Vexin-probucol self-assembled nanoparticles as well as preparation method and application of vitexin-probucol self-assembled nanoparticles

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to vitexin-probucol self-assembled nanoparticles as well as a preparation method and application thereof. The nanoparticle is formed by self-assembly of vitexin and probucol, and the mass ratio of vitexin to probucol is 2: (1-5). The nano particles are prepared by adopting a nano precipitation method, and the method specifically comprises the following steps: mixing vitexin and probucol, and dissolving and dispersing in a good solvent to obtain a mixed solution; and adding the obtained mixed solution into a poor solvent, and promoting the vitexin and probucol to be self-assembled to form the nanoparticles by utilizing a solvent replacement effect. The nanoparticles have the advantages of high drug loading capacity, synergistic anti-inflammation and anti-oxidation, long-acting circulation and the like, and can effectively treat atherosclerosis, reduce side effects and improve the treatment effect and safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a vitexin-probucol self-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (AS) is the leading cause of cardiovascular disease-related deaths worldwide. Its incidence continues to rise with the spread of risk factors such as population aging, obesity, and hyperlipidemia, becoming a major public health problem that seriously threatens human health. AS is a complex chronic inflammatory vascular disease, and its core pathological processes are closely related to treatment: In the early stages of the disease, vascular endothelial cells become dysfunctional under the stimulation of oxidized low-density lipoprotein (ox-LDL) and inflammatory factors. At the same time, oxidative stress is activated, and excessive reactive oxygen species (ROS) are produced, leading to lipid peroxidation, which further aggravates endothelial damage. Subsequently, monocytes in the blood adhere to the damaged endothelium and migrate to the vascular intima, differentiating into macrophages. Macrophages take up large amounts of ox-LDL and transform into foam cells. Foam cells aggregate to form lipid streaks, which gradually develop into fibrous plaques. Plaque rupture easily leads to thrombosis, resulting in fatal events such as acute myocardial infarction and cerebral infarction.

[0003] Currently, clinical treatment for ankylosing spondylitis (AS) primarily relies on drug intervention, with commonly used drugs including statins, fibrates, antiplatelet drugs, and antioxidants. Probucol (P) is a commonly used antioxidant in clinical practice, possessing both potent lipid-lowering and anti-AS effects. Its lipid-lowering mechanism is clear: firstly, it enhances the expression of low-density lipoprotein (LDL) receptors in the liver, promoting specific uptake and metabolism of LDL in plasma and accelerating the clearance of LDL from the bloodstream; secondly, it inhibits the activity of key cholesterol synthesis enzymes (such as HMG-CoA reductase), reducing endogenous cholesterol production in hepatocytes and lowering plasma total cholesterol and LDL-C levels; thirdly, its potent antioxidant properties inhibit the oxidation of LDL to ox-LDL, reducing damage to vascular endothelium from lipid peroxidation products, while simultaneously reducing the uptake efficiency of ox-LDL by macrophages, thus reducing lipid deposition on the vascular wall at its source. However, existing therapeutic drugs suffer from several insurmountable drawbacks: First, poor targeting; after entering the body, the drugs are mostly distributed systemically, with only a small amount reaching the lesion site of AS, resulting in low treatment efficiency. Second, limited drug loading capacity; traditional formulations struggle to effectively deliver high doses of drugs, and vitexin and probucol exhibit poor water solubility and low bioavailability, further limiting efficacy. Third, significant systemic side effects; drugs not distributed on a specific target are prone to toxicity to normal tissues and organs. For example, probucol may cause liver damage, muscle pain, and other adverse reactions, and long-term use is poorly tolerated by patients. Therefore, it is urgent to find new methods to address the problems of poor targeting, low bioavailability, and safety concerns regarding carrier materials inherent in traditional drugs.

[0004] Nanocarriers, with their unique size and surface modification advantages, have shown significant potential in improving drug water solubility, prolonging blood circulation time, and enhancing bioavailability. For example, patent CN106309373A discloses a probucol solid dispersion and its preparation method, which uses hot-melt extrusion technology to prepare a probucol solid dispersion from probucol, a hydrophilic polymer carrier material, and a surfactant, thereby improving the dissolution and bioavailability of probucol. However, existing nanodelivery systems still face key technological bottlenecks: on the one hand, most nanocarriers rely on exogenous carriers such as polymers, liposomes, and inorganic nanomaterials, and some carrier materials have poor biocompatibility, which may trigger immune responses or accumulate in vivo, posing potential safety risks; on the other hand, the targeting precision is insufficient. Existing targeting strategies are mostly based on single receptor recognition, making it difficult to achieve a specific response to the inflammatory microenvironment of AS lesions, resulting in limited enrichment efficiency of nanoparticles at the lesion site and failing to fully exert their therapeutic effects.

[0005] Compared to traditional nanomedicine delivery systems, self-assembled carrier-free nanomedicine delivery systems offer advantages such as high drug loading capacity and the elimination of the need for inert carriers, effectively avoiding the toxicity issues that may arise from traditional carriers. Through non-covalent interactions between drug molecules, such as hydrophobic interactions, π-π stacking, hydrogen bonding, electrostatic forces, and coordination interactions, they achieve self-assembly to form stable nanostructures, demonstrating great potential in treating various diseases such as cancer, inflammation, and ischemic stroke. For example, patent CN120478673A discloses a biomimetic carrier-free nanomedicine coated with a macrophage membrane, its preparation method, and its application. This invention prepares PR@PBNPs from puerarin and probucol using TCM-SAN (Traditional Chinese Medicine Component Self-Assembled Nanoparticles) technology, achieving efficient carrier-free loading of PR and PB. Then, macrophage membranes are used to de-coat PR@PBNPs to prepare MPR@PBNPs. This patent involves the self-assembly of probucol and puerarin, requiring coating with a macrophage membrane for better targeting and aggregation to the AS lesion area, thus improving therapeutic efficacy, which is relatively cumbersome.

[0006] Vitexin (V) is a natural flavonoid compound extracted from plants of the Vitex genus. It possesses clear anti-inflammatory, antioxidant, and vascular endothelial cell protective bioactivities and can target APEX1, which is highly expressed on inflamed endothelial cells. This invention reveals that the synergistic effect of vitexin and probucol can cover multiple key stages in the pathological process of ankylosing spondylitis (AS), providing a new approach for the targeted delivery of nanomedicines. Summary of the Invention

[0007] In view of this, the present invention combines the anti-inflammatory and antioxidant activity of vitexin with the lipid-lowering, anti-lipid peroxidation and self-assembly properties of probucol, and makes full use of the targeting site advantages of inflammatory endothelial cells to construct carrier-free self-assembled nanoparticles. This invention aims to solve the problems of poor targeting, low bioavailability and safety risks of carrier materials in traditional drugs, so as to achieve precise targeting of drugs to inflammatory endothelium and improve the treatment effect of AS.

