A multifunctional nano-coated stent for preventing and treating in-stent restenosis and a preparation method and application thereof

Abstract

The application provides a multifunctional nano-coated stent for preventing and treating in-stent restenosis and a preparation method and application thereof. Based on the multifunctional nano interface of EGCG-Cys, the liposome targeting VCAM-1 is used to deliver MnO2 nanoscale enzyme and FGF21 growth factor, so that the triple treatment effects of active oxygen scavenging, immune regulation and endothelial regeneration can be realized at the vascular lesion site. The EC-MFVL-WE43 stent creates a new paradigm for the design of interventional instruments, realizes the spatiotemporal precise release of drugs through the oxidative stress triggered degradation mechanism, and verifies the excellent anti-restenosis and anti-thrombosis performance in mouse, rabbit and pig models. Mechanism research shows that the nano-coated stent can reprogram macrophages to M2 repair type polarization, and effectively inhibit the abnormal proliferation of smooth muscle cells, providing a biological activity solution to break the vicious cycle of "inflammation-oxidation-restenosis".

Description

Technical Field

[0001] This invention relates to the field of polymer materials, and in particular to a multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis, its preparation method, and its application. Background Technology

[0002] Cardiovascular disease (CVD) is the leading cause of death worldwide, with coronary artery disease (CAD), especially acute myocardial infarction, being a major driver of CVD deaths. Recent data shows that CAD causes approximately 17.9 million deaths annually, accounting for 30% of all deaths globally. Atherosclerosis (AS) is the fundamental pathological process of CVD; the rupture of unstable atherosclerotic plaques leading to acute coronary artery obstruction is the primary cause of the high mortality rate from CAD. Percutaneous coronary intervention (PCI) is the preferred treatment, rapidly restoring blood flow to narrowed coronary arteries, salvaging ischemic myocardium, and reducing mortality. In 2023, the number of PCI procedures in China exceeded 1.4 million, a 10% increase compared to 2022. Stent implantation, as the main method of PCI, is more effective than simple balloon angioplasty in preventing vascular recoil. However, in-stent restenosis (ISR) remains a significant challenge affecting the long-term efficacy of PCI and leading to CAD recurrence. Current strategies for preventing and treating ISR are still not ideal, with an ISR incidence rate exceeding 10% after stent implantation. More worryingly, approximately 25% of ISR cases result in acute myocardial infarction due to delayed intervention, which is often fatal. Therefore, in-depth research on ISR is urgently needed to improve the clinical success rate of PCI in CAD patients. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis.

[0004] Another object of the present invention is to provide a method for preparing the above-described multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis.

[0005] Another object of the present invention is to provide the application of the above-described multifunctional nanocoated stent for the prevention and treatment of in-stent restenosis.

[0006] The objective of this invention is achieved through the following technical solution: A multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis is prepared by the following steps: (1) Preparation of manganese dioxide particles Dissolve potassium permanganate in a solution, add reaction reagents, stir to precipitate, centrifuge and wash the product, dry to obtain manganese dioxide particles; (2) Preparation of MF-FGF21 Manganese dioxide particles were added to water, the pH was adjusted, and the mixture was sonicated. EDC and NHS were added, and the mixture was shaken to activate the manganese dioxide particles. After centrifugation and washing, FGF21 solution was added, and the mixture was incubated. After centrifugation, the particles were inactivated and washed to obtain MF-FGF21. (3) Preparation of liposome solution Cholesterol, DPPC, DEPE-PEG2K and DSPE-PEG2K-MAL were dissolved in chloroform, and a thin film was formed by rotary evaporation. The film was then hydrated by adding MF-FGF21 suspension to obtain a liposome solution. (4) Preparation of MFVL The VHPKQHR-peptide was dissolved in PBS, mixed with TCEP solution, and placed at room temperature. Then it was mixed with the liposome solution prepared in step (3), the mixture was sonicated, incubated overnight, filtered, and purified to obtain MFVL. (5) Stent pretreatment The metal stent was ultrasonically cleaned with water and acetone, dried, and then immersed in Tris buffer solution. After that, it was removed and cleaned. EGCG and Cys were added to the Tris buffer solution and dispersed by ultrasonic vibration to obtain a pretreatment solution. The cleaned metal stent was then immersed in the pretreatment solution and magnetically stirred. After the reaction was completed, the stent was removed and cleaned for later use. (6) Nanocoating loading MFVL was dispersed in Tris buffer to obtain a dispersion. The pretreated metal stent was immersed in the dispersion and loaded by shaking. After loading, the stent was removed, washed, and dried to obtain a multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis.

[0007] The solution mentioned in step (1) is hydrochloric acid or water.

[0008] When the solution is hydrochloric acid, the ratio of potassium permanganate to hydrochloric acid is 1-2 mg:1 mL; preferably 2 mg:1 mL.

[0009] The concentration of the hydrochloric acid is 0.1–0.2 mol / L; preferably 0.1 mol / L.

[0010] The reaction reagents in step (1) are citric acid or sodium hydroxide.

[0011] The final concentration of the citric acid is 50-200 mM; preferably 100 mM.

[0012] The mass ratio of sodium hydroxide to potassium permanganate is 1-2:1-2; preferably 7:8.

[0013] The stirring reaction conditions described in step (1) are stirring at room temperature for 0.5 to 24 hours.

[0014] The centrifugation conditions described in step (1) are 800–1200 rpm for 5–20 min.

[0015] The pH adjustment mentioned in step (2) is to adjust the pH to 5-7.

[0016] The final concentration of EDC in step (2) is 1 to 3 mg / mL.

[0017] The final concentration of NHS in step (2) is 3 to 5 mg / mL.

