Copper-based nanoparticle qu@cu-ss31 and preparation and use thereof

Abstract

This invention belongs to the field of nanobiomedicine technology, specifically relating to a mitochondrial-targeting copper-based nanoparticle (Qu@Cu-SS31) and its application in the preparation of drugs for treating myocardial infarction. The nanoparticles consist of quercetin (Que) and copper ions (Cu²⁺). + The nanoparticles, consisting of a self-assembly of the mitochondrial-targeting peptide SS31, exhibit a uniform, near-spherical morphology with an average particle size of 17.2 ± 1.6 nm. These nanoparticles specifically target cardiomyocyte mitochondria, responsively releasing Cu²⁺. + Furthermore, Qu@Cu-SS31 restores cardiomyocyte copper homeostasis by downregulating the copper metabolism negative regulator COMMD1, the copper uptake protein CTR1, and the copper chaperone protein CCS. Based on this, Qu@Cu-SS31 exhibits remarkable SOD-like and CAT-like enzyme activities, efficiently scavenging various reactive oxygen species / reactive nitrogen species and significantly reducing oxidative stress. Simultaneously, it inhibits NLRP3 inflammasome activation and Caspase-1-mediated pyroptosis, regulates the Bcl-2 / Bax balance to inhibit apoptosis, and promotes vascular endothelial growth factor expression and angiogenesis.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and nanomedicine, specifically relating to a mitochondrial-targeting copper nanomodulator, its preparation method, and its application in the preparation of drugs for treating myocardial infarction (MI). Background Technology

[0002] Myocardial infarction is one of the leading causes of disability and death among cardiovascular diseases worldwide. Its core pathology involves acute coronary artery obstruction leading to myocardial ischemia and hypoxia, followed by reperfusion injury. This process triggers mitochondrial dysfunction, explosive production of reactive oxygen species, calcium overload, uncontrolled inflammatory responses, and activation of various cell death mechanisms (such as apoptosis, necrosis, and pyroptosis), collectively resulting in large-scale cardiomyocyte death and heart failure. Although reperfusion therapy is a crucial clinical approach, it itself exacerbates the damage and cannot effectively repair already necrotic myocardial tissue.

[0003] The trace element copper plays a crucial role in maintaining cardiac function. Copper is an essential cofactor for mitochondrial electron transport chain complex IV (cytochrome c oxidase, COX) and cytoplasmic / mitochondrial antioxidant enzyme (copper-zinc superoxide dismutase, SOD1). Serum and myocardial tissue copper levels in patients with myocardial infarction often exhibit abnormal fluctuations, suggesting that copper metabolism disorders are involved in disease progression. COMMD1 (Copper Metabolism Domain Containing 1) is a recently discovered negative regulator of copper metabolism, limiting intracellular copper accumulation by promoting the ubiquitination and degradation of the copper-transporting ATPase ATP7B. Through single-cell transcriptomics, clinical sample, and animal model analysis, the inventors have discovered for the first time that COMMD1 is significantly upregulated after myocardial infarction and is a key driver of copper homeostasis dysregulation; however, its specific mechanism of action and targeted intervention strategies in myocardial infarction have not been previously reported.

[0004] Currently, there is a lack of clinical treatments that can precisely target mitochondria and synergistically regulate copper homeostasis and oxidative stress. Summary of the Invention

[0005] In view of this, the present invention aims to develop a therapeutic drug that can precisely target mitochondria and synergistically regulate copper homeostasis and oxidative stress. Nanotechnology provides new ideas for precise intervention, especially the mitochondrial-targeting peptide SS31, which can accumulate in the inner mitochondrial membrane by binding to cardiolipin and scavenging ROS. However, SS31 itself does not have the ability to regulate copper metabolism.

[0006] Quercetin, a naturally derived antioxidant and anti-inflammatory substance, possesses unique biological activities and physiological functions, showing potential in scavenging free radicals, reducing oxidative stress, and inhibiting inflammatory responses. When combined with copper, based on the complementarity of their chemical structures and biological functions, there is theoretically a possibility of a synergistic effect. This synergistic effect is expected to significantly enhance its antioxidant and anti-inflammatory capabilities in the myocardial infarction repair process.