[0008] One of the objectives of this invention is to provide a vitexin-probucol self-assembled nanoparticle.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A vitexin-probucol self-assembled nanoparticle, wherein the nanoparticle is self-assembled from vitexin and probucol, and the mass ratio of vitexin to probucol is 2:4~8.

[0011] Preferably, the mass ratio of vitexin to probucol is 2:5.

[0012] Preferably, the driving force for self-assembly includes any one or more of hydrogen bonding, hydrophobic interaction, and π-π conjugation.

[0013] Preferably, the nanoparticles have a Zeta potential of -20 to -40 mV, exhibiting a stable negative potential characteristic. A strong negative potential can reduce the aggregation of nanoparticles through electrostatic repulsion, thereby improving their stability in blood circulation.

[0014] Preferably, the zeta potential of the nanoparticles is -22.5 ± 1.7 mV.

[0015] Preferably, the nanoparticles have a particle size of 80-150 nm. This particle size range avoids rapid clearance by the kidneys while allowing them to pass through the high permeability (EPR effect) of vascular endothelium.

[0016] Preferably, the nanoparticles have a particle size of 114.7 ± 9.0 nm.

[0017] The second objective of this invention is to provide a method for preparing vitexin-probucol self-assembled nanoparticles.

[0018] To achieve the above objectives, the present invention adopts the following technical solution:

[0019] The preparation method of vitexin-probucol self-assembled nanoparticles includes the following steps:

[0020] (1) Mix vitexin, probucol and DSPE-PEG-OH, dissolve and disperse them in a good solvent to obtain a mixed solution;

[0021] (2) Add the mixed solution obtained in step (1) to a poor solvent and use the solvent displacement effect to promote the self-assembly of vitexin and probucol to form nanoparticles.

[0022] Preferably, the good solvent includes any one or two of DMF and DMSO; the bad solvent includes any one or two of ddH2O and PBS.

[0023] More preferably, the good solvent is DMF and the bad solvent is dd H2O.

[0024] Preferably, the undesirable solvent is added in excess.

[0025] Preferably, the volume ratio of the good solvent to the bad solvent is 1:4-10, more preferably 1:4.

[0026] Preferably, the mass ratio of vitexin, probucol and DSPE-PEG-OH is 2:4~8:0.5-1, more preferably 2:5:0.7.

[0027] Preferably, 2 mg of vitexin, 5 mg of probucol, and 0.7 mg of DSPE-PEG-OH are mixed and added to 1 mL of a good solvent, and stirred until completely dissolved. Then, under continuous stirring at room temperature, the mixed solution is slowly added dropwise to 4 mL of an excess of poor solvent ddH2O, and the solvent displacement effect is used to promote the self-assembly of vitexin and probucol to form nanoparticles.

[0028] Preferably, after the addition of the nanoparticles in step (2) is completed, the mixture is stirred for a certain period of time to stabilize the conformation of the nanoparticles.

[0029] Preferably, after the solvent replacement reaction in step (2) is completed, the resulting reaction solution is subjected to dialysis to obtain vitexin-probucol self-assembled nanoparticles.

[0030] Preferably, the dialysis time is 4-12 hours, and the molecular weight cutoff of the dialysis bag is 3000-4000 Da.

[0031] Preferably, the dialysis bag has a molecular weight cutoff of 3500 Da.

[0032] Preferably, the dialysis medium is deionized water.

[0033] A third objective of this invention is to provide a drug for treating atherosclerosis.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] A drug for treating atherosclerosis, wherein the active ingredient of the drug includes the aforementioned nanoparticles.

[0036] The fourth objective of this invention is to provide the application of vitexin-probucol self-assembled nanoparticles in the preparation of a drug for treating atherosclerosis.

[0037] To achieve the above objectives, the present invention adopts the following technical solution:

[0038] Application of vitexin-probucol self-assembled nanoparticles in the preparation of drugs for the treatment of atherosclerosis.

[0039] Preferably, the use of vitexin-probucol self-assembled nanoparticles in the preparation of drugs for inhibiting endothelial cell uptake of ox-LDL and / or endothelial cell apoptosis during the development of atherosclerosis.

[0040] The beneficial effects of this invention are as follows:

[0041] 1. To address the problems of poor drug targeting, low drug loading, and significant side effects in the treatment of atherosclerosis and its associated stroke and microcirculatory disorders, this invention combines the self-assembly technology of active ingredients in traditional Chinese medicine with the inflammatory endothelial targeting mechanism to construct carrier-free self-assembled nanoparticles (VPNPs) of vitexin and probucol. Utilizing the anti-inflammatory and antioxidant properties of vitexin, it self-assembles with probucol, which has potent anti-AS activity, to form a stable nanodelivery system. Relying on the affinity between vitexin and the specific receptor APEX1, which is highly expressed on inflammatory endothelial cells, the drug is precisely enriched at the AS lesion site. By synergistically inhibiting oxidative stress, inflammatory response, and foam cell formation, it achieves the goal of highly efficient and safe treatment of AS, while simultaneously solving the technical pain points of low bioavailability and insufficient targeting of traditional drugs.

[0042] 2. The construction of carrier-free self-assembled nanoparticles is the core prerequisite for the synergistic delivery of vitexin and probucol. This invention uses the nanoprecipitation method to prepare VPNPs. The solvent displacement effect promotes the spontaneous assembly of the two small molecule drugs into nanoparticles. This method does not require additional carrier materials and has the advantages of simple operation and high biocompatibility potential.

[0043] 3. To screen for the optimal formulation, this invention systematically investigated the effects of different mass ratios of vitexin to probucol (1:5, 2:5, 4:5, 5:5, 5:4, 5:2, 5:1) on the physicochemical properties and drug loading performance of VPNPs. Dynamic light scattering analysis showed that VPNPs prepared with different mass ratios all exhibited characteristic particle size distribution peaks, but significant differences in dispersibility existed. Among them, when the mass ratio of vitexin to probucol was 2:5, the hydration kinetic diameter distribution of VPNPs was the most concentrated, and the corresponding polydispersity index (PDI) was the lowest (<0.2), indicating that the uniformity and dispersibility of the nanoparticles were optimal at this ratio, with no obvious aggregation.