[0018] The shaking condition described in step (2) is to shake for 20 to 40 minutes.

[0019] The mass ratio of manganese dioxide particles to FGF21 in step (2) is 10:1.

[0020] The inactivation in step (2) is performed by using 1wt% BSA for 20-40 minutes.

[0021] The molar ratio of cholesterol, DPPC, DEPE-PEG2K and DSPE-PEG2K-MAL in step (3) is 4-6:6-8:1-2:0.1-0.3; preferably 4:6:1:0.2.

[0022] The hydration in step (3) involves adding a MF-FGF21 suspension preheated to 50-60°C and shaking it in a water bath at 150-200 rpm for 30-90 minutes.

[0023] The mass ratio of MF-FGF21 to DSPE-PEG2K-MAL in step (3) is 50:1.

[0024] The VHPKQHR-peptide mentioned in step (4) is a short peptide with the amino acid sequence VHPKQHR.

[0025] The molar ratio of VHPKQHR-peptide to TCEP in step (4) is 1:2.

[0026] The placement conditions described in step (4) are to place the item at room temperature for 20 to 40 minutes.

[0027] The filtration described in step (4) involves filtering 40 to 60 times using a 200 nm filter.

[0028] The purification described in step (4) is performed using an S-100 dextran gel column.

[0029] The metal bracket mentioned in step (5) is a WE43 metal bracket.

[0030] The concentration of the Tris buffer solution in step (5) is 2 mg / mL and the pH is 8.5.

[0031] The immersion in Tris buffer solution in step (5) is for 1 to 3 hours.

[0032] The molar ratio of EGCG to Cys in step (5) is 1:2.

[0033] The magnetic stirring conditions described in step (5) are magnetic stirring for 10 to 15 hours.

[0034] The concentration of MFVL in the dispersion described in step (6) is 1 to 2 mg / mL.

[0035] The oscillation conditions described in step (6) are oscillation at 100-150 rpm for 10-15 hours.

[0036] The concentration of the Tris buffer in step (6) is 10 mM and the pH is 8.5.

[0037] The above-mentioned multifunctional nano-coated stents for the prevention and treatment of in-stent restenosis are used in the treatment of atherosclerosis.

[0038] The above-mentioned multifunctional nano-coated stents for the prevention and treatment of in-stent restenosis are used in the relief of in-stent restenosis. Attached Figure Description

[0039] Figure 1 The results are the peroxidase-like activity analysis results of the manganese dioxide particles in Example 1.

[0040] Figure 2 These are the characterization results related to the load-bearing effect of manganese dioxide particles and EC-MFVL-WE43 stent in Example 3.

[0041] Figure 3 This is a graph showing the experimental results of the EC-MFVL-WE43 scaffold in Example 4, which induced endothelial functional remodeling in vitro.

[0042] Figure 4 This is a diagram showing the experimental results of how the EC-MFVL nanocoating in Example 4 can regulate the characteristic oxidized lipid microenvironment of atherosclerotic lesions in vitro.

[0043] Figure 5 This is a graph showing the results of the in vivo anti-atherosclerotic and antioxidant effects of the MFVL nanoplatform in Example 5.

[0044] Figure 6 This is a graph showing the experimental results of the in vivo anti-in-stent restenosis efficacy of the EC-MFVL nanocoated stent in Example 6. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0047] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0048] Example 1: Preparation of manganese dioxide particles 1.1 Preparation of MnO2NFs 80 mg KMnO4 was dissolved in 40 mL of dilute hydrochloric acid (0.1 mol / L) to obtain a uniform purple-red solution. Then, 1 mL of citric acid (100 mM) was added to the solution and stirred at room temperature for 30 min until a brown suspension appeared in the purple-red solution. The reaction product was then centrifuged and washed three times (1000 rpm, 10 min), and then dried under vacuum at -50 °C to obtain MnO2NFs.

[0049] 1.2 Preparation of MnO2SP KMnO4 (700 mg) was dispersed in 10 mL of deionized water, and the mixture was stirred for 5 minutes to produce a deep purple solution. NaOH (800 mg) was then added to the KMnO4 solution, and the mixture was stirred for 24 hours until the solution turned brown. The obtained MnO2SP precipitate was centrifuged, washed three times with DI water, and then dried in a vacuum desiccator at room temperature. Finally, the product was calcified at 500 °C for 3 hours to obtain MnO2SP.

[0050] 1.3 Peroxidase-like activity analysis of manganese dioxide particles The peroxidase-like activity of MNM was studied by catalytic oxidation of TMB in the presence of H2O2. The specific steps are as follows: A certain amount of MnO2NFs and MnO2SP were suspended in phosphate-citrate buffer (0.15 M) at 100 µg / mL. 200 μL of each sample was added to each well of a 96-well plate along with 2 μL of TMB solution (1.25 mM, ethanol) and 4 μL of H2O2. The plate was then shaken in the dark to provide a homogeneous mixture, and the UV-Vis spectra (500–800 nm) of the samples were recorded using a microplate reader. After 10 minutes of reaction, the absorbance at 652 nm was measured using UV-Vis spectroscopy. The relative activity (%) was used as an indicator of optimized experimental conditions, calculated by dividing the current absorbance by the maximum absorbance at 652 nm relevant to the specific experimental conditions.

[0051] Subsequently, the oxidase-like activities of the two nanozymes in the absence of H2O2 were evaluated using TMB substrates. Following the method described above, 200 μL samples suspended in phosphate-citrate buffer and 2 μl TMB were used for the experiment. After incubation for 10 minutes, the UV-Vis absorption spectra (500-800 nm) of each sample were recorded.

