Nano-drug delivery system for treating myocardial fibrosis as well as preparation method and application of nano-drug delivery system

Abstract

The invention discloses a nano-drug delivery system for treating myocardial fibrosis as well as a preparation method and application of the nano-drug delivery system, and belongs to the technical field of biological medicines. According to the system, ZIF-8 is taken as a core carrier, the loading efficiency of pirfenidone is improved through high specific surface area and adjustable aperture, PCM myocardial targeting peptide is covalently coupled on the surface, and the limitation of tissue selectivity is broken through. Through coordination complexation, pi-pi conjugate accumulation and electrostatic interaction synergy, stable drug loading and microenvironment response release are guaranteed; a one-pot method and a carbodiimide method are adopted to wrap PFD and modify PCM on the surface of ZIF-8 to form a nano-drug delivery system; according to the system, the fibrosis process is intervened through multiple mechanisms, the enrichment degree and the acting time of the medicine at the diseased region are improved, the prepared medicine integrates the carrier structure advantage and the targeting peptide function, efficient medicine carrying, precise delivery and controllable release are achieved, a new scheme is provided for myocardial fibrosis treatment, and the system has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a nanomedicine delivery system for the treatment of myocardial fibrosis, its preparation method, and its application. Background Technology

[0002] Myocardial fibrosis is the core pathological mechanism of heart failure, characterized by excessive collagen deposition, abnormal fibroblast activation, and extracellular matrix remodeling in myocardial tissue. This ultimately leads to increased myocardial stiffness, impaired cardiac systolic and diastolic function, and consequently, serious cardiovascular diseases such as heart failure. Currently, drug therapy remains the dominant treatment strategy for myocardial fibrosis. However, existing drugs have significant limitations in efficacy, safety, and targeted delivery, making it difficult to meet clinical needs. Traditional anti-fibrotic drugs, represented by angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and aldosterone receptor antagonists (MRAs), can indirectly inhibit the progression of myocardial fibrosis by regulating the renin-angiotensin-aldosterone system (RAAS). However, long-term use may induce unpredictable secondary reactions such as electrolyte disturbances, renal impairment, edema, and arrhythmias, limiting the feasibility of long-term use. Pirfenidone, the only approved oral medication for the treatment of idiopathic pulmonary fibrosis, has also shown preliminary efficacy against myocardial fibrosis. It exerts its therapeutic effect by inhibiting the expression of pro-inflammatory factors (such as tumor necrosis factor-α and interleukin-6) and pro-fibrotic cytokines (such as TGF-β1), and by blocking collagen synthesis. However, this drug suffers from poor solubility (only 0.8 mg / mL in water), low absorption efficiency (oral bioavailability of approximately 50%), and a short half-life (approximately 2.4 hours), resulting in insufficient in vivo exposure and difficulty in achieving effective therapeutic concentrations in target tissues (such as fibrotic myocardium). To overcome these limitations of small-molecule drugs, researchers have attempted to utilize nanocarrier technology to achieve targeted delivery and controlled release of the drug.

[0003] Traditional metal-organic framework (MOF) materials, such as zeolite imidazole ester framework material (ZIF-8), are widely used in drug delivery due to their high specific surface area, tunable pore size, and excellent drug loading capacity. However, existing MOF carriers still face the following key challenges in the treatment of myocardial fibrosis: lack of targeting and in vivo stability, making it impossible to accurately deliver drugs to fibrotic myocardial tissue; poor in vivo stability, easily degrading in the physiological environment, leading to premature drug release or carrier failure; and difficulty in controlling drug release, making on-demand dosing impossible, affecting therapeutic efficacy and safety; existing MOF carriers also struggle to accurately respond to the multiple characteristics of the fibrotic myocardial microenvironment (such as weakly acidic pH), resulting in a mismatch between drug release and disease progression, thus affecting therapeutic efficacy. Summary of the Invention

[0004] In addressing the current limitations of conventional myocardial fibrosis treatments, such as significant side effects from traditional drugs, low bioavailability of small-molecule drugs, and poor targeting, instability, and limited release mechanisms of traditional MOF carriers, which fail to meet the demands of the fibrotic myocardial microenvironment, this invention aims to provide a nanomedicine delivery system for myocardial fibrosis treatment, along with its preparation method and applications. By introducing a targeting peptide chain, the system achieves specific recognition of fibrotic myocardial tissue, ensuring precise drug release within the target tissue microenvironment. Simultaneously, the carrier synthesis process is optimized to enhance biocompatibility, providing a highly efficient, precise, and safe nanomedicine delivery system for myocardial fibrosis treatment.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a nanomedicine delivery system for the treatment of myocardial fibrosis. The nanomedicine delivery system uses ZIF-8, whose surface is modified with cardiomyocyte-specific targeting peptides, as a core carrier. The core carrier encapsulates a drug for the treatment of myocardial fibrosis. The sequence of the cardiomyocyte-specific targeting peptide PCM is shown in SEQ ID NO.1.

[0006] Preferably, the amino acid sequence of the cardiomyocyte-specific targeting peptide PCM is: WLSEAGPVVTVRALRGTGSW (as shown in SEQ ID NO.1). The drug used for the treatment of myocardial fibrosis is any one of pirfenidone, sunitinib, and nintedanib.

[0007] Preferably, the drug used for the treatment of myocardial fibrosis is pirfenidone.

[0008] The myocardial targeting peptide is covalently coupled to the surface of the ZIF-8 core carrier.

[0009] Preferably, the core carrier synergistically encapsulates the drug through coordination complexation, π-π conjugated stacking, and electrostatic interactions.

[0010] The targeting peptide PCM specifically recognizes and binds to receptors on the surface of cardiovascular disease-related cells.