[0044] 4. Compared with existing drugs and preparations for treating atherosclerosis, the vitexin-probucol self-assembled nanoparticles of the present invention have the following significant advantages: ① Precise targeting: Achieves specific targeting of inflammatory endothelial cells at the site of AS lesions, improves the enrichment efficiency of drugs at the lesion site, and reduces side effects caused by systemic distribution; ② High drug loading: The carrier-free self-assembly mode avoids the limitation of exogenous carriers, significantly increases the drug loading of vitexin and probucol, and enhances the therapeutic effect; ③ Synergistic effect: Fully utilizes the anti-inflammatory and antioxidant activity of vitexin and the anti-lipid peroxidation and anti-foam cell formation effects of probucol to achieve multi-target synergistic treatment, covering key links in the pathological process of AS; ④ High safety: Without exogenous carrier materials, it has good biocompatibility, reduces the risk of immune response and in vivo accumulation, and the targeted release of drugs reduces toxicity to normal tissues; ⑤ Good stability: It has good stability in both in vivo and in vitro environments, making it easy to store, transport, and apply clinically.

[0045] 5. The carrier-free self-assembled nanoparticles of vitexin-probucol of the present invention exhibit good long-term circulation characteristics in mice, effectively preventing rapid clearance by the body and significantly prolonging the drug's residence time in the bloodstream. This provides an important pharmacokinetic basis for the sustained release and treatment of the drug at the lesion site.

[0046] 6. This invention utilizes a self-assembled carrier-free nanomedicine delivery system and the drug's targeting properties to the inflamed endothelium. This not only achieves carrier-free and efficient loading of vitexin and probucol, but also effectively targets the inflamed site without the need for cell membrane coating, thereby improving the therapeutic effect while effectively reducing drug costs and manufacturing difficulty. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the assembly of vitexin-probucol self-assembled nanoparticles (VPNPs).

[0048] Figure 2 Figures show the characterization results of VPNPs; where A is a schematic diagram of VPNPs prepared by the nanoprecipitation method; B and C are the DLS particle size distribution (B) and PDI distribution (C) of VPNPs prepared at different mass ratios of Vitexin and Probucol, respectively; D is the detection results of particle size, PDI and Zeta potential of VPNPs at a 2:5 ratio, scale bar = 400 nm; E is the transmission electron microscope image of VPNPs at a 2:5 ratio, scale bar = 400 nm; F and G are the comparison diagrams of UV absorption peaks (F) and FT-IR (G) of Vitexin powder, Probucol powder, Vitexin+Probucol mixed powder and VPNPs lyophilized powder, respectively.

[0049] Figure 3Figure 1 shows the results of VPNPs cytotoxicity against HUVECs. In Figure 2, A shows the results of CCK-8 assay on the cytotoxicity of vitexin to HUVECs after 24 h of treatment with different concentrations of vitexin; B shows the results of CCK-8 assay on the cytotoxicity of probucol to HUVECs after 24 h of treatment with different concentrations of vitexin; C shows the results of CCK-8 assay on the cytotoxicity of a mixture of vitexin and probucol to HUVECs after 24 h of treatment with different concentrations of vitexin; D shows the results of CCK-8 assay on the cytotoxicity of VPNPs to HUVECs after 24 h of treatment with different concentrations of VPNPs. Data are expressed as mean ± standard deviation (n=3), ns≥ 0.05, *P<0.05, ****P<0.0001.

[0050] Figure 4 The figures show the results of the detection of the cellular uptake characteristics of VPNPs on HUVECs; where A is a representative image of normal and activated HUVECs phagocytosing VPNPs at different concentrations (50, 100, 150 µg / mL) observed by CLSM; B is a representative image of normal and activated HUVECs phagocytosing VPNPs at different incubation times (1, 3, 6 h) observed by CLSM; in the figures, blue represents DAPI-labeled cell nuclei, green represents cytoskeleton stained with cyclophosphamide, and red represents DiD-labeled nanomedicines, with a scale bar of 25 µm.

[0051] Figure 5 Figure 1 shows the results of verifying the anti-inflammatory and antioxidant effects of VPNPs in HUVECs. A shows Oil Red O staining images of intracellular lipid deposition (low-density lipoprotein uptake) after co-incubation with HUVECs in different treatment groups. Red areas represent lipid deposition, and the scale bar is 100 µm. B shows the Image J quantitative Oil Red O positive area. Data in the figures are expressed as mean ± standard deviation (n=3), with P < 0.0001. C shows the fluorescence staining images of live / dead cells after co-incubation with HUVECs in different treatment groups. Red represents apoptotic cells, and green represents live cells, with the scale bar at 100 µm.

[0052] Figure 6 Figure A shows the results of flow cytometry analysis of the effects of different treatment groups on HUVECs cell apoptosis and the quantitative results from Image J. Figure B shows the results of flow cytometry analysis of the effects of different treatment groups on HUVECs cell apoptosis; Figure B shows the quantitative results of Image J on the effects of different treatment groups on HUVECs cell apoptosis. Data in the figures are expressed as mean ± standard deviation (n=3), ****P<0.0001.

[0053] Figure 7Figure 1 shows the results of the long-term circulating experiment of VPNPs in vivo; where A is the fluorescence image of the circulating blood of VPNPs in c57 mice after 48 hours, and the nanoparticles are DiD labeled; B is the quantitative fluorescence result of mouse blood at different time points; data are expressed as mean ± standard deviation (n=3).

[0054] Figure 8 Fluorescence intensity maps of DiD-VPNPs in the carotid arteries of mice in different treatment groups (n=5). Detailed Implementation

[0055] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0056] This invention combines the self-assembly technology of active ingredients in traditional Chinese medicine with the inflammatory endothelial targeting mechanism to construct carrier-free self-assembled nanoparticles (VPNPs) of vitexin and probucol. After intravenous injection, these VPNPs, due to their suitable particle size and negative surface potential, remain stable in the bloodstream, avoiding rapid clearance or aggregation. When the nanoparticles flow through the lesion site of ankylosing spondylitis (AS), their surface functional groups specifically bind to inflammatory endothelial cells. Simultaneously, leveraging the EPR effect of the vascular endothelium at the lesion site, the nanoparticles achieve highly efficient enrichment in the AS plaque region, with significantly higher enrichment concentrations than traditional free drugs and non-targeted nanoparticle formulations.

[0057] Nanoparticles enriched at lesion sites slowly release vitexin and probucol under stimulation in an inflammatory microenvironment (such as acidic pH and high ROS levels). These two substances synergistically exert multiple therapeutic effects: ① Anti-inflammatory effect: Inhibits the activation of inflammatory signaling pathways, reduces the expression of inflammatory factors (TNF-α, IL-6, IL-1β) and adhesion molecules (VCAM-1, ICAM-1), alleviates inflammatory infiltration of the vascular endothelium, and delays the progression of the inflammatory response; ② Antioxidant stress: Clears excess ROS in the body, inhibits lipid peroxidation, protects vascular endothelial cell function, and prevents endothelial cell apoptosis; ③ Inhibits foam cell formation: Probucol inhibits the uptake of ox-LDL by macrophages, while vitexin promotes the reverse transport of cholesterol within macrophages. Together, they block the formation and proliferation of foam cells, reducing lipid deposition in the vascular endothelium; ④ Stabilizes atherosclerotic plaques: By reducing inflammation, decreasing lipid accumulation, and inhibiting foam cell apoptosis, the proportion of the lipid core in the plaque is reduced, enhancing the stability of the fibrous cap and reducing the risk of plaque rupture.