[0052] Experimental results are as follows Figure 1 As shown, MnO2NFs exhibits relatively higher peroxidase-like activity than MnO2SP. In the absence of H2O2, both MnO2NFs and MnO2SP catalyze the rapid oxidation of TMB, with MnO2NFs exhibiting stronger oxidase-like activity. Therefore, MnO2NFs was used in subsequent experiments.

[0053] Example 2: Preparation of a multifunctional nano-coated scaffold 2.1 Preparation of MF-FGF21 A certain amount of MnO2NFs was weighed and added to water (0.5 mg / mL, 1 mL). The pH was adjusted to 6.0 by sonication with K2CO3 at 37 °C (100 W) for 1 h. EDC (final concentration 2 mg / mL) and NHS (final concentration 4 mg / mL) were added to the solution, and the mixture was gently shaken for 30 min to activate the MnO2NFs. The mixture was centrifuged and washed three times before use. Then, FGF21 (100 μg / mL, 0.5 mL) in PBS was mixed with the activated MnO2NFs and incubated at 37 °C for 1 h. After centrifugation three times to remove unbound fractions, MnO2NFs-FGF21 (MF) was obtained. The product was inactivated with 1% BSA for 30 min, then dispersed in 1 mL PBS, and then dispersed by sonication (100 W, 3 min) at room temperature. The particles were washed and collected by high-speed centrifugation (1000 rpm for 10 min), then dried by freeze dryer, and stored at 4°C for later use.

[0054] In addition, following the steps above, FGF21 was replaced with an equal amount of deionized water to prepare activated MnO2NFs.

[0055] 2.2 Preparation of VHPK-Lipo (1) Cholesterol (3.87 mg), DPPC (11 mg), DEPE-PEG2K (5 mg), and DSPE-PEG2K-MAL (2 mg) were dissolved in chloroform (3 mL) at a molar ratio of 4:6:1:0.2. The solution was then transferred to a round-bottom flask and connected to a rotary evaporator. The water bath temperature was set to 45°C. Most of the solvent was evaporated at a low vacuum at a moderate rotation speed. After the liquid volume decreased significantly, the vacuum was gradually increased to the maximum value of the system. Rotation was continued until a uniform lipid film without any trace of flowing liquid formed on the inner wall of the flask. The high vacuum was then maintained and the flask was rotated for at least 30 minutes to ensure that the chloroform was completely removed.

[0056] (2) After the dried lipid film formed by rotary evaporation is cooled to room temperature, 1 mL of deionized water preheated to 50-60°C is slowly added along the bottle wall. The bottle mouth is then sealed with sealing film and placed in a constant temperature water bath shaker at 50-60°C. The shaker is gently oscillated at 150-200 rpm for 1 hour. Violent vortexing or shaking should be avoided during this process to ensure that the lipid film can be fully and gently hydrated and swollen to obtain a liposome solution.

[0057] (3) Dissolve VHPKQHR-peptide (about 2 mg) in PBS and mix it with 0.5 M TCEP solution (the molar ratio of VHPKQHR-peptide to TCEP is 1:2). Then place it at room temperature for about 30 minutes, activate VHPKQHR peptide with TCEP in PBS, and then mix it with the liposome solution prepared in step (2). Sonicate the mixture at 45 °C for 0.5 h, and then incubate it at 4 °C overnight. Filter the suspension through a 200 nm filter 50 times at 50 °C, and then purify the nanoparticles through an S-100 dextran gel column to remove free components and obtain VHPK-Lipo.

[0058] 2.3 Preparation of MFVL The preparation method is the same as described in 2.2, except that the addition of 1 mL of deionized water in step (2) is changed to the addition of 1 mL of MF-FGF21 suspension (100 mg / mL) for hydration. Then, MFVL (MnO2NFs-FGF21@VHPK-Liposomes) is prepared using the same method.

[0059] In addition, MnO2NFs@VHPK-Lipo was prepared by replacing the MF-FGF21 suspension with an activated MnO2NFs suspension.

[0060] 2.4 Stent Pretreatment Before preparing the EGCG-Cys layer, the WE43 metal sheet was ultrasonically cleaned three times (10 min each time) with acetone and ultrapure water (UP), and then dried with N2 for 2 h before use. The WE43 metal pad was immersed in Tris buffer solution (2 mg / mL, pH 8.5) for 2 h, and then washed three times (3 min each time) with ultrapure water. Tris buffer solution (10 mM, pH 8.5) was prepared, and EGCG and Cys were added to the prepared Tris buffer solution to dissolve them, so that the molar ratio of EGCG to Cys was 1:2 (EGCG 0.4 mM, Cys 0.8 mM). The prepared solution was placed in an ultrasonic oscillator for 3 min to ensure that all solutes were uniformly dispersed and completely dissolved. The WE43 metal pad was immersed in the solution and magnetically stirred thoroughly at room temperature for 12 h. The sample was then removed and ultrasonically cleaned with deionized water for 1 min. The prepared sample was then ready for use.

[0061] 2.5 nm coating load First, MFVL liposomes were dispersed in Tris buffer (10 mM, pH 8.5) at a concentration of 1 mg / mL. The WE43 metal sheet with EGCG-Cys coating was then immersed in the MFVL liposome dispersion and shaken for 12 h (in horizontal reciprocating shaking mode, at a speed of 120 rpm, at room temperature of 25°C for 12 hours). Unbound MFVL was removed by washing several times, and the sheet was freeze-dried for storage to obtain the MFVL-loaded WE43 metal sheet.