[0011] This invention provides a method for preparing the above-mentioned nanomedicine delivery system for the treatment of myocardial fibrosis, comprising the following steps: Step 1: After mixing and reacting zinc nitrate methanol solution and 2-methylimidazolium methanol solution, the mixture is allowed to stand, centrifuged, washed, and vacuum dried to obtain ZIF-8 powder. Step 2: Dissolve ZIF-8 powder and drug, stir the mixture to react, centrifuge, wash, and vacuum dry to obtain Z@ drug; Step 3: Mix the carbodiimide compound, hydroxysuccinimide compound, and myocardial targeting peptide, add 2-(N-morpholino)ethanesulfonic acid (MES) solution, react at room temperature, adjust the pH value after reaction, add Z@ drug modified with PEG-PEI 25K, stir reaction, centrifuge, wash, and vacuum dry to obtain a nanomedicine delivery system for the treatment of myocardial fibrosis.

[0012] In step 1, the molar ratio of zinc nitrate to 2-methylimidazole is 1:1~2; in step 2, the mass ratio of ZIF-8 to pirfenidone is 2:1, and the stirring reaction is carried out at room temperature for 1~3 hours.

[0013] Preferably, in step 1, the vacuum drying temperature is 60~70℃ and the drying time is 10~14h.

[0014] Preferably, in step 2, the solvent used for dissolution is methanol, the solvent used for washing is methanol, and the freeze-drying time is 10-14 hours.

[0015] In step 3, the mass ratio of the carbodiimide compound, the hydroxysuccinimide compound, and the myocardial targeting peptide is 2:4:1, the room temperature reaction time is 20-26 h, and the stirring reaction is carried out at room temperature for 3-5 h.

[0016] Preferably, in step 3, the pH value is adjusted to 7-9 after the reaction, and the freeze-drying time is 10-14 hours.

[0017] This invention provides the application of the above-mentioned nanomedicine delivery system for the treatment of myocardial fibrosis in the preparation of drugs for treating myocardial fibrosis.

[0018] The nanomedicine delivery system inhibits the progression of myocardial fibrosis by suppressing the activity of connective tissue growth factor, downregulating the transcriptional levels of key factors in the TGF-β signaling pathway and its downstream target gene Col-I, and inhibiting the expression of myocardial fibrosis markers.

[0019] The present invention provides a medicament for treating myocardial fibrosis, the medicament comprising the aforementioned nanomedicine delivery system for treating myocardial fibrosis.

[0020] Compared with the prior art, the present invention achieves the following technical effects: This invention provides a nanomedicine delivery system for the treatment of myocardial fibrosis. By constructing a nanocarrier system with myocardial targeting function, it achieves precise drug delivery and release control. Using ZIF-8 as the core carrier, its high specific surface area and tunable pore size enhance the loading efficiency of drugs for myocardial fibrosis treatment, overcoming the poor solubility of small molecule drugs. PCM myocardial targeting peptides are modified on the carrier surface, which specifically recognize receptors on the surface of cardiovascular disease-related cells, overcoming the limitation of traditional nanocarriers lacking tissue selectivity.

[0021] The cardiac target peptide PCM is immobilized on the surface of the ZIF-8 carrier via covalent coupling. Compared to non-covalent modification methods such as physical adsorption or electrostatic interaction, covalent coupling achieves a persistent binding between the target peptide and the carrier by forming stable chemical bonds (such as amide bonds), effectively preventing the target peptide from detaching during in vivo circulation, thereby maintaining the stability of the nanoparticles in the blood and the continuity of their targeting function. Coordination complexation enhances the binding stability between the drug and the carrier framework through chemical bonds between metal ions and drug molecules, thus adapting to the structural stability requirements under different pH environments. π-π conjugated stacking utilizes the electron cloud interaction between the drug's aromatic ring and the carrier ligand to increase drug loading and respond to the microenvironmental characteristics of changes in reactive oxygen species levels. Electrostatic interaction adjusts drug release kinetics through charge complementarity effects, achieving targeted controlled release under weakly acidic conditions. The synergistic effect of these three mechanisms overcomes the limitations of a single stimulus-response mode, enabling drug release to match both the high reactive oxygen species levels of fibrotic myocardial tissue and the weakly acidic microenvironment of the lesion area, thereby achieving precise controlled release of the drug in the target tissue.

[0022] This invention provides a method for preparing a nanomedicine delivery system for the treatment of myocardial fibrosis. The method involves synthesizing a ZIF-8 core carrier using a methanol system of zinc nitrate and 2-methylimidazole. By controlling the reaction conditions, a highly crystalline carrier material is obtained, providing a stable framework structure for subsequent drug loading. The drug molecules are physically embedded within the pores of the carrier through a stirred reaction of ZIF-8 with a methanol solution of pirfenidone, avoiding the influence of chemical modification on drug activity. An innovative carbodiimide / hydroxysuccinimide dual-activation system is employed to precisely modify the myocardial targeting peptide onto the carrier surface via covalent coupling. The choice of activator ensures efficient binding between the targeting peptide and the functional groups on the carrier surface, while avoiding non-specific adsorption. The methanol solvent system ensures the integrity of the ZIF-8 crystal structure. The stirring reaction time balances the drug loading rate and carrier stability. The stepwise activation-coupling operation achieves spatially oriented modification of the targeting peptide. Through the synergistic design of solvent system selection, reaction condition optimization, and functionalization modification sequence, the entire process ultimately yields a nanomedicine delivery system that combines targeting, drug loading stability, and biocompatibility.

[0023] The application provided by this invention utilizes a nanodelivery system with myocardial targeting capabilities in the preparation of anti-fibrotic drugs. Through the synergistic effect of the delivery system's targeted recognition capabilities and the drug's controlled-release properties, precise treatment is achieved. This delivery system intervenes in the fibrotic process through multiple mechanisms, including inhibiting connective tissue growth factor activity, downregulating the transcriptional levels of key factors in the TGF-β signaling pathway and its downstream Col-I gene, and inhibiting the expression of myocardial fibrosis markers. The synergistic effect of targeted delivery and multiple mechanisms of action significantly improves drug accumulation and duration of action at the lesion site, overcoming the insufficient efficacy caused by the non-specific distribution of traditional drugs.