[0058] To enhance understanding of the present invention, certain key technical and scientific terms will be clearly defined below. Unless otherwise specified herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be noted that the terminology used herein is intended to describe specific embodiments and not to be construed as limiting.

[0059] Vitexin (also known as Apogenin 8-C-glucoside) is a natural flavonoid compound. It is widely distributed in the leaves and stems of dozens of plants in nature, such as Vitex negundo, Vitex trifolia, Alpinia oxyphylla, Crataegus pinnatifida, Ficus microcarpa, Cyathea spinulosa leaves, Lygodium japonicum, Pteris vittata, Broussonetia papyrifera leaves, Hawthorn, and Isatis indigotica leaves. Currently, it is mainly extracted from Crataegus pinnatifida leaves. Vitexin has anti-tumor, blood-activating, qi-regulating, meridian-clearing, anti-inflammatory, antispasmodic, and antihypertensive effects, and is mainly used clinically to treat cardiovascular diseases. The chemical formula of vitexin is C6H2O. 21 H 20 O 10 It has a molecular weight of 432.38, a CAS registry number of 3681-93-4, and a structural formula as shown in Formula I below.

[0060]

[0061] I.

[0062] Self-assembly: refers to the process by which basic structural units such as molecules and nanomaterials spontaneously form ordered structures through non-covalent interactions (such as hydrogen bonds, hydrophobic interactions, π-π conjugation, etc.).

[0063] Carrier: In a drug delivery system, a carrier is a substance that can carry a drug to the target site and is usually used to improve the stability and bioavailability of the drug.

[0064] Hydrophobic interaction refers to the phenomenon that, in an aqueous medium, the folding of globular proteins tends to bury hydrophobic side chains or hydrophobic groups within the molecule. Essentially, nonpolar molecules or groups tend to avoid water molecules, creating an internal attraction effect, which is one of the key factors in the stability of nanoparticles.

[0065] π-π conjugation: refers to the interaction force between molecules with π electron systems formed by the overlap of electron clouds, which is an important factor in stabilizing nanoparticles.

[0066] In this embodiment of the invention, information on some experimental material manufacturers is shown in Table 1.

[0067] Table 1. Experimental Materials Information Table

[0068]

[0069] Example 1. Preparation and characterization of vitexin-probucol self-assembled nanoparticles (VPNPs)

[0070] 1. Preparation of VPNPs

[0071] This invention uses a nanoprecipitation method to prepare VPNPs, and its synthesis schematic diagram is shown below. Figure 2 As shown in Figure A. The specific preparation steps are as follows:

[0072] ① Raw material preparation: Accurately weigh vitexin and probucol according to different mass ratios (1:5, 2:5, 4:5, 5:5, 5:4, 5:2, 5:1), and prepare 0.7 mg of DSPE-PEG-OH. The specific dosages of vitexin and probucol are shown in Table 2.

[0073] Table 2

[0074]

[0075] ② Dissolution and dispersion: Add the above mixed raw materials to 1 mL of good solvent DMF and stir until completely dissolved to form a homogeneous mixed solution.

[0076] ③ Nanoprecipitation: Under continuous stirring at room temperature, the mixed solution was slowly added dropwise to 4 mL of excess unsuitable solvent ddH2O. The solvent displacement effect was used to promote the self-assembly of vitexin and probucol to form nanoparticles (VPNPs).

[0077] ④ Subsequent stabilization: After the droplet addition is complete, continue stirring for a certain period of time to stabilize the conformation of the nanoparticles.

[0078] ⑤ Purification: Transfer the reaction solution to a dialysis bag and perform dialysis with deionized water as the dialysis medium for 5 hours. The molecular weight cutoff of the dialysis bag is 3500 Da to remove unassembled small molecule raw materials and good solvents.

[0079] ⑥ Store at room temperature or freeze-dried for subsequent characterization.

[0080] 2. Characterization of VPNPs

[0081] 2.1 Stability determination of VPNPs with different proportions

[0082] ① Prepare the sample solution according to the experimental requirements, ensuring that it is homogeneous and free of impurities.

[0083] ② Instrument warm-up: Turn on the DLS instrument 30 minutes in advance to stabilize the optical path and temperature.

[0084] ③ Before each measurement: Observe whether there are any visible changes in the sample, such as precipitation or turbidity. If so, record and analyze them.

[0085] ④ Measurement operation:

[0086] Before use, the cuvette should be rinsed three times with ultrapure water to avoid residual impurities interfering with the results. Then, take 2 mL of sample and add it to the cuvette.

[0087] Select the "Particle Size Distribution" mode and record the average particle size (DLS) and polydispersity index (PDI).

[0088] ⑤ Sample preservation: Seal the samples during the test interval and store them at room temperature, avoiding vibration or contamination.

[0089] ⑥ Data analysis and stability assessment.

[0090] The results showed that when the mass ratio of Vitexin to Probucol was 2:5, the Zeta potential of VPNPs was -22.5 ± 1.7 mV, exhibiting a stable negative potential characteristic. Negatively charged nanoparticles in aqueous solution can effectively reduce particle adhesion and aggregation through electrostatic repulsion, thereby improving the colloidal stability of the nanosystem and ensuring that VPNPs maintain a dispersed state in aqueous solution and subsequent physiological environments.

[0091] 2.2 Particle size potential and morphology characterization of VPNPs

[0092] Preheat the DLS instrument 30 minutes in advance. Under room temperature conditions, use DLS to determine the hydration dynamic diameter, PDI and Zeta potential of VPNPs in aqueous solution. Use transmission electron microscopy (TEM) to observe the morphological characteristics of VPNPs surface.