[0007] This invention provides a copper-based nanoparticle, Qu@Cu-SS31, characterized in that it is composed of quercetin and copper ions (Cu²⁺). + It is composed of mitochondrial-targeting peptide SS31; the copper-based nanoparticles Qu@Cu-SS31 are uniformly spherical with an average particle size of 10 to 24 nanometers.

[0008] This invention provides a method for preparing copper-based nanoparticles Qu@Cu-SS31, characterized by the following steps: quercetin and copper salt undergo a self-assembly reaction in a methanol solution containing polyvinylpyrrolidone under an inert atmosphere and low temperature; mitochondrial-targeting peptide SS31 is added for surface modification; the copper-based nanoparticles are obtained by dialysis purification and lyophilization.

[0009] The present invention also provides the use of copper-based nanoparticles Qu@Cu-SS31 in the preparation of drugs for the prevention and / or treatment of myocardial infarction.

[0010] The copper-based nanoparticles Qu@Cu-SS31 release copper ions Cu² in response to mitochondria. + It also downregulated COMMD1 protein expression to restore copper homeostasis in cardiomyocytes.

[0011] The copper-based nanoparticles Qu@Cu-SS31 can scavenge reactive oxygen species and reactive nitrogen species, including superoxide anions (O2-). ), hydroxyl radicals ( OH), hydrogen peroxide (H2O2), and ABTS+ DPPH free radicals.

[0012] The copper-based nanoparticles Qu@Cu-SS31 exhibit superoxide dismutase and catalase-mimicking activities.

[0013] The copper-based nanoparticles Qu@Cu-SS31 inhibit cardiomyocyte pyroptosis by suppressing NLRP3 inflammasome activation, Caspase-1 cleavage, and GSDMD-N formation.

[0014] The copper-based nanoparticles Qu@Cu-SS31 inhibit cardiomyocyte apoptosis by regulating the Bcl-2 / Bax protein balance.

[0015] The copper-based nanoparticles Qu@Cu-SS31 promote angiogenesis in the myocardial infarction area by upregulating hypoxia-inducible factor HIF-α and vascular endothelial growth factor VEGF.

[0016] The copper-based nanoparticles Qu@Cu-SS31 are used to reduce the concentration of calcium ions in cells at sites of cardiac damage, reduce the generation of reactive oxygen species, enhance the activity of antioxidant enzymes, and maintain the stability of mitochondrial membrane potential.

[0017] The effective concentration of the copper-based nanoparticles Qu@Cu-SS31 in the drug for the prevention and / or treatment of myocardial infarction is 1 μg / mL to 100 μg / mL at the time of administration.

[0018] To achieve the above objectives, the present invention adopts the following technical solution: The copper-based nanoparticles Qu@Cu-SS31 provided by this invention are composed of quercetin (Que) and copper ions (Cu²⁺). + The nanoparticles are formed through self-assembly of the mitochondrial-targeting peptide SS31. These nanoparticles are uniformly spherical with an average diameter of 17.2 ± 1.6 nm (range 10.0–24.0 nm), exhibiting good dispersibility and biocompatibility. Modification with the SS31 peptide endows them with excellent mitochondrial targeting capabilities.

[0019] The preparation method of the Qu@Cu-SS31 nanoparticles includes the following steps: under an inert atmosphere and low temperature conditions, quercetin and copper salt (such as copper chloride dihydrate) undergo a self-assembly reaction in a methanol solution containing polyvinylpyrrolidone (PVP), followed by the addition of mitochondrial-targeting peptide SS31 for surface modification. After the reaction is completed, the product is purified by dialysis and lyophilized to obtain the final product.

[0020] The Qu@Cu-SS31 nanoparticles exert a synergistic protective effect in the treatment of myocardial infarction through the following multiple mechanisms: 1. Targeted Reconstruction of Copper Homeostasis: Nanoparticles are precisely targeted to the mitochondria of cardiomyocytes via the SS31 peptide, and responsively release Cu²⁺ in the acidic microenvironment formed during reperfusion. + It significantly downregulates the expression of the copper negative regulator protein COMMD1, while also downregulating the copper uptake protein CTR1 and the copper chaperone protein CCS, thereby restoring copper homeostasis in damaged cardiomyocytes and providing a material basis for subsequent antioxidant activity.