[0062] In addition, following steps 2.4 and 2.5, the WE43 metal sheet was replaced with a WE43 scaffold to obtain a WE43 scaffold loaded with MFVL. For subsequent experimental comparison, following the scaffold loading method, the MFVL liposomes were replaced with the same concentrations of VHPK-Lipo, FGF21, and MnO2NFs@VHPK-Lipo to prepare the corresponding loaded scaffolds.

[0063] Example 3: Characterization experiments related to nanocarriers 3.1 Transmission electron microscopy analysis The morphology of MnO2NFs, MF-FGF21, VHPK-Lipo and MFVL prepared in Examples 1 and 2 was observed under a transmission electron microscope (TEM).

[0064] The results are as follows Figure 2 As shown in Figure a, nanoscale morphology analysis using transmission electron microscopy (TEM) reveals significantly different structural features in each group of samples: Group I (MnO2 NFs) and Group II (MnO2 NFs-FGF21) exhibit typical nanoflower-like structures, while Group III (VHPK-Lipo) and Group IV (MnO2 NFs-FGF21@VHPK-Lipo) display spherical liposome structures. Notably, Group IV exhibits a unique core-shell structure, successfully encapsulating MnO2 nanoflowers within MFVL liposomes.

[0065] 3.2 Verification of cholesterol removal effect To evaluate the cholesterol scavenging efficiency of MnO2 NFs-FGF21@VHPK-Lipo (MFVL) nanoparticles, an in vitro reaction system constructed using FITC-labeled cholesterol crystals and Cy5.5-labeled MFVL nanoparticles was used for verification.

[0066] Experimental results are as follows Figure 2 As shown in Figure b, confocal imaging at 0 hours reveals strong spatial colocalization, indicating a high cholesterol binding affinity. After 24 hours, the fluorescence intensity of FITC-cholesterol decreased by more than 70%, confirming that MFVL nanoparticles possess potent catalytic activity and cholesterol scavenging ability.

[0067] 3.3 Detection of Oxidative Stress Hydrolysis Response Effect Experimental results are as follows Figure 2 As shown in Figure c, MFVL nanoparticles also exhibit a significant hydrogen peroxide concentration-dependent hydrolysis reaction: the degree of degradation is extremely low (<25%) under low oxidative stress conditions (0.01~0.1 mM H2O2), while it decomposes rapidly (>75%) within 20 minutes under high hydrogen peroxide concentration (3 mM), and then enters a stable plateau period.

[0068] 3.4 Load Effect Detection As a small-molecule NIR-II probe, BBT-2FT has the advantages of deep tissue penetration and low background noise. Our previous studies have confirmed that BBT-2FT can be efficiently prepared into nanoformations and successfully applied to the real-time tracking of atherosclerotic plaques, making it very suitable for integration into multifunctional diagnostic and therapeutic applications. Following the preparation method in Example 2, in step (1) of 2.2, 0.22 mg of BBT-2FT was added while dissolving the raw material in chloroform, and then EGCG-Cys-MFVL nanoparticles carrying the probe were prepared and further loaded onto a scaffold for characterization.

[0069] Experimental results are as follows Figure 2 As shown in Figure d, the visible light and NIR-II imaging results of the integrated platform demonstrate that the WE43 scaffold surface has been successfully functionalized with EGCG-Cys-MFVL nanoparticles. The coated scaffold exhibits significantly enhanced contrast under visible light and displays a strong NIR-II fluorescence signal, indicating that the nanoparticles are stably attached to the scaffold surface, providing a reliable foundation for achieving precise optical tracking.

[0070] 3.5 ROS Response Performance Testing Experimental results are as follows Figure 2 As shown in Figure e, under different oxidative conditions, the cumulative release curves of MFVL in the EGCG-Cys-MFVL-coated stent exhibit a clear H2O2-responsive release characteristic. Higher concentrations of H2O2 (200 μM) significantly accelerated drug release, with near-complete release (approximately 100%) within 20 days; while moderate (100 μM) and low (50 μM) concentrations of H2O2 resulted in slower and more sustained release kinetics. These results indicate that the EGCG-Cys-based nanocoating possesses redox-sensitive degradation capabilities, enabling environmentally adaptive drug release in response to oxidative stress levels. This characteristic is of significant importance for targeted vascular therapy of inflammatory lesions. 3.6 Other characterization experiments After surface modification of the WE43 scaffold with EGCG-Cys and EC-MFVL coatings, its water contact angles were significantly reduced from ~65° to ~15° and ~10°, respectively. Figure 2 The image (f) indicates a significant enhancement in the hydrophilicity of the material. This finding is consistent with previous reports on the excellent biocompatibility of EGCG-based coatings, and the significant improvement in hydrophilicity further supports the application potential of EGCG-Cys functionalization in promoting vascular implant integration and optimizing bio-interfaces. Scanning electron microscopy (SEM) images show (f) Figure 2 The EGCG-Cys-MFVL scaffold surface exhibits a uniform nanostructured coating, contrasting sharply with the smooth morphology of the bare scaffold. Energy-dispersive X-ray spectroscopy (EDS) elemental distribution analysis confirmed the successful introduction and uniform distribution of C, S, and Mn elements, indicating that the EGCG-Cys-MFVL nanocoating has been stably anchored to the scaffold surface. A persistent Mg signal reflects the presence of the underlying scaffold substrate, while an enhanced Mn signal highlights the successful integration of MnO2 nanodomains. These results validate the structural integrity and compositional precision of the nanocoating, providing strong support for its feasibility as a multifunctional vascular interface. The relevant experimental methods can be found in the literature "Qin Y, Zhu Y, Lu L, Wu H, Hu J, Wang F, Zhang B, Wang J, Yang X, Luo R, Chen J, Jiang Q, Yang L, Wang Y, Zhang X. Tailored extracellular matrix-mimetic coating facilitates reendothelialization and tissue healing of cardiac occluders. Biomaterials. 2025 Feb;313:122769. doi: 10.1016 / j.biomaterials.2024.122769. Epub 2024 Aug 23. PMID: 39208698.".