[0024] The present invention provides a drug for treating myocardial fibrosis. By constructing a nanomedicine delivery system with myocardial targeting function, drug molecules are efficiently loaded onto a ZIF-8 carrier, and the surface modification of the myocardial targeting peptide PCM achieves precise localization to the lesion tissue. The high specific surface area and tunable pore structure of the core carrier ZIF-8 ensure efficient drug loading. Its crystal framework synergistically stabilizes drug molecules through coordination complexation, π-π conjugated stacking, and electrostatic interactions. The myocardial targeting peptide PCM is covalently modified onto the carrier surface, specifically recognizing and binding to receptors on the surface of cardiomyocytes, overcoming the limitations of non-specific drug distribution in traditional methods. Pirfenidone, as an anti-fibrotic active ingredient, exerts its therapeutic effect by inhibiting pro-inflammatory factors and the TGF-β signaling pathway. This delivery system integrates the structural advantages of the carrier material with the biorecognition function of the targeting peptide, improving drug stability while achieving target tissue enrichment. Furthermore, it optimizes drug controlled release behavior through a microenvironment-responsive release mechanism, ultimately forming a therapeutic system with efficient drug loading, precise delivery, and controlled release characteristics. Attached Figure Description

[0025] Figure 1 The images show SEM and TEM images of ZIF-8, ZIF-8@PFD, and Z@PFD@PCM NPs before and after composite formation, as well as AFM images of Z@PFD@PCM NPs. Figure 2 The physicochemical properties of ZIF-8, Z@PFD and Z@PFD@PCM of the present invention are characterized, wherein (a) is an X-ray diffraction (XRD) pattern, (b) is a Fourier transform infrared (FTIR) pattern, (c) is a Zeta potential pattern, (d) is a comparison of PFD loading efficiency under different Z / PFD ratios, and (e) is a line graph showing the change of PFD loading efficiency over time under different pH values ​​(pH=7.4 and pH=5.5). Figure 3 To illustrate the interaction of PFD within the ZIF-8 framework from different perspectives, (a) shows the interaction between the carbonyl (-C=O) functional group in the PFD molecule and the Zn in the ZIF-8 skeleton. 2+Ion formation coordination diagrams: (b) shows the π-π stacking interaction between PFD and the imidazole ligand of ZIF-8; (c) shows the multiple electrostatic interactions between PFD and atoms in the ZIF-8 pores. Figure 4 The images shown are fluorescence images of different fields of view or sample areas under an inverted fluorescence microscope according to the present invention. (a) is a Z@PFD image labeled with red fluorescence (DiI), (b) is a polypeptide image labeled with green fluorescence (FITC), and (c) is an image after the two are coupled. Figure 5 The figure shows the experimental results of the effects of Z@PFD@PCM NPs of the present invention on blood and cells. (a) shows the hemolysis rate after treatment with water (H2O), physiological saline (Saline), and different concentrations of Z@PFD@PCM NPs (1-5 mg / mL). (b) shows the percentage of cell viability after treatment with different concentrations of Z@PFD@PCM NPs (10-80 μg / mL). Figure 6 The cell uptake (DAPI, DiI, Merge) of the Z@PFD and Z@PFD@PCM nanoparticles of this invention at 0.5 hours and 1 hour. Figure 7 To statistically analyze the mRNA levels of fibrosis markers Col-1 and TGF-β1 in different treatment groups of this invention using RT-qPCR, the groups were: Sham (sham operation group), Model (model group), Z@PFD group, and Z@PFD@PCM group. (a) represents the relative expression level of Col-1 mRNA, and (b) represents the relative expression level of TGF-β1 mRNA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference, and β-actin was used as an internal control. Figure 8The images show the M-mode echocardiograms of mice under different treatment groups in this invention: Sham (sham-operated group), Model (model group), Z@PFD group, and Z@PFD@PCM group. (a) is a representative M-mode echocardiogram image; (b) is the left ventricular ejection fraction (LVEF) of mice; (c) is the statistical value of left ventricular fractional shortening (LVFS); (d) is the statistical value of left ventricular internal dimension at diastole (LVIDd); and (e) is the statistical value of left ventricular internal dimension at systole (LVIDs). *p<0.05, ns indicates no significant difference. Figure 9 To assess cardiac fibrosis in different treatment groups (Sham, Model, Z@PFD, Z@PFD@PCM) of this invention, (a) shows the results of hematoxylin-eosin (HE) staining and Masson staining of cardiac tissue in each group, and (b) shows the statistical analysis of myocardial fibrosis area in each group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 10 The effects of different treatment groups (Sham, Model, Z@PFD, Z@PFD@PCM) on myocardial fibrosis and angiogenesis were shown. (a) Immunohistochemical staining showed the expression of Col-1, Col-3 and CD-34, (b) the percentage of Col-1 positive area, (c) the percentage of Col-3 positive area, and (d) the percentage of CD-34 positive area. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference. Figure 11 Representative immunofluorescence images of IL-1β and IL-6 in different treatment groups (Sham, Model, Z@PFD, Z@PFD@PCM), and statistical analysis of the positivity rates of IL-1β and IL-6. (a) Immunofluorescence staining of IL-6 and IL-1β, (b) Percentage of IL-6 positive area, (c) Percentage of IL-1β positive area, (d) Statistical data on SOD activity. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference; Figure 12 To analyze the mRNA levels of fibrosis markers TGF-β1, Col-1, CTGF, α-SMA, IL-6, and IL-1β in different treatment groups (Sham, Model, Z@PFD, Z@PFD@PCM) using RT-qPCR, (a) is a bar chart showing the relative mRNA levels of TGF-β1, (b) Col-1, (c) CTGF, (d) α-SMA, (e) IL-6, and (f) IL-1β. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference, and β-actin was used as an internal control. Figure 13 The results of H&E staining of major organs of mice in different treatment groups (Sham, Model, Z@PFD, Z@PFD@PCM) are shown. (a) H&E staining of major organs (Lung, Liver, Brain, Spleen, Kidney) of mice in the Sham (sham-operated group) and Z@PFD@PCM groups are shown. (b) Statistical analysis of ALT activity in mouse blood is shown. (c) Statistical analysis of Cr content in mouse blood is shown. Figure 14 This diagram shows the molecular docking between PFD molecules and different proteins. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0028] Example 1 This embodiment provides a targeted nanomaterial based on a metal-organic framework (MOF). A nanomedicine delivery system is formed by simultaneously encapsulating and modifying pirfenidone (PFD) and the polypeptide PCM on the surface of ZIF-8 using a one-pot method and a carbodiimide method. The specific steps are as follows: (1) Synthesis of ZIF-8 Weigh 148.74 g of zinc nitrate (Zn(NO3)2·6H2O, 0.5 mol), dissolve it in 10 mL of methanol to prepare a 0.1 mol / L zinc nitrate methanol solution; weigh an equimolar amount of 2-methylimidazole (2-MIM) and dissolve it in 10 mL of methanol; quickly pour the zinc nitrate methanol solution into the 2-methylimidazole methanol solution, and stir on a magnetic stirrer at 500 r / min for 5 min to ensure thorough mixing and reaction of the two solutions; immediately centrifuge (8000 r / min, 10 min), collect the precipitate, and wash it three times with methanol to remove unreacted reagents and impurities; vacuum dry the washed precipitate at 65 °C (above the boiling point of methanol) for 12 h to obtain ZIF-8 powder.