[0021] 2. Highly effective antioxidant: Qu@Cu-SS31 possesses broad-spectrum enzyme-like activity, mimicking the functions of SOD and CAT, and efficiently scavenging superoxide anions (O2). - ), hydroxyl radicals ( It contains various reactive oxygen species and reactive nitrogen species such as OH and hydrogen peroxide (H2O2), which significantly reduce oxidative stress damage to cardiomyocytes.

[0022] 3. Anti-inflammatory and immunomodulatory effects: This material can inhibit the activation of the NLRP3 inflammasome, block the Caspase-1-mediated pyroptosis pathway (manifested as a reduction in GSDMD-N and IL-1β), downregulate pro-inflammatory factors TNF-α and IL-6, upregulate anti-inflammatory factor IL-10, and improve the cardiac inflammatory microenvironment.

[0023] 4. Anti-cell death: By upregulating Bcl-2 and downregulating Bax, it regulates the Bcl-2 / Bax balance and inhibits apoptosis in the mitochondrial pathway.

[0024] 5. Promotes Repair and Regeneration: Qu@Cu-SS31 can activate the HIF-α / VEGF signaling pathway, promote angiogenesis in the infarct border area, and improve blood supply; at the same time, it inhibits myocardial fibrosis and promotes tissue repair. In animal models of myocardial infarction, Qu@Cu-SS31 treatment can significantly increase left ventricular ejection fraction (LVEF) and short-axis shortening rate (LVFS), reduce infarct area, promote angiogenesis, and demonstrate good in vivo biocompatibility.

[0025] Preferably, the effective concentration of the drug is 1 μg / mL to 100 μg / mL.

[0026] Compared with existing technologies, the present invention achieves the following beneficial effects: This invention relates to the synthesis of copper-based nanoparticles (Qu@Cu-SS31) and their application in promoting myocardial infarction repair. The invention provides a novel use for copper-based nanoparticles (Qu@Cu-SS31): through their antioxidant properties, they effectively neutralize free radicals, reduce oxidative stress, inhibit the production of key inflammatory factors such as TNF-α, IL-1β, and IL-6, regulate the polarization state of macrophages, stimulate the release of angiogenic factors (such as VEGF), and promote angiogenesis, thereby improving blood flow to ischemic areas of the myocardium and further promoting myocardial infarction repair.

[0027] This invention incorporates copper in a loaded form into a quercetin nanoparticle system to achieve controlled release of copper at specific sites and targeted therapeutic effects, thereby effectively reducing the systemic side effects that may be caused by traditional treatments and significantly improving the safety and effectiveness of the treatment process.

[0028] This invention combines SS31 with a copper-quercetin nanosystem to construct a multifunctional nanomodulator, Qu@Cu-SS31, which possesses mitochondrial targeting, copper homeostasis reprogramming, and broad-spectrum ROS scavenging functions for precise repair of myocardial infarction. Copper-based nanoparticles (Qu@Cu-SS31) with excellent antioxidant properties were developed. Their stability and biocompatibility were optimized through reaction condition regulation to achieve both antioxidant activity and nanoscale bioactivity. The application of this nanoparticle in reactive oxygen species scavenging and its potential value in myocardial infarction repair are explored.

[0029] This invention provides a new treatment option for clinical medicine and has broad application prospects. Attached Figure Description

[0030] The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Figure 1 The images show the transmission electron microscope (TEM) image, particle size distribution, EDS elemental distribution, Zeta potential, XRD, FT-IR, and UV-vis spectra of the copper-based nanoparticles (Qu@Cu-SS31) of this invention.

[0032] Figure 2 ROS removal data for copper-based nanoparticles (Qu@Cu-SS31) in Example 2 of this invention.

[0033] Figure 3 This invention describes the H2O2 scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) in Example 3 of this invention, as well as their application as superoxide dismutase (SOD) and catalase (CAT).

[0034] Figure 4 The images show the cellular uptake fluorescence (over time) and mitochondrial colocalization confocal image of the copper-based nanoparticles (Qu@Cu-SS31) in Example 4 of this invention.

[0035] Figure 5 The results of live / dead cell staining and flow cytometry apoptosis detection of copper-based nanoparticles (Qu@Cu-SS31) in Example 5 of this invention are shown.