[0071] Example 4 Cell Experiment 4.1 Cell experiments to verify the properties of biological interfaces Figure 3 To evaluate the in vitro efficacy of EGCG-Cys-MFVL nanocoatings in preventing in-stent restenosis, a functionalized disc model was first prepared to simulate stent surface conditions. After functionalization with the EGCG-Cys-MFVL nanocoating, a uniform, firmly adhered film with characteristic color formed on the surface of WE43 alloy, confirming the success of the surface modification. Figure 3(a-b) Macrophages, smooth muscle cells, and endothelial cells are key immunomodulatory mediators in the pathogenesis of in-stent restenosis. This pathological cascade is characterized by pro-inflammatory macrophage infiltration, abnormal smooth muscle cell proliferation, and endothelial dysfunction, collectively driving neointimal hyperplasia and vascular occlusion. To recreate the immune microenvironment associated with in-stent restenosis in vitro, RAW264.7 mouse macrophages, MOVAS-1 mouse aortic smooth muscle cells, and human umbilical vein endothelial cells were used as representative cell models to evaluate the biointerface properties of the nanocoating.

[0072] Three days of in vitro biological evaluation confirmed that this multifunctional nanointerface can elicit selective cellular responses. Crystal violet staining results showed ( Figure 3 (c) On the EGCG-Cys-MFVL-modified substrate, the proliferation of human umbilical vein endothelial cells (HUVECs) was significantly enhanced, indicating that this interface created a pro-endothelial microenvironment conducive to angiogenesis. Conversely, the clonogenic ability of MOVAS cells was significantly inhibited, suggesting that smooth muscle proliferation was effectively suppressed. Consistent with this, wound healing assays showed that the EGCG-Cys-MFVL surface accelerated the migration of HUVECs while significantly attenuating the migration of MOVAS cells. Cytoskeleton staining further revealed ( Figure 3 Compared with the control group and the EGCG-Cys group, MOVAS cells cultured on EGCG-Cys-MFVL-coated WE43 pads (group 3) showed decreased F-actin expression and reduced cell density with prolonged culture time. In conclusion, this nanocoating effectively disrupts the cytoskeleton structure and inhibits smooth muscle cell spreading, demonstrating its excellent anti-proliferation and anti-restenosis potential.

[0073] 4.2 Cell model to simulate the microenvironment of atherosclerosis This experiment further validated the anti-atherosclerotic potential of the EC-MFVL nanocoating in vitro. Macrophage-derived and smooth muscle cell-derived foam cells were used as cell models of immune and vascular components to construct a biomimetic atherosclerotic microenvironment. The specific steps are as follows: 5×10 4MOVAS and RAW264.7 cells were seeded onto cell slides in six-well plates and incubated for 24 h. Then, treatment drugs were added according to the groups: Group 1 (Ctrl), Group 2 (FGF21), Group 3 (MFL), and Group 4 (MFVL), to achieve a final particle concentration of 20 μg mL⁻¹. Intracellular ROS levels were then detected using the DCFH-DA probe. DCFH-DA was diluted 1:1000 with PBS to a final concentration of 10 μmol L⁻¹. The cell culture medium was removed, and 1 mL of diluted DCFH-DA (10 μM) was added to each well of the six-well plate to fully cover the cells. The cells were incubated at 37°C for 20 min. Cells were washed three times with PBS to remove any uninfiltrated DCFH-DA. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min and rinsed twice with PBS. Finally, the slides were mounted with DAPI-containing mounting medium and observed and images were acquired under a laser confocal microscope. Prepare the ORO staining working solution according to a 3:2 ratio of Oil Red O solution and Oil Red O diluent. Mix well, let stand for 10 min, then filter through filter paper or a 0.45 µm syringe filter. Use within two hours. Fix with 4% paraformaldehyde for 15 min, wash with PBS, incubate with 60% isopropanol for 1 min, cover with Oil Red O staining solution in the dark for 15 min, separate with 60% isopropanol for 5 s, counterstain cell nuclei with hematoxylin for 1 min, observe under a microscope, and measure the percentage of ORO+ area using ImageJ software (V1.8.0).

[0074] Experimental results are as follows Figure 4 As shown, the results indicate that the EC-MFVL nanocoating (Group 4) significantly inhibited the uptake of oxLDL, intracellular lipid accumulation, and oxidative stress levels in macrophage-derived and smooth muscle cell-derived foam cells. Figure 4 (a-f) This therapeutic effect is closely related to the highly efficient targeting ability of MFVL nanoparticles, and the modification with VHPK peptides further enhances this ability. VHPK has a high affinity for vascular cell adhesion molecule-1 (VCAM-1), a key inflammatory marker that is significantly highly expressed in foam cells of atherosclerotic plaques. The ligand-receptor interaction between VHPK and VCAM-1 can promote the selective aggregation of nanoparticles in the inflammatory microenvironment enriched by foam cells and their efficient internalization by cells, thereby significantly improving the precision and efficacy of local biological interface regulation.