[0029] (2) Synthesis of Z@PFD NPs Weigh 185.22 g of PFD and 370.44 g of ZIF-8 powder, disperse them in 5 mL of methanol, and stir on a magnetic stirrer at 300 r / min for 2 h to allow PFD molecules to enter the pore size and channel structure of ZIF-8. After the reaction is complete, centrifuge (8000 r / min, 10 min), collect the precipitate, wash it three times with methanol to remove unencapsulated PFD molecules, and freeze-dry the washed precipitate for 12 h to obtain Z@PFD NPs powder.

[0030] (3) Synthesis of Z@PFD@PCM NPs Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 10 mg), N-hydroxysuccinimide (NHS, 20 mg), and 5 mg of PCM peptide. Finally, add 10 mL of 2-(N-morpholino)ethanesulfonic acid (MES) solution at pH 5.5 and react at room temperature for 20–26 h. Then adjust the pH to 8.0, add 30 mg of Z@PFD modified with PEG-PEI 25K, stir at room temperature for 3–5 h, centrifuge, place at -80℃ overnight, and freeze dry for 12 h to obtain Z@PFD@PCM powder.

[0031] Small amounts of ZIF-8, Z@PFD NPs, and Z@PFD@PCM NPs powders were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS) elemental mapping, atomic force microscopy (AFM), powder X-ray diffraction (XRD), and zeta potential metering. Detailed results are shown in the appendix. Figures 1-2 As shown.

[0032] SEM characterization analysis revealed that ZIF-8 exhibited a highly regular cubic morphology with a smooth surface and sharp edges, free from obvious defects or damage. However, after composite formation, fine particles appeared on the ZIF-8 surface. This is because, during the reaction, bifenidone molecules were restricted by the pore size when trying to enter the specific pore structure of ZIF-8. Some bifenidone molecules could not smoothly enter the pores and could only aggregate near the ZIF-8 surface. When these aggregated bifenidone molecules reached a certain concentration, they formed fine particles. TEM images of Z@PFD@PCM NPs showed a uniform contrast distribution, indicating that PDA molecules were successfully encapsulated in the ZIF-8 pores without significant aggregation. However, the Z@PFD@PCM NPs tended to become more spherical from their initial cubic shape. This may be because the PCM peptides, after being adsorbed onto the ZIF-8 surface through electrostatic interactions, altered the surface energy of ZIF-8. To reduce surface energy, the ZIF-8 crystals tended to adjust their morphology to minimize surface area. Driven by this force, the originally sharp-edged cubic morphology gradually transforms into a circular shape. This is because the surface area to volume ratio of a circle is relatively small, reducing surface energy and thus making the system more stable. Combined with EDS elemental mapping, Zn, C, O, and N elements are uniformly distributed, while PDA characteristic elements (such as O and N) exhibit co-localization within the cube, confirming the effective drug loading. Furthermore, atomic force microscopy (AFM) visually reveals varying degrees of roughness on the Z@PFD@PCM NPs surface, indicating that drug or peptide modification or structural alteration has led to increased surface inhomogeneity. AFM provides nanometer-level spatial resolution, clearly observing the morphology and size of individual Z@PFD@PCM NPs, showing that the Z@PFD@PCM NPs are relatively uniform in size. AFM accurately measured the height at different locations on the sample surface, revealing that the thickness of the Z@PFD@PCM NPs is not entirely uniform, suggesting that the peptide has caused new surface undulations on the ZIF-8 surface. Compared to ZIF-8 and Z@PFD, the powder X-ray diffraction pattern of Z@PFD@PCM NPs showed an extraneous diffraction peak at 2θ=11.04°, which is due to the coating of the PCM peptide. Fourier transform infrared spectroscopy showed that, compared to ZIF-8, Z@PFD NPs exhibited a higher peak at 1660 cm⁻¹. -1 An absorption peak appears at 2854 cm⁻¹, corresponding to the characteristic absorption peak of the PFD carbon group, compared to Z@PFD NPs. Z@PFD@PCM NPs show an absorption peak at 2854 cm⁻¹. -1 and 1742cm -1Absorption peaks appeared at the locations, corresponding to the characteristic peaks h'h of the -NH and carboxyl groups of the PCM peptide, respectively. Combined images showed that the size of Z@PFD@PCM NPs was ~300.47 nm, while the zeta potentials of ZIF-8, Z@PFD NPs, and Z@PFD@PCM NPs were 33.9 mV, -5.15 mV, and 15.7 mV, respectively. Among them, ZIF-8 had a larger absolute value of zeta potential, indicating its relative stability in solution and resistance to aggregation. This also indicates successful loading of the drug and peptide. Since most cell surfaces carry a negative charge, positively charged nanoparticles can utilize this electrostatic interaction to increase accumulation in the lesion area, improve the local drug concentration, thereby enhancing the cell-killing effect and reducing side effects on normal tissues. See appendix Figure 3 To further explore the adsorption mechanism of the target molecule PFD within the ZIF-8 pores, this invention constructs a reasonable model of the PFD molecule and the ZIF-8 framework based on density functional theory (DFT) to simulate their interaction environment during actual adsorption. The constructed model is then calculated using DFT, yielding key data such as adsorption energies and interatomic distances under different interaction modes between PFD and the ZIF-8 framework. The interaction modes between the two are systematically analyzed. The calculation results show that there are three main adsorption sites between PFD and the ZIF-8 framework: firstly, the carbonyl (-C=O) functional group in the PFD molecule interacts with the Zn group in the ZIF-8 framework. 2+ First, the ions form a coordination complex, with an adsorption energy of -105.36 kJ / mol calculated by DFT. The Zn-O bond length is 2.016 Å, indicating a strong coordination complex between the two. Second, there is a π-π conjugated stacking effect between the benzene ring structure of PFD and the five-membered ring of the imidazole ligand of ZIF-8, with an adsorption energy of -101.41 kJ / mol and a π-π conjugated plane spacing of 3.768 Å, which is consistent with the characteristic distance of typical aromatic ring π-π interactions, indicating that this interaction is significant in the adsorption process of PFD and ZIF-8. Third, the atoms within the PFD and ZIF-8 cage form multiple electrostatic interactions, with an adsorption energy of -100.02 kJ / mol calculated by DFT. The interatomic interaction distances are 2.349 Å, 3.001 Å, 3.031 Å, and 3.209 Å, respectively. The different distance distributions indicate the existence of alternating strong and weak electrostatic interactions, further illustrating that electrostatic interactions play an important role in the adsorption process. These results corroborate existing research findings on the adsorption mechanism of MOF materials, indicating that the adsorption process of PFD by ZIF-8 is the result of coordinated complexation, π-π conjugate stacking, and multiple electrostatic interactions.