[0036] Figure 6 The copper metabolism proteins (ATP7B, COMMD1, CCS, CTR1) of the copper-based nanoparticles (Qu@Cu-SS31) in Example 6 of this invention were quantified by Western blot and ELISA.

[0037] Figure 7 The intracellular ROS (DCFH-DA, DHE) and mitochondrial membrane potential (JC-1) of the copper-based nanoparticles (Qu@Cu-SS31) in Example 7 of this invention were detected.

[0038] Figure 8 The pyroptosis / apoptosis-related proteins (GSDMD, Caspase-1, Bax, Bcl-2, p-Akt, etc.) and inflammatory factors in copper-based nanoparticles (Qu@Cu-SS31) in Example 8 of this invention were analyzed by Western blot and ELISA.

[0039] Figure 9 The copper-based nanoparticles (Qu@Cu-SS31) in Example 9 of this invention were used in cardiac function echocardiography (LVEF, LVFS) and TTC staining of infarct area in a mouse model of myocardial infarction.

[0040] Figure 10 The results of TUNEL apoptosis staining, Masson fibrosis staining, and CD31 angiogenesis staining of copper-based nanoparticles (Qu@Cu-SS31) in mice with myocardial infarction are shown in Example 9 of this invention. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Those skilled in the art should understand that the embodiments are merely to help understand the present invention and should not be considered as specific limitations on the embodiments of the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] This invention provides copper-based nanoparticles (Qu@Cu-SS31) and their application in promoting myocardial infarction repair.

[0043] This invention relates to the copper-loaded quercetin (Qu@Cu-SS31) nanomaterial. Its morphology is as follows... Figure 1 They are regular and uniform spherical nanoparticles.

[0044] The present invention does not impose any special limitations on the preparation method of the copper-based nanoparticles (Qu@Cu-SS31).

[0045] This invention prepared a first mixed solution of copper-based nanoparticles (Qu@Cu-SS31), ABTS, and PBS, and studied its ability to scavenge RNS. The results showed that the copper-based nanoparticles (Qu@Cu-SS31) could effectively scavenge RNS generated by ABTS, thereby causing the prepared solution to decolorize, as evidenced by a decrease in the absorbance at 734 nm.

[0046] This invention prepares a second mixed solution using copper-based nanoparticles (Qu@Cu-SS31), DPPH, and PBS, and studies its ability to scavenge RNS. The results show that the copper-based nanoparticles (Qu@Cu-SS31) can effectively scavenge RNS generated by DPPH, thereby causing the prepared solution to decolorize, as evidenced by a decrease in the absorbance at 517 nm.

[0047] Therefore, the present invention provides the RNS scavenging ability of copper-based nanoparticles (Qu@Cu-SS31).

[0048] This invention combines H2O2 (0.2 mM) with Fe 2+ The scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) was investigated after generating a large amount of hydroxyl radicals (•OH) via the Fenton reaction in an acetate buffer system at pH=4 (0.2 mM). The results showed that copper-based nanoparticles (Qu@Cu-SS31) can effectively scavenge •OH.

[0049] Therefore, the present invention provides the •OH scavenging ability of copper-based nanoparticles (Qu@Cu-SS31).

[0050] This invention uses a total SOD activity assay kit to evaluate the effect of copper-based nanoparticles (Qu@Cu-SS31) on superoxide anion (SOD). • O2 The scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) as superoxide dismutase (SOD) was investigated. Results showed that copper-based nanoparticles (Qu@Cu-SS31) exhibit SOD-like enzyme activity, resulting in decolorization of the prepared solution, manifested as a decrease in absorbance at 560 nm.

[0051] Therefore, this invention provides the application of copper-based nanoparticles (Qu@Cu-SS31) as superoxide dismutase (SOD).

[0052] This invention uses a hydrogen peroxide detection kit to evaluate the scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) for H2O2. The results show that copper-based nanoparticles (Qu@Cu-SS31) can effectively scavenge H2O2, as evidenced by a decrease in the absorbance at 583 nm.

[0053] Therefore, the present invention provides the ability of copper-based nanoparticles (Qu@Cu-SS31) to remove H2O2.