[0075] Foam cells, primarily derived from macrophages and phenotypically regulated smooth muscle cells stimulated by oxLDL, are core mediators of neointimal proliferation and structural remodeling in in-stent restenosis. Their continuous accumulation drives lipid deposition, oxidative stress, and chronic inflammation, thereby exacerbating the restenosis-promoting microenvironment. Compared to FGF21 (Group 1), EGCG-Cys (Group 2), and MFVL alone (Group 3), the EGCG-Cys-MFVL nanointerface exhibited significantly lower Dil-oxLDL fluorescence intensity (…). Figure 4 Medium g~j), very little Oil Red O positive lipid deposition ( Figure 4 (k-l), and significantly reduced intracellular reactive oxygen species levels ( Figure 4 (m~n). These results fully demonstrate that this multifunctional coating has excellent performance in the simultaneous regulation of lipid accumulation and oxidative stress, and is particularly suitable for microenvironments rich in foam cells.

[0076] Example 5: Mouse animal experiment 5.1 Laboratory Animals All experimental animals used in this study were male ApoE- / - mice aged 4–6 weeks, purchased from Shrek Animal Co., Ltd., Shanghai, China. All experimental procedures complied with the requirements of the Medical Ethics Committee of Lishui University School of Medicine (No. 2024YD0163). In the mouse animal experiments described in this study, 40 mg / kg... Mice were anesthetized by intraperitoneal bolus injection of 1% sodium pentobarbital. Sufficient sedation was confirmed by the absence of paw withdrawal reflex. Finally, all mice were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital (100 mg / kg).

[0077] 5.2 Modeling and Grouping Reference Figure 5 The method shown in Figure a was used to model and treat mice. The mice were fed a high-fat diet for 30 days beforehand. Then, on the 7th and 14th days, different nano-formulations were injected intravenously. The mice were sacrificed on the 28th day and histological analysis was performed, including Oil Red O staining and reactive oxygen species detection.

[0078] 5.3 Sample Collection and Testing 5.3.1 ORO staining Oil Red staining of aortic tissue in mice was performed. After one month of treatment in different groups (Group 1: Ctrl group, Group 2: VHPK-Lipo group, Group 3: MVL group, Group 4: FGF21 group, Group 5: MFVL group), mice were euthanized and fixed in a supine position. The thoracic cavity was then opened to expose the heart. A cannula was inserted into the left ventricle, and pre-cooled physiological saline (0.9% NaCl, 4℃) was perfused for 2 minutes until complete perfusion. The aorta was observed and images were acquired under a stereomicroscope. Subsequently, the aorta from the base of the heart to the bifurcation of the common iliac artery was completely dissected. Based on the number of samples and the required volume of staining working solution per sample, ORO staining working solution was prepared at a ratio of 3:2 (Oil Red O solution and Oil Red O diluent). The solution was mixed, allowed to stand for 10 minutes, and then filtered through filter paper or a 0.45 µm syringe filter. It was used within two hours.

[0079] 5.3.2 Whole aorta staining Immediately after dissection, the mouse aorta was immersed in 4% paraformaldehyde and fixed at 4°C for 24 h. After removal, it was rinsed with PBS for 5 min × 3 times. It was then dehydrated with 60% isopropanol for 1 min, and then immersed in ORO working solution in the dark for 15-30 min (room temperature). After separation with 60% isopropanol for 5 s, it was rinsed with PBS until no dye leakage occurred. The aorta was then longitudinally cut open and photographed under saline. For frozen section staining: the dissected mouse aorta was embedded in OCT and frozen at -80°C. 6 μm thick sections were cut and attached to glass slides, fixed with 4% paraformaldehyde for 15 min, rinsed with PBS, incubated with 60% isopropanol for 1 min, covered with Oil Red O stain in the dark for 15 min, separated with 60% isopropanol for 5 s, and counterstained with hematoxylin for 1 min. The results were observed under a microscope. The percentage of ORO+ area was measured using ImageJ software (V1.8.0) to quantify atherosclerotic lesions in the mouse aorta.

[0080] 5.3.3 Assessment of Oxidative Stress Levels After treating AS model ApoE- / - mice according to different groups (Group 1: Ctrl group, Group 2: VHPK-Lipo group, Group 3: MVL group, Group 4: FGF21 group, Group 5: MFVL group) for one month, the mice were sacrificed. The entire aorta, from the heart to the iliac artery branches, was rapidly dissected immediately after opening the thoracic cavity and stored in 4% paraformaldehyde. Then, sections were prepared using a cryostat. Mouse aortic sections were stained with 1 mL of 1000-fold diluted DCFH-DA (10 μM) for 20 min, washed twice with PBS to remove unbound DCFH-DA probes, and then mounted with mounting medium containing DAPI. Images were observed and acquired under a laser confocal microscope.

[0081] 5.4 Experimental Results To evaluate the in vivo therapeutic effects of the MFVL nanoplatform, this study comprehensively evaluated its role in atherosclerotic plaque regression and oxidative stress regulation in an ApoE- / - mouse model. The specific results are as follows: Three-dimensional microscopic observation of the aortic root showed that, compared with the control group and other intervention groups, the plaque burden in the MnO2 NFs-FGF21@VHPK-Lipo (group 5) treatment group was significantly reduced. Figure 5 (b)

[0082] Oil Red O staining of the isolated aorta and cross-sectional staining of the aortic root further confirmed that lipid deposition was significantly reduced after MFVL administration. Figure 5 (c-d). Quantitative analysis showed that the aortic plaque area in group 5 (7.5 ± 1.2%) was significantly lower than that in group 1 (control group, 34.6 ± 3.1%, p<0.0001), group 2 (VHPK-Lipo, 38.2 ± 2.7%, p<0.0001), and group 3 (FGF21, 26.1 ± 2.4%, p<0.001). Figure 5 (f). Similarly, the Oil Red O positive area in the aortic root section was significantly smaller in group 5 (0.42 ± 0.13 mm²) than in groups 1 to 3 (p<0.001). Figure 5 (g).