[0033] Example 2 To enhance the myocardial targeting capability of nanoparticles, this invention covalently modifies the surface of a Z@PFD nanocarrier with the myocardial targeting peptide PCM, and evaluates the efficiency of PCM peptide modification on the Z@PFD nanocarrier using CHP binding rate measurement. The PCM peptide was labeled with FITC to exhibit green fluorescence; Z@PFD nanodroplets were labeled with DiI to exhibit red fluorescence. After the labeled PCM peptide and Z@PFD nanodroplets reacted fully, the samples were observed under an inverted fluorescence microscope. Images of the PCM peptide (green fluorescence) and the Z@PFD nanoprobe (red fluorescence) were acquired under blue and green light excitation channels, respectively, and then the images were merged. The results are shown in the appendix. Figure 4 As shown in the image, the red and green fluorescence in the orange merged image exhibit ideal co-localization, directly confirming the efficient binding of Z@PFD nanoprobes and PCM peptides, suggesting that the constructed Z@PFD@PCM NPs possess cardiomyocyte targeting potential. This study used an EDC / NHS bifunctional linker to mediate the coupling reaction, primarily based on three considerations: first, the EDC / NHS linker system has no cytotoxic effects in biological systems, ensuring safety for application in organisms; second, the byproducts generated in the reaction have good water solubility, facilitating subsequent purification and washing; and third, the NHS / EDC-mediated covalent bonding mode can precisely regulate the surface modification process of biomolecules while ensuring the structural stability of the nanoparticle surfactant layer, providing a reliable guarantee for the construction of the targeted delivery system and ensuring that the nanoparticles maintain their structural and functional integrity during delivery, thereby better exerting their cardiomyocyte targeting effect.

[0034] Example 3 Simultaneous hemolytic and cytotoxicity assays were conducted to verify the blood compatibility and cellular safety of the Z@PFD@PCM nanomedicine delivery system. This comprehensive validation of the Z@PFD@PCM nanomedicine delivery system provides crucial safety assessment data for its subsequent clinical application in anti-fibrotic therapy. Hemolytic assay: The nanoparticle solution was co-incubated with erythrocytes, and the rupture of erythrocytes was observed and recorded. The hemolysis rate was measured to assess the effect of the nanocarrier on erythrocytes. Cytotoxicity assay: The nanocarrier was exposed to normal cells at the same concentration range (1-5 mg / mL), and its effect on normal cell viability was detected to determine its cytotoxicity. Results are shown in the appendix. Figure 5As shown, when the nanoparticle concentration is within the high-dose range of 1-5 mg / mL, the hemolysis rate remains consistently below the international safety threshold of 5%, and no significant erythrocyte rupture is observed. The Z@PFD@PCM nanomedicine delivery system does not induce significant erythrocyte rupture, demonstrating good blood compatibility. Simultaneously, cytotoxicity experiments show that this nanocarrier has less than 10% impact on the viability of normal cells within the same concentration range. These results fully demonstrate that the Z@PFD@PCM nanomedicine delivery system not only possesses excellent blood compatibility but also exhibits low cytotoxicity, providing crucial safety data support for its clinical translation in the field of antifibrotic therapy. Furthermore, from the perspective of tolerability at both the blood system and cellular levels, it further confirms its application potential in related disease interventions.

[0035] (1) In vitro cardioprotective effect See appendix Figure 6 The intracellular cardioprotective properties of the Z@PFD@PCM nanomedicine delivery system were investigated using immortalized fibroblasts. Fluorescence microscopy was used to observe Z@PFD@PCM nanoparticles (e.g., DiI-labeled). Samples containing only Z@PFD and Z@PFD@PCM were observed at different time points (0.5 h and 1 h). The dynamic process of nanoparticle entry into cells was studied by detecting changes in intracellular fluorescence intensity over time. The results showed that intracellular fluorescence intensity increased in a time-dependent manner, indicating that PCM modification targeting the nanoparticle surface can promote cell internalization through a fusion mechanism.