[0054] In this invention, mouse cardiomyocytes HL-1 were seeded in 96-well plates at approximately 9000 cells / well. After culturing for 24 hours to allow cell adhesion, fresh culture medium containing 0.5 mM H2O2 and different concentrations of copper-based nanoparticles (Qu@Cu-SS31) was added. After another 12 hours of conditioning, the culture medium was removed, and the cells were stained using the ROS fluorescent probe DCFH-DA. The results showed that the copper-based nanoparticles (Qu@Cu-SS31) significantly scavenged ROS in mouse cardiomyocytes HL-1, exhibiting a good scavenging effect. In some embodiments of this invention, the amount of copper-based nanoparticles (Qu@Cu-SS31) used was 0-100 µg / mL.

[0055] In some embodiments of the present invention, the cardiomyocytes referred to are mouse cardiomyocytes HL-1.

[0056] This invention provides the synthesis of copper-based nanoparticles (Qu@Cu-SS31) and their application in promoting myocardial infarction repair. This invention offers a novel use for copper-based nanoparticles (Qu@Cu-SS31): through their antioxidant properties, they effectively neutralize free radicals, reduce oxidative stress, inhibit the production of key inflammatory factors such as TNF-α, IL-1β, and IL-6, regulate the polarization state of macrophages, stimulate the release of angiogenic factors (such as VEGF), and promote angiogenesis, thereby improving blood flow to ischemic areas of the myocardium and further promoting myocardial infarction repair. This invention provides a new treatment option for clinical medicine and has broad application prospects.

[0057] The raw materials used in this invention are not subject to any special restrictions and can be commercially available.

[0058] To further illustrate the present invention, the application of the copper-based nanoparticles (Qu@Cu-SS31) provided by the present invention will be described in detail below with reference to the embodiments, but it should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1 Preparation of copper-based nanoparticles (Qu@Cu-SS31): 20 mg of quercetin (Que) was dissolved in 1 mL of methanol solution, 20.3 mg of copper chloride dihydrate (CuCl2·2H2O) was dissolved in 1 mL of methanol solution, and 132.2 mg of polyvinylpyrrolidone (PVP-K30) was dissolved in 50 mL of methanol solution. The methanol solution containing Cu ions and the methanol solution containing PVP-K30 were stirred vigorously at 25 °C for 5 min. Then, the quercetin methanol solution was added and stirred for 3 h to obtain the initial product Qu@Cu. Subsequently, 5.24 mg of EDC and 3.15 mg of NHS were added and stirred for 1 h. 23.1 mg of SS31 was added and stirred again for 3 h. Then, the mixture was dialyzed through a 3500 Da dialysis bag for 24 h to obtain the final product Qu@Cu-SS31.

[0060] Depend on Figure 1 It is known that the copper-based nanoparticles (Qu@Cu-SS31) of this embodiment are uniformly dispersed spherical nanoparticles. Dynamic light scattering (DLS) determined the average hydrated particle size to be 17.2 nm, with Qu@Cu at -18.3 ± 1.5 mV and Qu@Cu-SS31 at -9.8 ± 1.2 mV (the positively charged amino group of SS31 neutralizes the surface negative charge). EDS surface scanning showed that C, O, N, and Cu were uniformly distributed. XPS Cu2p spectra showed main peaks at 934.7 eV (2p3 / 2) and 954.5 eV (2p1 / 2), accompanied by satellite peaks (943.2 eV), confirming that Cu(II) is dominant. FT-IR showed that Que was at 3420 cm⁻¹. - ¹The (-OH) peak shifts to 3350-3550 cm⁻¹ - ¹And the strength decreases, suggesting the interaction between -OH and Cu² + Coordination. Qu@Cu-SS31 at 1650 cm - ¹The presence of an amide I band confirms SS31 connectivity. UV-vis: Que at 290 nm; Qu@Cu at 310 nm shows an MLCT band; Qu@Cu-SS31 redshifts to 375 nm with increased absorbance, and Que exhibits a characteristic absorption peak at 290 nm; with Cu² + After coordination, Qu@Cu exhibits a metal-ligand charge transfer band at 310 nm; the absorption peak of Qu@Cu-SS31 further redshifts to 375 nm and its intensity increases.