[0083] Regarding the assessment of oxidative stress levels, DCFH-DA fluorescence staining results showed that the average fluorescence intensity of group 5 was 6.3 ± 1.0 × 10⁻⁶. 3 Compared to the first group (26.9 ± 2.6 × 10), 3 (p<0.0001), Group 2 (25.7 ± 3.1×10) 3 (p<0.0001) and the third group (18.8 ± 2.3 × 10 3 The values ​​of p < 0.001 all decreased sharply, indicating that the MFVL system has a strong antioxidant capacity. Figure 5 (e and h). In summary, these results demonstrate that the MnO2 NFs-FGF21@VHPK-Lipo nanoplatform can significantly reduce atherosclerotic plaque formation and effectively alleviate oxidative stress in vivo, highlighting its potential as a multifunctional therapeutic agent in advanced vascular nanomedicine.

[0084] Example 6: Animal experiments with pigs and rabbits 6.1 Laboratory Animals The experimental animals used in this study were all male New Zealand White rabbits (2.5-3 kg) purchased from Shrek Animal Co., Ltd. in Shanghai, China; the miniature pigs (weighing about 40 kg, 3-4 months old) were purchased from commercial bases with miniature experimental pig production licenses.

[0085] 6.2 Modeling and Experimentation 6.2.1 Rabbit Model Experiment This study used 12 healthy male New Zealand white rabbits (approximately 3 months old, weighing 2.5–3 kg) for in vivo vascular stent implantation experiments. The experimental animals were randomly divided into two groups (n=6 in each group): an EC-MFVL-WE43 coated stent group and a WE43 bare stent control group. All stents were sterilized with ethylene oxide vapor at 37°C for 12 hours before the procedure.

[0086] The experiment involved general anesthesia via intravenous injection of sodium pentobarbital (25 mg / ml, 0.7 ml / kg) to expose the common carotid artery (CCA) and its branches (internal and external carotid arteries). A stent was delivered to the target segment of the CCA via the external carotid artery (ECA) using a balloon catheter, inflated at 8 atm and maintained for 40 seconds to allow the stent to adhere to the carotid wall. After catheter withdrawal, the ECA was ligated, and ampicillin sodium solution (10 mg / ml, 1 ml / kg) was administered intramuscularly to prevent infection. Postoperatively, dual antiplatelet therapy with aspirin (100 mg / kg) and clopidogrel (3 mg / kg) was administered for 3 consecutive days.

[0087] Carotid CTA follow-up examinations were performed regularly within one month after stent implantation, and the imaging data were analyzed using RadiAnt DICOMViewer (version 2020.2.3). Three months after implantation, the stent-arterial complex was removed, and euthanasia was performed via sodium pentobarbital overdose. The harvested tissue was divided into three segments for immunofluorescence staining, immunohistochemical staining, and scanning electron microscopy (SEM) analysis, respectively.

[0088] 6.2.2 Pig Model Experiment Male miniature pigs (approximately 35 kg in weight and 6 months in age) were quarantined and observed for 8 days prior to the experiment to confirm the absence of infectious diseases. Twelve hours before stent implantation, the pigs underwent oral antiplatelet pretreatment with aspirin (75 mg) and clopidogrel (100 mg). Under aseptic conditions, systemic anticoagulation was achieved by injecting heparin (200 IU / kg) via the ear vein, followed by puncture of the right femoral artery to establish vascular access.

[0089] Following the injection of contrast agent (200 μg / stent), under coronary angiography guidance, WE43 stents and EC-MFVL-WE43 coated stents were randomly implanted into major coronary artery branches (right coronary artery and left anterior descending artery) via a 6F guiding catheter along a 0.014-inch guidewire. A 12 atm high-pressure balloon was used for 30 seconds to fully apposition the stent to the coronary wall. Coronary angiography was repeated to assess stent patency and location. Postoperatively, patients received intramuscular penicillin (20,000 units / ml, 1 ml / kg) daily for 7 consecutive days and continued oral antiplatelet therapy.

[0090] One month and three months after stent implantation, vascular patency was assessed using optical coherence tomography (OCT), and the imaging data were analyzed using RadiAnt DICOM Viewer (version 2020.2.3). Three animals from each group were sacrificed, and the stent-arterial segment was collected for histological analysis (HE staining). The sacrifice method was an overdose of sodium pentobarbital.

[0091] 6.3 Experimental Results To evaluate the in vivo anti-in-stent restenosis (ISR) effect of the EGCG-Cys-MFVL nanocoated WE43 stent, this study conducted multimodal imaging assessments in three types of vascular interventional models: rat, rabbit, and pig. Figure 6 (a). In a rat carotid artery stent model, MRI imaging showed that, compared with the WE43 group and the AS group, the EGCG-Cys-MFVL-WE43 group had significantly reduced signal loss and better preservation of luminal continuity, suggesting that ISR was effectively suppressed. Figure 6 (b) In the rabbit model, three-dimensional reconstruction based on serial CTA showed that the bare WE43 group developed progressive luminal occlusion within 28 days, while the nano-coated stent group maintained good vascular patency and showed minimal ISR progression. Figure 6 (c). Quantitative analysis further showed that the restenosis volume in the EGCG-Cys-MFVL-WE43 group was significantly smaller than that in the WE43 group on day 28 (p<0.01, n = 5).