[0093] The results are as follows Figure 2As shown in BE, the optimal preparation ratio of VPNPs was determined to be 2:5 (Vitexin to Probucol mass ratio). At this ratio, the hydration kinetic diameter of the VPNPs was 114.7 ± 9.0 nm, and the polydispersity index (PDI) was 0.084 ± 0.022. The PDI value being below the critical value of 0.2 indicates that the nanoparticles have a narrow particle size distribution, good system uniformity and dispersibility, and no obvious particle aggregation. Furthermore, the particle size range of 100–200 nm is suitable for the physicochemical requirements of targeted drug delivery for atherosclerosis. This particle size effectively prevents the nanoparticles from being rapidly cleared by the mononuclear-macrophage system in vivo and possesses the ability to achieve targeted enrichment at lesion sites through the vascular endothelial space. Combined with TEM morphology images, VPNPs at a 2:5 ratio exhibited regular morphology and clear outlines, with no obvious adhesion or aggregation between particles, forming quasi-spherical or spherical self-assembled nanostructures. The morphological uniformity shown by TEM, along with the low PDI and narrow particle size distribution results measured by DLS, corroborates the findings, proving that vitexin and probucol at a 2:5 ratio can spontaneously assemble into carrier-free nanoparticles with regular morphology through solvent displacement effect of nanoprecipitation. This verifies the feasibility of the preparation method and the rationality of the optimal ratio in this study.

[0094] 2.3 UV absorption spectra of VPNPs

[0095] To verify the self-assembly effect of VPNPs, this invention diluted vitexin, probucol, a physical mixture of the two, and VPNPs solution to 50 µg / mL, and detected the UV absorption peaks within a specific wavelength range. The changes in characteristic peaks were used to analyze intermolecular interactions. Studies have shown that if a π-π conjugated system exists in the self-assembled nanoparticles, their UV absorption peaks will exhibit a redshift, and changes in the absorption peaks can be used to assess whether the nanoparticles have successfully assembled. Specific steps include: diluting high-concentration vitexin solution, probucol solution, vitexin+probucol mixed solution, and VPNPs solution to samples with a concentration of 50 µg / mL; sequentially detecting the UV-Vis spectra of each sample in the 185-500 nm range using a UV-Vis spectrophotometer; and finally processing the data using GraphPad Prism software.

[0096] The results are as follows Figure 2As shown in Figure F, both vitexin and probucol monomers exhibit their typical UV absorption peaks. The absorption peaks of the physical mixture are a simple superposition of the monomer peaks, with no significant changes in peak position, shape, or intensity. However, the characteristic UV absorption peaks of VPNPs show a red shift compared to the physical mixture, with a broadened peak shape and enhanced absorption intensity. These changes in characteristic peaks demonstrate that during the self-assembly of vitexin and probucol to form VPNPs, π-π conjugation occurs between molecules, forming a new π-π conjugated system, indicating that the two are not simply physically mixed. Furthermore, this result, from a UV spectroscopy perspective, confirms the successful preparation of carrier-free self-assembled vitexin-probucol nanoparticles and the existence of non-covalent intermolecular interactions.

[0097] 2.4 Infrared absorption spectra of VPNPs

[0098] To verify the composition and intermolecular forces of VPNPs, Fourier transform infrared spectroscopy (FTIR) was used to analyze the samples. FTIR absorption spectroscopy can detect shifts in functional group vibrational frequencies, peak shapes, and intensity changes, analyzing the interactions between Vitexin and Probucol molecules (such as hydrogen bonds and hydrophobic interactions), thereby verifying the successful assembly and structural characteristics of VPNPs. The specific experimental steps are as follows:

[0099] ① Sample pretreatment: Take appropriate amounts of Vitexin powder, Probucol powder, Vitexin + Probucol mixed powder and VPNPs lyophilized powder respectively.

[0100] ② Tablet preparation: Using the KBr tableting method, 1 mg of each dried sample was weighed and thoroughly ground with 100 mg of spectrally pure KBr powder under an infrared lamp until a homogeneous and fine mixture was formed, ensuring uniform dispersion of the sample. The mixture was transferred to a tableting mold to form circular tablets, and blank KBr tablets were prepared as a control.

[0101] ③ Instrument Debugging and Testing: Conduct tests at room temperature to ensure the instrument's optical path is stable and the baseline is flat. Perform infrared spectral scanning, selecting a beam range of 500-4000 cm⁻¹. -1 Record the infrared absorption spectra of each sample.

[0102] ④ Data processing: Baseline correction and smoothing were performed on the raw spectral data using GraphPad Prism software.

[0103] The results are as follows Figure 2 As shown in G:

[0104] ① Hydroxyl group (-OH): The phenolic hydroxyl / alcoholic hydroxyl groups of vitexin and probucol are both at 3200~3600 cm⁻¹. -1The wavenumber range exhibits strong and sharp characteristic absorption peaks. In this range, the peaks of the physically mixed powder are simply superpositions of the individual drug peaks, with no significant changes in peak shape or position. However, the absorption peaks of VPNPs in this range change from sharp to broad, and the peak position exhibits a red shift of 30–50 cm⁻¹. -1 This change is a result of the formation of intermolecular hydrogen bonds. The hydroxyl group acts as a hydrogen bond donor / acceptor in the intermolecular binding of vitexin and probucol. The hydrogen bond effect reduces the vibrational frequency of the hydroxyl group, resulting in peak broadening and red shift of the peak position.

[0105] ② Carbonyl group (C=O): The C=O group of vitexin flavonoids is located at 1630~1680 cm⁻¹. -1 A strong characteristic absorption peak exists in the wavenumber range. In physically mixed powders, this peak position is consistent with that of vitexin monomer, with no shift and no decrease in peak intensity; in VPNPs, this peak undergoes a blue shift of 20–40 cm⁻¹. -1 Furthermore, the absorption intensity is significantly reduced because the weak intermolecular interaction between the carbonyl group and the active hydrogen of probucol changes the electron cloud density distribution of the carbonyl group, causing the carbonyl vibration frequency to increase, which ultimately manifests as a blue shift in peak position and a decrease in peak intensity.

[0106] ③ Aromatic ring carbon-carbon double bond (C=C): The C=C ratios of the aromatic ring / benzene ring of vitexin flavonoids and the aromatic ring of probucol are both between 1500 and 1600 cm. -1 The wavenumber range has characteristic absorption peaks. In the physically mixed powder, the peaks in this range are superimposed by the two, with sharp peak shapes and clear peak boundaries. In VPNPs, the peak shape in this range is broadened, the peak position is slightly red-shifted, and the peak intensity is reduced, indicating that there is a π-π conjugation between the aromatic rings of vitexin and probucol. This, together with the hydrophobic interaction, changes the vibrational environment of the C=C aromatic ring, leading to changes in its spectral characteristics.

[0107] ④ Ether bond (COC): The ether bond of probucol and the phenol-oxygen bond of vitexin are both between 1000 and 1200 cm. -1 The wavenumber range has a characteristic absorption peak, and the peak does not change significantly in physically mixed powders; in VPNPs, the peak position shifts slightly and the peak intensity decreases slightly, indicating that the ether bond participates in the intermolecular hydrophobic interaction and together with the hydrogen bond, it constructs an intermolecular binding system.