[0061] Example 2 The scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) on RNS: 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a very stable nitrogen-centered free radical with a strong absorption at 517 nm and a purple color in its alcoholic solution. Upon addition of different concentrations of Qu@Cu-SS31, the absorption gradually disappears due to its single-electron pairing with DPPH. The degree of fading is positively correlated with the antioxidant capacity of Qu@Cu-SS31, thus allowing for rapid analysis using UV light. 2,2-Aza-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) reacts with K2S2O8 to generate a stable blue-green ABTS free radical (ABTS). + •) Solution. Similar to DPPH•, the solution showed varying degrees of fading after the addition of different concentrations of Qu@Cu-SS31. Rapid analysis was performed using UV detection of absorbance at 734 nm.

[0062] Scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) for •OH: H2O2 (0.2 mM) was reacted with Fe... 2+ (0.2 mM) In an acetate buffer system at pH=4, a large amount of hydroxyl radicals (•OH) were generated by the Fenton reaction, and then copper-based nanoparticles (Qu@Cu-SS31) of different concentrations were added and the volume was adjusted to 3 mL with PBS solution at pH=7.4.

[0063] Depend on Figure 2 It is known that the copper-based nanoparticles (Qu@Cu-SS31) of this invention can effectively remove RNS and •OH.

[0064] Example 3 Application of copper-based nanoparticles (Qu@Cu-SS31) as superoxide dismutase (SOD): Evaluation of the effect of Qu@Cu-SS31 on superoxide anion (SOD) using a total SOD activity assay kit. • O2 The ability to clear xanthine. Utilizing the xanthine / xanthine oxidase reaction system to generate... • O2 Nitroblue tetrazolium (NBT) was reduced to blue formazan, which exhibited strong absorption at 560 nm. The effects of different absorption intensities at 560 nm on Qu@Cu-SS31 were analyzed. • O2 Its ability to clear debris.

[0065] H2O2 scavenging ability of copper-based nanoparticles (Qu@Cu-SS31): The scavenging ability of copper-based nanoparticles (Qu@Cu-SS31) for H2O2 was evaluated using a hydrogen peroxide detection kit. Ferric ions are generated by oxidizing divalent iron ions with hydrogen peroxide, which then reacts with xylenol orange in a specific solution to form a purple product. The scavenging ability of Qu@Cu-SS31 for H2O2 was analyzed by examining the differences in absorption intensity at 570 nm.

[0066] Depend on Figure 3 It is understood that the copper-based nanoparticles (Qu@Cu-SS31) of the present invention have the ability to scavenge H2O2 and can be used as superoxide dismutase (SOD) and catalase (CAT).

[0067] Example 4 Copper-based nanoparticles (Qu@Cu-SS31) targeting mouse cardiomyocytes HL-1: Mouse cardiomyocytes (HL-1) were seeded in 6-well plates and incubated with FITC-labeled Qu@Cu-SS31 (50 μg / mL) for 1, 2, and 4 h. To investigate whether oxidative stress damage affected the uptake efficiency of Qu@Cu-SS31 by HL-1 cells, a pretreatment group was set up: cells were co-incubated with 500 μmol H2O2 and Qu@Cu-SS31 for 8 h. After incubation, fluorescence microscopy was used to observe and acquire images, and the intracellular fluorescence intensity was assessed.

[0068] Mitochondrial colocalization of Qu@Cu-SS31 nanoparticles was observed using confocal laser scanning microscopy. The specific procedure was as follows: HL-1 cells were divided into different treatment groups and incubated with FITC-labeled Qu@Cu-SS31 for 0, 2, 4, and 8 h, respectively. Mitotracker Deep Red (100 nM) was then added to stain the mitochondria. Images were acquired using confocal microscopy and analyzed using ImageJ software (version 1.53e, National Institutes of Health).

[0069] Depend on Figure 4 It is known that the copper-based nanoparticles (Qu@Cu-SS31) of this invention can efficiently target mitochondria in HL-1 cells.