[0092] In a porcine coronary artery stent model, high-resolution CTA imaging after 3 months of high-fat diet feeding showed that the WE43 group exhibited significant neointimal hyperplasia (red arrow), while the EGCG-Cys-MFVL-WE43 group maintained a clear luminal structure. Figure 6 (d). The corresponding intravascular ultrasound cross-sectional and longitudinal scan results further validated the above findings. Figure 6(e). Quantitative IVUS analysis showed that the minimum lumen area of ​​the EGCG-Cys-MFVL-WE43 group (6.42 ± 0.41 mm²) was significantly greater than that of the WE43 stent group (3.87 ± 0.35 mm², p<0.001, n = 4), and the neointimal area was smaller (p<0.01).

[0093] In summary, these results fully demonstrate that the multifunctional nanocoating has excellent anti-ISR properties and can achieve continuous luminal patency in various animal models by regulating the bioactivity of the vascular microenvironment.

[0094] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis, characterized in that... The preparation method includes the following steps: (1) Preparation of manganese dioxide particles Dissolve potassium permanganate in a solution, add reaction reagents, stir to precipitate, centrifuge and wash the product, dry to obtain manganese dioxide particles; (2) Preparation of MF-FGF21 Manganese dioxide particles were added to water, the pH was adjusted, and the mixture was sonicated. EDC and NHS were added, and the mixture was shaken to activate the manganese dioxide particles. After centrifugation and washing, FGF21 solution was added, and the mixture was incubated. After centrifugation, the particles were inactivated and washed to obtain MF-FGF21. (3) Preparation of liposome solution Cholesterol, DPPC, DEPE-PEG2K and DSPE-PEG2K-MAL were dissolved in chloroform, and a thin film was formed by rotary evaporation. The film was then hydrated by adding MF-FGF21 suspension to obtain a liposome solution. (4) Preparation of MFVL The VHPKQHR-peptide was dissolved in PBS, mixed with TCEP solution, and placed at room temperature. Then it was mixed with the liposome solution prepared in step (3), the mixture was sonicated, incubated overnight, filtered, and purified to obtain MFVL. (5) Stent pretreatment The metal stent was ultrasonically cleaned with water and acetone, dried, and then immersed in Tris buffer solution. After that, it was removed and cleaned. EGCG and Cys were added to the Tris buffer solution and dispersed by ultrasonic vibration to obtain a pretreatment solution. The cleaned metal stent was then immersed in the pretreatment solution and magnetically stirred. After the reaction was completed, the stent was removed and cleaned for later use. (6) Nanocoating loading MFVL was dispersed in Tris buffer to obtain a dispersion. The pretreated metal stent was immersed in the dispersion and loaded by shaking. After loading, the stent was removed, washed, and dried to obtain a multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis.

2. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The solution mentioned in step (1) is hydrochloric acid or water; When the solution is hydrochloric acid, the ratio of potassium permanganate to hydrochloric acid is 1-2 mg: 1 mL; The concentration of the hydrochloric acid is 0.1–0.2 mol / L; The reaction reagent in step (1) is citric acid or sodium hydroxide; The final concentration of the citric acid is 50–200 mM; The mass ratio of sodium hydroxide to potassium permanganate is 1-2:1-2.

3. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The stirring reaction conditions described in step (1) are stirring at room temperature for 0.5 to 24 hours; The centrifugation conditions described in step (1) are 800–1200 rpm for 5–20 min.

4. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The pH adjustment mentioned in step (2) is to adjust the pH to 5-7; The final concentration of EDC mentioned in step (2) is 1 to 3 mg / mL; The final concentration of NHS in step (2) is 3-5 mg / mL; The shaking condition described in step (2) is shaking for 20 to 40 minutes; The mass ratio of manganese dioxide particles to FGF21 in step (2) is 10:1; The inactivation in step (2) is performed by using 1wt% BSA for 20-40 minutes.

5. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The molar ratio of cholesterol, DPPC, DEPE-PEG2K and DSPE-PEG2K-MAL in step (3) is 4-6:6-8:1-2:0.1-0.3; The hydration in step (3) involves adding a MF-FGF21 suspension preheated to 50-60°C and shaking it on a shaker at 150-200 rpm for 30-90 minutes in a water bath at 50-60°C. The mass ratio of MF-FGF21 to DSPE-PEG2K-MAL in step (3) is 50:

1.

6. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The VHPKQHR-peptide mentioned in step (4) is a short peptide with the amino acid sequence VHPKQHR; The molar ratio of VHPKQHR-peptide to TCEP in step (4) is 1:2; The placement conditions described in step (4) are to place the item at room temperature for 20 to 40 minutes; The filtration described in step (4) involves filtering 40 to 60 times using a 200 nm filter; The purification described in step (4) is performed using an S-100 dextran gel column.

7. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The metal bracket mentioned in step (5) is a WE43 metal bracket; The concentration of the Tris buffer solution mentioned in step (5) is 2 mg / mL, and the pH is 8.5; The immersion in Tris buffer solution in step (5) is for 1 to 3 hours; The molar ratio of EGCG to Cys in step (5) is 1:2; The magnetic stirring conditions described in step (5) are magnetic stirring for 10 to 15 hours.

8. The multifunctional nano-coated stent for preventing and treating in-stent restenosis according to claim 1, characterized in that: The concentration of MFVL in the dispersion described in step (6) is 1-2 mg / mL; The oscillation conditions described in step (6) are oscillation at 100-150 rpm for 10-15 hours; The concentration of the Tris buffer in step (6) is 10 mM and the pH is 8.

5.

9. The application of the multifunctional nano-coated stent for preventing and treating in-stent restenosis as described in any one of claims 1 to 8 in the treatment of atherosclerosis.

10. The use of the multifunctional nano-coated stent for the prevention and treatment of in-stent restenosis as described in any one of claims 1 to 8 in alleviating in-stent restenosis.

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