[0108] In summary, the characteristic changes in peak position, peak shape, and absorption intensity of the above bonds are not caused by simple physical mixing, which confirms that vitexin and probucol molecules form non-covalent interactions through hydrogen bonds (with hydroxyl groups as the core), π-π conjugation, and hydrophobic interactions.

[0109] Example 2. Cytotoxicity and cellular uptake characteristics of VPNPs on HUVECs

[0110] 1. Cytotoxicity

[0111] To evaluate the cytotoxicity of vitexin, probucol, a mixture of vitexin and probucol, and VPNPs, and to determine the safe concentration range for subsequent cell experiments, this invention used the CCK-8 cell proliferation-toxicity assay kit to detect the effects of four samples at different concentrations (0–300 µg / mL) on the viability of human umbilical vein endothelial cells (HUVECs). The specific steps are as follows:

[0112] ① HUVECs cultured to 90% confluence were digested with trypsin and seeded into 96-well plates. The concentration of the HUVECs cell suspension was 1×10⁻⁶. 4 Cells / mL, add 100 µL of cell suspension to each well; simultaneously set up blank wells (add only 100 µL PBS, no cells) and negative control groups (add 100 µL of cell suspension, no drugs), and set up 3 parallel replicates for each experimental group, blank well, and negative control group; place the plated 96-well plate in an incubator and incubate for 12-24 h until the cells are completely attached.

[0113] ② Preparation of drug concentration gradients: Take appropriate amounts of vitexin, probucol, their physical mixture powder (mass ratio of 2:5), and VPNPs solution with a concentration of 1 mg / mL, respectively, and prepare sterile drug stock solutions using ECM special culture medium. Then, dilute them stepwise to 0, 10, 30, 50, 100, 150, 200, and 300 µg / mL drug working solutions, and prepare them immediately before use.

[0114] ③ Drug incubation treatment: Remove the original culture medium from each well of the 96-well plate, wash once with PBS, add 100 µL of PBS or culture medium to the blank wells and negative control group, and add 100 µL of different concentrations of vitexin, probucol, a mixture of the two, and VPNPs working solution to the experimental groups respectively; after the drug addition is completed, put the 96-well plate back into the incubator and incubate at 37℃ and 5% CO2 for 24 h.

[0115] ④ CCK-8 reagent incubation: Remove the culture medium from the well plate, wash once with PBS, dilute the CCK-8 solution with ECM serum-free medium to make its volume 10% of the total volume of the culture medium in each well, add 100 µL of CCK-8 detection reagent to each well (avoid generating air bubbles), gently shake the 96-well plate to ensure that the reagent is in full contact with the cells, and continue to incubate in the incubator in the dark for 1-2 h.

[0116] ⑤ Absorbance (OD value) detection: Remove the 96-well plate and measure the absorbance (OD value) of each well using a microplate reader at 450 nm (the detection wavelength specified by the CCK-8 kit). Record the experimental data. Calculate the relative cell viability of each group based on the measured OD values, using the following formula:

[0117] Cell viability (%) = (OD value of drug-treated wells - OD value of blank wells) / (OD value of negative control group - OD value of blank wells) × 100%

[0118] ⑥ Data Processing and Plotting: GraphPad Prism software was used for statistical analysis of the experimental data. Data were plotted as mean ± standard deviation. The values ​​are represented as follows: (x) ; with drug concentration as the x-axis and cell viability (%) as the y-axis, cytotoxicity curves of the four drugs on HUVECs were plotted, and statistical difference analysis between groups was performed. P>0.05 was used to indicate no statistically significant difference (ns).

[0119] Experimental results are as follows Figure 3 As shown, within a concentration gradient range of 0–150 µg / mL, the relative cell viability of HUVECs treated with vitexin, probucol monomer, their physical mixture, and VPNPs did not show a significant decrease, and there was no statistically significant difference in cell viability among the concentration groups (*P>0.05, ns). None of the four samples exhibited significant in vitro cytotoxicity to HUVECs. Therefore, this experiment determined 0–150 µg / mL as the safe concentration range for subsequent in vitro cell experiments with vitexin, probucol, their mixture, and VPNPs, eliminating the interference of cytotoxicity on subsequent experimental results and ensuring that the results of subsequent experiments investigating VPNPs cell uptake, lysosomal escape, and anti-inflammatory activity represent drug-specific biological effects.

[0120] 2. In vitro phagocytosis assay of normal / activated endothelial cells

[0121] To investigate the phagocytic characteristics of VPNPs by HUVECs under normal and inflammatory activation states, this invention uses CLSM for qualitative observation to analyze the uptake efficiency of DiD-labeled VPNPs by cells at different concentrations (50, 100, 150 µg / mL) and different incubation times (1, 3, 6 h). The specific steps include the following:

[0122] ① HUVECs cultured to 90% confluence were digested with trypsin and seeded into 12-well plates. The concentration of the HUVECs cell suspension was 2 × 10⁻⁶. 5 Add 1 mL of cell suspension to each well and place the 12-well plate in an incubator for 12-24 h until the cells are fully adhered.

[0123] ② Inflammatory activation treatment of endothelial cells: After the plated cells were completely adhered, TNF-α working solution with a concentration of 10 ng / mL was added to the 12-well plate of the TNF-α activation group, and an equal volume of ECM complete culture medium was added to the normal cell group; both groups were incubated in a 37℃, 5% CO2 incubator for 12 h to construct an inflammatory activated endothelial cell model. After completion, the culture medium containing TNF-α was discarded, and the cells were washed once with PBS.

[0124] ③ Preparation of DiD-labeled VPNPs working solution: VPNPs were prepared into a sterile stock solution using ECM-specific medium, and then serially diluted to 50, 100, and 150 µg / mL working solutions. The solution was prepared and used immediately. DiD labeling ensures that the nanoparticle labeling efficiency is >90%, and red fluorescence is used to track the intracellular uptake of VPNPs.

[0125] ④ Drug co-incubation with cells (with concentration gradient and time gradient): Add 1 mL of the above-mentioned working solution of DiD-VPNPs at different concentrations to 12-well plates of normal / activated HUVECs that have adhered to the plate, and incubate for 3 h; at the same time, set a time gradient and place the cells after adding 100 µg / mL DiD-VPNPs in an incubator for co-incubation for 1 h, 3 h and 6 h respectively. After incubation, wash once with PBS.

[0126] ⑤ CLSM Sample Preparation and Observation: This step is used to qualitatively observe the phagocytosis of VPNPs of different concentrations and incubation times by normal / activated HUVECs. The entire process should be performed in the dark (to prevent fluorescence quenching).