[0070] Example 5 In vitro therapy with copper-based nanoparticles (Qu@Cu-SS31): HL-1 cells were seeded in 6-well plates, and an oxidative stress injury model was established after cell adhesion. Experimental groups included a normal control group, an H2O2 injury model group, and treatment groups with different concentrations of Qu@Cu-SS31. Except for the normal control group, cells in all other groups were first treated with 400 μmol H2O2 for 6 h to induce oxidative stress injury, and then cultured in fresh medium containing Qu@Cu-SS31 for another 8 h. Cell viability was assessed using the Calcein-AM / PI double staining method. After treatment, the medium was aspirated, and the cells were gently washed twice with PBS. A staining working solution containing Calcein-AM (2 μmol) and PI (4 μmol) was added, and the cells were incubated at 37°C in the dark for 30 min. After incubation, excess dye was removed by washing with PBS, and images were immediately observed and acquired using a fluorescence microscope.

[0071] Quantitative detection was performed using Annexin V-FITC / PI double staining combined with flow cytometry. Cells from each group were collected, washed with pre-chilled PBS, resuspended in Binding Buffer, and then Annexin V-FITC and PI staining solutions were added sequentially. Cells were incubated at room temperature in the dark for 15 min. Immediately after staining, flow cytometry was used to analyze apoptosis rates using FlowJo software.

[0072] Depend on Figure 5 It is known that the copper-based nanoparticles (Qu@Cu-SS31) of the present invention can effectively alleviate H2O2-induced cardiomyocyte damage and improve cell survival rate in vitro.

[0073] Example 6 In vitro regulation of copper homeostasis by Qu@Cu-SS31: An in vitro oxidative damage model was established by treating HL-1 cardiomyocytes with H2O2. The experiment included a control group, an H2O2 model group, a Que group, a Qu@Cu group, and a Qu@Cu-SS31 group. Western blot was used to detect the expression levels of key copper metabolism proteins ATP7B, COMMD1, CCS, and CTR1 in each group.

[0074] Depend on Figure 6 It is understood that the copper-based nanoparticles (Qu@Cu-SS31) in this embodiment of the invention exert cardioprotective effects by intervening in the copper metabolism network.

[0075] Example 7 In vitro ROS scavenging effect of Qu@Cu-SS31: The intracellular ROS scavenging capacity of Qu@Cu-SS31 was evaluated using an H2O2-damaged HL-1 cell model. Cells were treated with Que, Qu@Cu, and Qu@Cu-SS31, respectively. Intracellular ROS levels in each group were detected using DCFH-DA, DHE, and JC-1 probes. Images were acquired using fluorescence microscopy, and fluorescence signal intensity was quantitatively analyzed by flow cytometry to assess its ROS scavenging capacity.

[0076] Depend on Figure 7 It is known that the copper-based nanoparticles (Qu@Cu-SS31) in this embodiment of the invention alleviate oxidative stress damage and protect mitochondrial function by scavenging ROS.

[0077] Example 8 Qu@Cu-SS31's anti-apoptotic, anti-inflammatory, and anti-apoptotic effects in vitro: The expression changes of apoptosis, inflammation, and copper metabolism-related molecules were detected by Western blot and qPCR.

[0078] After cell treatment, cells were washed twice with pre-cooled PBS, and lysed for 30 min on ice with RIPA lysis buffer containing protease and phosphatase inhibitors. Cells were centrifuged at 12,000 ×g for 15 min at 4 °C, and the supernatant was collected as total protein. Protein concentration was determined by BCA method, and equal volumes of protein were subjected to SDS-PAGE electrophoresis, transferred to a membrane, and blocked. Primary antibodies against apoptosis (Bax, Bcl-2), pyroptosis (Caspase-1, GSDMD), inflammation (TNF-α, IL-1β, IL-6), copper metabolism (ATP7B, COMMD1, CCS, CTR1), antioxidant (SOD1, NRF2), signaling pathways (Akt, p-Akt, p-p38 MAPK), and internal control (β-Actin) were added, and incubated overnight at 4 °C. The following day, cells were incubated with secondary antibodies and ECL were developed. Grayscale analysis was performed using ImageJ software, and the relative expression level was expressed as the target protein / internal control grayscale ratio.

