoxygenase inhibitors, anti-inflammatory drugs, and calcium overload inhibitors. Then, under water bath conditions of 45-55℃, a thin film is formed by vacuum rotary evaporation, and then hydrated with deionized water; Then, under ice bath conditions, PFP was added and emulsified using an acoustic vibratory analyzer; then, after low-temperature centrifugation, washing, and resuspension, liposome nanoparticles containing the phase change material PFP were obtained. S2. Liposome nanoparticles coated with TA-Ce metal polyphenol network: TA and (NH4)2Ce(NO3)6 were added to the liposome nanoparticles prepared in S1, which were encapsulated with phase change material PFP, and the solution pH was adjusted to 8.0 by adding NaOH. After washing and resuspending at low temperature, nanoparticles for treating myocardial ischemia-reperfusion injury were obtained.

4. The method for preparing nanoparticles for treating myocardial ischemia-reperfusion injury according to claim 3, characterized in that: In S2, acoustic vibration is carried out under ice bath conditions, with the vibration height 5mm from the bottom of the container, the vibration power 40W, and the vibration lasting for 6 minutes in a 5s on, 5s off sequence.

5. The nanoparticles for treating myocardial ischemia-reperfusion injury, their preparation method, and their application according to claim 3, characterized in that: In S2, the pH adjustment rate is 400 μL / s.

6. The application of TA-Ce metal polyphenol network coating in the preparation of nanoparticles for treating myocardial ischemia-reperfusion injury, characterized in that: A TA-Ce metal polyphenol network coating is wrapped around the outside of the nanoparticles.

7. The application of the nanoparticles for treating myocardial ischemia-reperfusion injury as described in claim 1 or 2 in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

8. The application of the nanoparticles for treating myocardial ischemia-reperfusion injury as described in claim 1 or 2 in the preparation of ultrasound contrast agents, characterized in that: Ultrasound contrast agents are used to treat myocardial ischemia-reperfusion injury.

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