[0036] Immunofluorescence assay: A model group and a Z@PFD@PCM group were set up, and immunofluorescence assays were performed on both groups to observe the expression and distribution of connective tissue growth factor (CTGF) and its downstream components Col-I and Col-III. The effect of Z@PFD@PCM nanoparticles on myocardial fibrosis was evaluated by comparing the fluorescence intensity and distribution patterns of these factors in the two groups. In the immunofluorescence assay, the model group showed a large amount of fluorescent deposition in the myocardial interstitium, mainly distributed in the intercellular spaces of cardiomyocytes, revealing the occurrence of myocardial fibrosis. Connective tissue growth factor (CTGF) plays an important role as a key pro-fibrotic cytokine in the fibrotic process. CTGF can upregulate the expression of Col-I and Col-III and promote their transcription and translation by binding to specific reactive elements in the Col-I promoter region. In the Z@PFD@PCM group, the fluorescence intensity of CTGF decreased from 80% to 57% compared to the model group, and the distribution pattern changed from a large amount of reticular deposition in the model group to a scattered point distribution. This directly reflects that Z@PFD@PCM has an inhibitory effect on CTGF. Z@PFD@PCM may reduce the synthesis and deposition of downstream Col-I and Col-3 extracellular matrix components by inhibiting the expression of CTGF or blocking its signaling pathway. CTGF upregulates the expression of Col-I and Col-III. The inhibitory effect of Z@PFD@PCM on CTGF is further reflected in the expression of downstream collagen. The positive area of ​​Col-I decreased from 18.7% to 7.2% (P<0.01); the positive area of ​​Col-3 decreased from 18.7% to 7.2% (P<0.01). This indicates that Z@PFD@PCM nanoparticles inhibit CTGF, thereby reducing the synthesis and deposition of downstream Col-I and Col-III extracellular matrix components, thus inhibiting myocardial fibrosis and demonstrating its potential cardioprotective function.

[0037] Cellular qPCR: To further verify the inhibitory effect of Z@PFD@PCM on myocardial fibrosis at the gene level, the mRNA expression levels of type I collagen (Col-I) and transforming growth factor-β (TGF-β1) were detected using real-time quantitative PCR (qPCR). Results are shown in the appendix. Figure 7As shown in the figure, the relative expression levels of Col-I and TGF-β1 mRNA in the model group were 4.3-fold and 2.7-fold higher than those in the control group, respectively (P<0.001), indicating a significant upregulation of pro-fibrotic genes. After treatment with Z@PFD@PCM, the expression level of Col-I mRNA decreased by 55.81% compared with the model group, and the expression level of TGF-β1 mRNA decreased by 63.04%, recovering to 1.3-fold and 1.5-fold higher than those in the control group, respectively, consistent with the protein expression trend detected by immunofluorescence. These results suggest that Z@PFD@PCM may inhibit the progression of myocardial fibrosis at the gene level by downregulating the transcriptional levels of key factors in the TGF-β signaling pathway and its downstream target gene Col-I.

[0038] (2) In vivo study of intervention in myocardial fibrosis in mice Further application experiments were conducted on the nanomedicine delivery system provided by this invention to investigate its therapeutic effect on a drug-induced mouse model of myocardial fibrosis. Specifically, thirty mice were randomly divided into three groups (5 mice per group): sham-operated group, myocardial fibrosis group, myocardial fibrosis + Z@PFD group, and myocardial fibrosis + Z@PFD@PCM group. Echocardiography was used to assess cardiac function, including ejection fraction, left ventricular ejection fraction, and diastolic / systolic left ventricular diameter (LVIDd / LVIDs), as detailed in the appendix. Figure 8 As shown in the figure. Echocardiographic results showed that cardiac function was significantly impaired in mice in the myocardial fibrosis group compared to the sham-operated group. Cardiac function recovered after treatment with nanoparticles of different compositions. Quantitative analysis showed that cardiac function (EF = 58.34%, FS = 31.07%) was significantly decreased in the myocardial fibrosis group, while cardiac function (EF = 77.40%, FS = 44.66%) in the sham-operated group showed no significant change. For mice in the myocardial fibrosis group + Z@PFD group, the mean ejection fraction and left ventricular ejection fraction were 60.91% and 31.97%, respectively. Interestingly, the mean ejection fraction and left ventricular ejection fraction in the targeted Z@PFD@PCM group were 77.74% and 43.77%, respectively, higher than the non-targeted group and significantly higher than the sham-operated group. This is mainly attributed to the synergistic effect of the active targeting of the peptide and the enhanced penetration and retention (EPR) effect in ischemic cardiac tissue, thereby increasing drug accumulation. However, there was no statistically significant difference in diastolic / systolic left ventricular diameter (LVIDd / LVIDs), suggesting a time lag effect between functional improvement and structural repair. This may also be due to the small sample size, which resulted in the difference between groups not reaching a significant level. Further verification is needed by increasing the sample size or repeating the experiment.

[0039] Histopathological analysis of paraffin-embedded cardiac tissue further confirmed the therapeutic effect of the Z@PFD@PCM nanomedicine delivery system on myocardial fibrosis. Results are attached. Figure 9 As shown in the figure. H&E staining results revealed that the myocardial tissue in the model group exhibited typical pathological changes: disordered arrangement of cardiomyocytes, significantly irregular nucleus morphology, and extensive inflammatory cell infiltration, approaching the physiological level of the sham-operated group. In contrast, the Z@PFD@PCM treatment group showed significantly reduced cardiomyocyte damage and decreased inflammatory cell infiltration density. Further assessment of myocardial fibrosis using Masson staining revealed extensive blue collagen fiber deposition in the myocardial interstitium of the model group. Quantitative analysis using Image-Pro Plus software showed that the percentage of fibrosis area reached 12.63%, significantly higher than the 0.49% in the sham-operated group. Different nanoparticle interventions resulted in a dose-dependent reduction in myocardial collagen deposition, with the Z@PFD@PCM group showing the most significant effect, reducing the percentage of fibrosis area to 4.66%, only 36.8% of the model group. These results suggest that Z@PFD@PCM nanoparticles have a clear dose-response relationship and excellent therapeutic potential in inhibiting the progression of myocardial fibrosis.