[0127] This step involves qualitatively detecting the fluorescence intensity of DiD-VPNPs within normal / activated HUVECs to reflect the phagocytosis of nanoparticles by cells, and the entire process is performed in the dark.

[0128] Experimental results are as follows Figure 4 As shown in the image, qualitative observation results from CLSM images revealed that both normal HUVECs and TNF-α-activated inflammatory HUVECs could take up VPNPs (red fluorescent signal), and the fluorescent signal was mainly distributed intracellularly (distinguished from the blue nucleus and green cytoskeleton). The intracellular red fluorescence intensity in the activated group was significantly higher than that in the normal group. With increasing VPNP concentration (50→150 µg / mL) and prolonged incubation time (1→6 h), the intracellular fluorescence signal in both groups gradually increased. The activated group showed the most significant fluorescence enrichment at high concentrations (150 µg / mL) and long incubation times (6 h), suggesting that the phagocytosis of VPNPs by cells is concentration- and time-dependent.

[0129] Example 3. Validation of the anti-inflammatory and antioxidant effects of VPNPs in HUVECs

[0130] 1. In vitro VPNPs inhibit endothelial cell uptake of ox-LDL

[0131] Existing research indicates that during the development of atherosclerosis (AS), endothelial cells take up low-density lipoprotein (LDL) from peripheral blood and transport it to the arterial wall via endocytosis, thereby promoting lipid deposition at atherosclerotic sites and ultimately accelerating the development of AS plaques. Therefore, limiting endothelial cell uptake of LDL can prevent its accumulation at atherosclerotic sites. To clarify the effect of VPNPs on endothelial cell uptake of oxidized low-density lipoprotein (ox-LDL), this invention uses Oil Red O staining for qualitative observation and Image J quantitative analysis to explore its inhibitory effect on intracellular lipid deposition. The specific steps are as follows:

[0132] ① Cell plating and adherent culture: HUVECs in the logarithmic growth phase were digested with trypsin and the cell suspension concentration was adjusted to 1×10⁻⁶ cells / mL using ECM complete medium. 5 Cells were seeded at a density of 1 / mL into 24-well plates, with 1 mL of cell suspension added to each well. Control group (no ox-LDL induction, no drug treatment), Model group (ox-LDL induction, no drug treatment), Vitexin group, Probucol group, Vitexin+Probucol group, and VPNPs group were set up, with 3 replicates for each group. The 24-well plates were incubated at 37°C in a 5% CO2 incubator for 12–24 h until the cells were fully adhered.

[0133] ② Co-incubation of drugs with ox-LDL: Remove the original culture medium, wash once with PBS, and add culture medium containing ox-LDL at a concentration of 50 µg / mL and the corresponding drugs (Vitexin, Probucol, mixed solution, and VPNPs are all at a concentration of 50 µg / mL, refer to the safety concentration range above); each group is placed in an incubator and incubated at a constant temperature for 12 h to induce cells to take up ox-LDL and exert drug effects.

[0134] ③ Cell fixation: After incubation, remove the culture medium and wash twice with PBS; add Oil Red O fixative to each well and fix at room temperature for 30 min; discard the fixative and wash three times with PBS to remove residual fixative.

[0135] ④ Oil Red O staining: Add 60% isopropanol, remove the isopropanol after 20 s, add the prepared Oil Red O staining solution, soak for 20 min, remove the staining solution, add 60% isopropanol again, remove the isopropanol after 20 s, wash with water 3 times, and observe the lipid uptake of cells under an optical microscope.

[0136] ⑤ Image acquisition and quantitative analysis: Observe the intracellular lipid deposition in each group of cells under a microscope (the red area represents lipid deposition), and take pictures of the field of view where the cells are evenly distributed; use ImageJ software to perform quantitative analysis on the stained images, calculate the Oil Red O positive area of ​​each group, and statistically analyze the lipid deposition level of each group.

[0137] Experimental results are as follows Figure 5 As shown in AB. ① Qualitative observation results: Figure 5 The Oil Red O staining images shown in Figure A indicate that the Control group showed very little red lipid deposition in its cells, while the Model group (ox-LDL induced) showed widespread red fluorescence and significant lipid deposition. The Vitexin group, Probucol group, and a physical mixture of both showed a reduction in red deposition compared to the Model group, but some red deposition was still present. The VPNPs group showed a significant decrease in red fluorescence and a substantial reduction in lipid deposition, suggesting a superior inhibitory effect on ox-LDL uptake. ② Quantitative analysis results: Figure 5 Image J quantitative statistics (shown in B) showed that the proportion of Oil Red O positive area in the Model group was significantly higher than that in the Control group (****P<0.0001). The proportion of positive area in each drug treatment group was lower than that in the Model group, with the VPNPs group showing the most significant reduction, where the proportion of positive area was less than 30% of that in the Model group. These results indicate that VPNPs can effectively inhibit the uptake of ox-LDL by endothelial cells and reduce intracellular lipid accumulation, and the inhibitory effect is superior to that of vitexin, probucol monomer, and their physical mixture.

[0138] 2. Evaluation of the anti-apoptotic effect of VPNPs on endothelial cells in vitro

[0139] Oxidative stress-mediated apoptosis of endothelial cells in atherosclerotic sites is a key step in plaque formation and progression. This invention investigates the inhibitory effect of VPNPs on tert-butyl hydrogen peroxide (t-BHP)-induced apoptosis of HUVECs using live / dead cell fluorescence staining combined with Annexin V-FITC / PI double staining flow cytometry. The specific steps are as follows:

[0140] ① Cell plating and grouping: HUVECs in the logarithmic growth phase were harvested, digested with trypsin, and the cell suspension concentration was adjusted with ECM complete medium: 2 × 10⁶ cells / well for 6-well plates (for flow cytometry). 5 Add 2 mL per well; for 24-well plates (for live / dead staining), use 1 × 10⁻⁶ cells / mL. 5 Add 500 μL per well, per cell / mL.

[0141] Grouping is set up as follows: Control group (no t-BHP induction, no drug treatment), Model group (t-BHP induction, no drug treatment), Vitexin group, Probucol group, Vitexin+Probucol group, VPNPs group, with 3 parallel replicates in each group.

[0142] Incubate at 37°C and 5% CO2 for 12-24 hours until the cells are fully adhered.

[0143] ② Drug co-incubation: Remove the original culture medium, wash once with PBS, add ECM complete culture medium to the Control group; add culture medium containing the corresponding drug to the other groups, with a concentration of 100 μg / mL. Incubate at a constant temperature for 12 h.

[0144] ③ Apoptosis induction: Remove the original culture medium, wash once with PBS, and add 10 mM t-BHP to induce apoptosis for 1 h.