[0079] Total RNA was extracted using the TRIzol method. After determining its concentration and purity, 1 μg of RNA was reverse transcribed to synthesize cDNA. Using the cDNA as a template, amplification was performed on a real-time quantitative PCR instrument using SYBR Green premixed buffer. GAPDH was used as an internal control. - The relative expression levels of target genes were calculated using the ΔΔCt method. Genes detected included: inflammatory factors (TNF-α, IL-1β, IL-6), apoptosis-related genes (Bax, Bcl-2), pyroptosis-related genes (Caspase-1, GSDMD), and copper metabolism-related genes (COMMD1, CTR1, ATP7B). Depend on Figure 8It is known that the copper-based nanoparticles (Qu@Cu-SS31) of this invention can block oxidative stress-induced cell death, and the expression changes of pyroptosis and apoptosis-related markers were detected by Western blot.

[0080] Example 9 The therapeutic effect of Qu@Cu-SS31 in vivo: Mice were divided into five groups: sham surgery + 0.9% saline group, MI + 0.9% saline group, MI + Que group, MI + Qu@Cu group, and MI + Qu@Cu-SS31 group. Subsequently, one hour after surgery, 100 μg / mL Que, Qu@Cu, and Qu@Cu-SS31 were administered to each group via tail vein, while the sham surgery group and the MI control group were given an equal volume of saline.

[0081] Depend on Figure 9 and Figure 10 It is evident that the copper-based nanoparticles (Qu@Cu-SS31) of this invention have good in vivo therapeutic effects in a mouse model of acute myocardial infarction.

[0082] In summary, this invention provides the synthesis of copper-based nanoparticles (Qu@Cu-SS31) and their application in promoting myocardial infarction repair. This invention offers a novel use for copper-based nanoparticles (Qu@Cu-SS31): through their antioxidant properties, they effectively neutralize free radicals, reduce oxidative stress, inhibit the production of key inflammatory factors such as TNF-α, IL-1β, and IL-6, regulate the polarization state of macrophages, stimulate the release of angiogenic factors (such as VEGF), and promote angiogenesis, thereby improving blood flow to ischemic areas of the myocardium and further promoting myocardial infarction repair. This invention provides a new treatment option for clinical medicine and has broad application prospects.

[0083] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. Copper-based nanoparticles Qu@Cu-SS31, characterized in that, Composed of quercetin and copper ions Cu² + It is composed of mitochondrial-targeting peptide SS31; the copper-based nanoparticles Qu@Cu-SS31 are uniformly spherical with an average particle size of 10 to 24 nanometers.

2. The method for preparing copper-based nanoparticles Qu@Cu-SS31 according to claim 1, characterized in that, Includes the following steps: Quercetin and copper salt undergo a self-assembly reaction in a methanol solution containing polyvinylpyrrolidone under an inert atmosphere and low temperature conditions. Surface modification was performed by adding mitochondrial-targeting peptide SS31; The copper-based nanoparticles were obtained by dialysis purification and freeze-drying.

3. The use of the copper-based nanoparticles Qu@Cu-SS31 according to claim 1 in the preparation of drugs for the prevention and / or treatment of myocardial infarction.

4. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 release copper ions Cu² in response to mitochondria. + It also downregulated COMMD1 protein expression to restore copper homeostasis in cardiomyocytes.

5. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 can scavenge reactive oxygen species and reactive nitrogen species, including superoxide anions (O2-). ), hydroxyl radicals ( OH), hydrogen peroxide (H2O2), and ABTS+ DPPH free radicals.

6. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 exhibit superoxide dismutase and catalase-mimicking activities.

7. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 inhibit cardiomyocyte pyroptosis by suppressing NLRP3 inflammasome activation, Caspase-1 cleavage, and GSDMD-N formation.

8. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 inhibit cardiomyocyte apoptosis by regulating the Bcl-2 / Bax protein balance.

9. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 promote angiogenesis in the myocardial infarction area by upregulating hypoxia-inducible factor HIF-α and vascular endothelial growth factor VEGF.

10. The application as described in claim 3, characterized in that, The copper-based nanoparticles Qu@Cu-SS31 are used to reduce the concentration of calcium ions in cells at sites of cardiac damage, reduce the generation of reactive oxygen species, enhance the activity of antioxidant enzymes, and maintain the stability of mitochondrial membrane potential.

11. The application as described in claim 3, characterized in that, The effective concentration of the copper-based nanoparticles Qu@Cu-SS31 in the drug for the prevention and / or treatment of myocardial infarction is 1 μg / mL to 100 μg / mL at the time of administration.

Images