[0040] (3) Immunohistochemical analysis Immunohistochemical experiments further confirmed that the Z@PFD@PCM nanomedicine delivery system can significantly inhibit the expression of myocardial fibrosis markers. See Appendix for details. Figure 10 As shown in the figure, compared with the sham-operated group, the positive staining area of ​​Col-I (13.14%) and Col-III (19.52%) in the cardiac tissue of the model group was significantly increased, while the Z@PFD@PCM treatment group reduced the expression of Col-I and Col-III to 2.89% and 2.28%, respectively, suggesting that this nanosystem can effectively inhibit excessive deposition of extracellular matrix. CD34, as a marker transmembrane glycoprotein of hematopoietic stem cells and vascular endothelial progenitor cells, has attracted much attention for its dynamic expression in myocardial fibrosis. The expression level of CD34 in normal myocardial tissue is extremely low, but in the fibrotic microenvironment, bone marrow-derived or locally activated CD34+ cells can migrate to the injury site, and some cells participate in collagen deposition by differentiating into myofibroblasts (α-SMA+). In this study, immunohistochemical analysis showed that the number of CD34+ cells in the model group was significantly higher than that in the sham-operated group (9.03%), while Z@PFD@PCM treatment reduced it to 3.74%. Based on previous research, this effect may be related to PFD inhibiting the TGF-β1 / Smad signaling pathway—TGF-β1 can promote CD34 activation by activating Smad2 / 3. + Cells differentiate into myofibroblasts, and the targeted delivery of Z@PFD@PCM can enhance local drug concentration and block this differentiation process.

[0041] The above results not only confirm the multiple inhibitory effects of Z@PFD@PCM on myocardial fibrosis (including inhibition of collagen synthesis and regulation of progenitor cell differentiation), but also provide evidence for CD34... +The dual pro-inflammatory and pro-fibrotic role of cells in fibrosis provides new evidence and offers potential strategies for targeted intervention in the fibrotic microenvironment.

[0042] (4) Immunofluorescence analysis of IL-6 and IL-1β In recent years, numerous studies have confirmed that pro-inflammatory cytokines play a crucial driving role in the pathological process of cardiac fibrosis. Among them, IL-6 and IL-1β induce fibroblast activation and extracellular matrix deposition by activating signaling pathways such as NF-κB and JAK-STAT. To explore the regulatory effects of different treatment strategies on the inflammatory microenvironment, this study used immunofluorescence staining to visualize and analyze IL-6 and IL-1β proteins in cardiac tissue. The results are shown in the appendix. Figure 11 The results showed that, compared with the sham-operated group, the fluorescence area of ​​IL-6 and IL-1β in the cardiac tissue of the model group was significantly enhanced (IL-6: 7.24%; IL-1β: 4.98%), indicating a strong activation of the inflammatory response. After nanoparticle treatment, the expression levels of IL-6 and IL-1β in all treatment groups were significantly downregulated (IL-6: 3.86% in the non-targeted group; IL-1β: 2.99% in the targeted group; IL-6: 1.04% in the targeted group; IL-1β: 1.28%). Among them, the Z@PFD@PCM nanomedicine delivery system group showed the strongest anti-inflammatory effect, significantly better than other treatment groups. These results indicate that the Z@PFD@PCM nanomedicine delivery system may block the fibrosis process by targeting and inhibiting the IL-6 / IL-1β-mediated inflammatory cascade. Meanwhile, the results of SOD enzyme expression tests in each group showed that the sham-operated group, due to the absence of myocardial injury and low oxidative stress levels, maintained a normal physiological SOD enzyme content (132.9 U / mL). The model group, however, experienced a severe inflammatory response due to myocardial injury, leading to the generation of large amounts of free radicals and continuous consumption of SOD enzymes to combat oxidative stress; its content (66.23 U / mL) was significantly lower than that of the sham-operated group. After nanoparticle treatment, as the IL-6 and IL-1β-mediated inflammatory response was inhibited, oxidative stress levels decreased, SOD enzyme consumption decreased, and the content significantly rebounded compared to the model group. The non-targeted group showed no significant difference in SOD enzyme content compared to the model group, possibly due to the smaller number of animals and shorter treatment period. The Z@PFD@PCM nanomedicine delivery system group, exhibiting the strongest anti-inflammatory effect and the most significant improvement in oxidative stress, had an SOD enzyme content (125.8 U / mL). The U / mL level rebounded to levels closer to those of the sham-operated group. This result further validates the role of the targeted nanoparticles in indirectly regulating oxidative stress by inhibiting the inflammatory response, completes the evidence chain of the "inflammation-oxidative stress-fibrosis" regulatory chain, highlights its advantages in improving the pathological process of myocardium at multiple targets, and provides a reference for optimizing subsequent combined anti-inflammatory and antioxidant treatment strategies.

[0043] (5) Animal QPCR To deeply analyze the molecular pathological mechanisms in the progression of myocardial fibrosis and clarify the potential effects of targeted nanoparticle intervention, this study used real-time quantitative PCR technology to systematically detect the mRNA expression levels of key genes in fibrosis and inflammation-related signaling pathways. The results are shown in the appendix. Figure 12 The results showed that in cardiac tissue of a myocardial fibrosis model, the gene expression of pro-fibrotic factor TGF-β1, the major extracellular matrix component Col-1, the downstream effector molecule CTGF of TGF-β1, the myofibroblast marker α-SMA, and the pro-inflammatory cytokines IL-6 and IL-1β were all significantly upregulated. This is highly consistent with the molecular characteristics of inflammatory response activation and excessive extracellular matrix deposition in the pathological process of fibrosis in previous studies. Notably, after targeted nanoparticle therapy, the abnormally high expression of the above genes showed a significant downregulation trend, suggesting that the nanoparticles may play a role in delaying the progression of myocardial fibrosis by inhibiting the overactivation of fibrosis and inflammatory signaling pathways. These results not only further elucidate the molecular pathological mechanism of myocardial fibrosis development but also provide crucial molecular-level evidence for the application of targeted nanoparticles in the treatment of myocardial fibrosis.

[0044] To systematically evaluate the in vivo biosafety of the targeted nanoparticles, comprehensive samples were collected from vital organs such as the brain, liver, kidneys, spleen, and lungs, as well as blood, after the intervention. Multidimensional assessments were conducted through hematological analysis and histopathological examination. See the appendix for details. Figure 13 In the histopathological evaluation, hematoxylin and eosin (H&E) staining was used for microscopic observation of the morphology of various organs and tissues. The results showed that the liver lobule structure was intact, the hepatocyte morphology was normal, and no pathological changes such as swelling or necrosis were observed; the kidney glomeruli and renal tubules were clearly structured, and no protein casts or interstitial fibrosis were observed; the white pulp and red pulp of the spleen were clearly demarcated, and the lymphocytes were evenly distributed; the alveolar structure of the lungs was intact, and there were no abnormalities such as hemorrhage, edema, or inflammatory exudation. Hematological tests showed that liver and kidney function indicators such as alanine aminotransferase (ALT) and creatinine (Cr) in mice in each experimental group were within the normal physiological range. This indicates that during the experimental period, the targeted nanoparticles did not cause significant toxic effects on the mouse blood system, liver, and kidneys, demonstrating excellent biocompatibility and safety. This result is consistent with the previous conclusion that nanocarriers should not induce tissue inflammation or structural damage. This discovery not only conforms to the core principle of "safety-efficacy balance" in nanomedicine development, but also lays an important foundation for further exploration in the field of targeted therapy for myocardial fibrosis, and is expected to promote the application and development of nanoparticles in the treatment of cardiovascular diseases.

[0045] (5) Therapeutic mechanisms at the molecular and protein levels To investigate the mechanism of action of PFD, molecular docking and target prediction techniques were used to systematically analyze its potential molecular targets. (See appendix) Figure 14 The study found that PFD can specifically bind to key amino acid residues such as K1242, N1243, R1246, and A1245 on Col-1, and also interact with CTGF and TGF-β1, suggesting that it can inhibit the fibrosis process by blocking the cascade reaction of "TGF-β1→CTGF→collagen synthesis". Furthermore, the binding characteristics of PFD with IL-1β and IL-6 reveal its potential to modify cytokines and exert anti-inflammatory effects. In summary, PFD exhibits a synergistic "anti-fibrosis-anti-inflammatory" dual-effect. This finding not only provides a new perspective on elucidating the pharmacological mechanism of PFD, but also brings new directions for the development of multi-target precision treatment strategies for diseases such as pulmonary fibrosis and liver fibrosis.

[0046] To identify molecules involved in PFD-mediated regulation of myocardial fibrosis and inflammation, 4D Fast DIA proteomics was used to detect enriched proteins after treatment with the targeted nanoparticles Z@PFD@PCM. Based on bioinformatics analysis, a total of 5018 reliable proteins were identified, of which 4981 were quantifiable and comparable. Principal component analysis (PCA) showed a clear boundary between the Z@PFD@PCM treatment group and the control group.

[0047] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A nanodrug delivery system for myocardial fibrosis treatment, characterized by, The nano drug delivery system takes ZIF-8 with a myocardial cell specific targeting peptide as a core carrier, the core carrier is wrapped with a drug inside, the drug is a drug for myocardial fibrosis treatment, and the sequence of the myocardial cell specific targeting peptide is shown as SEQ ID NO.

1.

2. The nano-drug delivery system for the treatment of myocardial fibrosis according to claim 1, wherein, The drug for myocardial fibrosis treatment is any one of pirfenidone, sunitinib and nintedanib.

3. The nano-drug delivery system for the treatment of myocardial fibrosis according to claim 1, wherein, The myocardial targeting peptide is modified on the surface of the ZIF-8 core carrier through covalent coupling.

4. The nano drug delivery system for myocardial fibrosis treatment according to claim 1, wherein the core carrier cooperatively wraps the drug through coordination complexation, π-π conjugate stacking and electrostatic interaction.

5. A method for preparing a nanomedicine delivery system for the treatment of myocardial fibrosis as described in any one of claims 1 to 4, characterized in that, The method comprises the following steps: Step 1: mixing a zinc nitrate methanol solution with a 2-methyl imidazole methanol solution, standing, centrifuging, washing and vacuum drying to obtain a ZIF-8 powder; Step 2: dissolving the ZIF-8 powder and the drug, liquid stirring and reaction, centrifuging, washing and vacuum drying to obtain Z@drug; Step 3: mixing a carbodiimide compound, a hydroxysuccinimide compound and a myocardial targeting peptide, adding a 2-(N-morpholino) ethanesulfonic acid (MES) solution for reaction at room temperature, adjusting the pH value after reaction, adding the Z@drug modified with PEG-PEI 25K for stirring reaction, centrifuging, washing, vacuum drying and obtaining the nano drug delivery system for myocardial fibrosis treatment.

6. The method for preparing a nanomedicine delivery system for cardiovascular disease treatment according to claim 5, characterized in that, In step 1, the molar ratio of the zinc nitrate to the 2-methyl imidazole is 1:1-2; in step 2, the mass ratio of the ZIF-8 to the pirfenidone is 2:1, and the stirring reaction is room temperature stirring for 1-3 hours.

7. The method for preparing a nanomedicine delivery system for cardiovascular disease treatment according to claim 5, characterized in that, In step 3, the mass ratio of the carbodiimide compound, the hydroxysuccinimide compound and the myocardial targeting peptide is 2:4:1, the reaction time at room temperature is 20-26 hours, and the stirring reaction time at room temperature is 3-5 hours.

8. The nano drug delivery system for myocardial fibrosis treatment according to any one of claims 1-4 is applied to the preparation of a drug for treating myocardial fibrosis.

9. Use according to claim 8, characterized in that, The nano drug delivery system inhibits the myocardial fibrosis process by inhibiting the activity of connective tissue growth factor, down-regulating the transcription level of a key factor in a TGF-β signal pathway and a downstream target gene Col-I and inhibiting the expression of a myocardial fibrosis marker.

10. A medicament for treating myocardial fibrosis, characterized by, The drug comprises the nano drug delivery system for myocardial fibrosis treatment according to any one of claims 1-4.

Images