[0145] ④ Live / Dead Cell Fluorescence Staining and Quantification: After incubation, wash twice with PBS. Dilute the live / dead staining reagents (Calcein-AM solution and PI solution) according to the kit instructions, add 500 μL of the diluted staining solution to each well, incubate at room temperature in the dark for 15 min, and observe and photograph under a fluorescence microscope.

[0146] ⑤ Flow cytometry detection:

[0147] After incubation in 6-well plates, aspirate the culture medium, wash twice with PBS, add 0.25% trypsin digestion solution to each well, and digest until cells detach from the cell wall. Add an equal volume of ECM complete culture medium to terminate digestion. Centrifuge at 1000 r / min for 5 min to collect cells, discard the supernatant, and finally resuspend the cells in PBS, adjusting the concentration to 1×10⁻⁶. 6 Cells / mL. Add 100 μL of cell suspension to a flow cytometry tube, then add 100 μL of Annexin V-FITC and PI staining solution sequentially. Mix gently and incubate at room temperature in the dark for 15 min. Analyze the cells using a flow cytometer, and use FlowJo software to analyze the apoptosis rate (early apoptosis + late apoptosis) and generate a flow cytometry chromatogram.

[0148] ⑥ Data Processing: GraphPad Prism software was used to perform statistical analysis on the flow cytometry apoptosis rate data. Data are expressed as mean ± standard deviation. () indicates that statistical difference analysis was performed between groups.

[0149] Experimental results are as follows Figure 5 C and Figure 6 As shown. ① Qualitative observation results: Figure 5The live / dead fluorescence staining images shown in C indicate that in the Control group, green fluorescence (live cells) predominated, with very little red fluorescence (apoptotic cells); in the Model group (t-BHP induced), red fluorescence was widely distributed, while green fluorescence was significantly weakened, indicating significant cell apoptosis; in the vitexin group, probucol group, and the physical mixture of the two, red fluorescence was reduced compared to the Model group, but still present to some extent; in the VPNPs group, the proportion of green fluorescence was significantly increased, and red fluorescence appeared only in small amounts, suggesting that it had the best inhibitory effect on HUVEC apoptosis. ② Quantitative analysis results: Figure 6 Flow cytometry analysis showed that the total apoptosis rate (early apoptosis + late apoptosis) in the Model group was as high as 82.04%, which was significantly higher than that in the Control group (2.41%, ****P<0.0001). The apoptosis rate in each drug treatment group was significantly lower than that in the Model group. Among them, the apoptosis rate in the VPNPs group was the lowest (13.72%), which was significantly lower than that in the vitexin group (94.94%), the probucol group (46.03%), and the physical mixture group (67.64%).

[0150] The above results indicate that VPNPs can significantly inhibit t-BHP-induced apoptosis, and their therapeutic effect is superior to that of vitexin, probucol monomer, and physical mixtures of the two. This property is closely related to the pathological process of atherosclerosis—endothelial cell apoptosis disrupts vascular endothelial integrity, accelerates lipid deposition and plaque instability, and VPNPs can maintain vascular endothelial barrier function and delay the progression of atherosclerosis by inhibiting endothelial cell apoptosis.

[0151] Example 4. Long-term in vivo circulation of VPNPs

[0152] This invention investigates the blood circulation characteristics and long-term effects of VPNPs in mice by injecting DiD fluorescently labeled VPNPs into C57BL / 6 mice via tail vein injection, collecting blood samples from the tail vein at different time points and detecting fluorescence intensity. The specific steps are as follows:

[0153] ① Preparation of experimental animals: Select healthy SPF grade C57BL / 6 mice, acclimatize them for 1 week, allow them free access to food and water, maintain the temperature of the breeding environment at 22~25℃ and the humidity at 50%~60%, and maintain a circadian rhythm of 12h / 12h.

[0154] ② Preparation of fluorescently labeled samples: Prepare a DiD-labeled VPNPs solution (labeling efficiency > 90%), dilute it with PBS to the experimental concentration of 1 mg / mL, and use it immediately; at the same time, prepare an equal volume of PBS + DiD solution as a negative control.

[0155] ③ Tail vein injection: After weighing the mice, calculate the dosage according to their body weight, and slowly inject DiD-VPNPs solution or PBS+DiD control solution into the tail vein. 200 µL is injected into each mouse, and 3 parallel mice are set up in each group.

[0156] ④ Blood sample collection: Blood samples were collected at six time points: 0.5 h, 4 h, 8 h, 12 h, 24 h, and 48 h after injection. Before collection, the tail tip of the mouse was disinfected, the tail was cut off and about 50 µL of blood was collected and placed in a heparin anticoagulation centrifuge tube, and gently inverted to mix.

[0157] ⑤ Blood sample processing: Store anticoagulated blood samples at 4°C and transfer them to a 96-well plate for testing, with 3 replicates per sample.

[0158] ⑥ Measure the fluorescence intensity (au) of plasma samples from each well and record the experimental data. Plot the in vivo drug concentration-time curve using GraphPad Prism software with blood collection time as the x-axis and plasma fluorescence intensity as the y-axis to analyze the long-acting circulation characteristics of VPNPs in vivo; data are expressed as mean ± standard deviation (A / B). )express.

[0159] ⑦ Post-experimental treatment: After blood collection, the tail tip of the mouse was disinfected to stop bleeding. After the experiment, the mouse was euthanized and the carcass was disposed of in accordance with animal ethics requirements.

[0160] Experimental results are as follows Figure 7 As shown. ① Qualitative observation results: Figure 7 The mouse blood fluorescence imaging shown in Figure A indicates that significant red fluorescence signals were detected in the VPNPs group from 0.5 to 24 hours after injection; while only weak fluorescence was detected in the PBS+DiD control group, with almost no visible fluorescence signal. This suggests that VPNPs can achieve long-term circulation in mice, significantly prolonging the in vivo retention time of the fluorescent label. ② Quantitative analysis results: Figure 7 Quantitative analysis of blood fluorescence signals shown in Figure B revealed that the fluorescence intensity in the VPNPs group peaked at 0.5 h after injection, then slowly declined, maintaining a high fluorescence signal level until 24 h, and quenching at 48 h; while the fluorescence intensity in the control group remained at an extremely low level at all time points after injection.

[0161] The above results indicate that VPNPs possess excellent long-acting circulating properties in vivo, effectively preventing rapid clearance by the body and significantly prolonging the drug's residence time in the bloodstream. This characteristic enhances the targeted accumulation efficiency of the drug at atherosclerotic lesion sites, providing an important pharmacokinetic basis for its sustained in vivo anti-atherosclerotic effect.

[0162] Example 5

[0163] I. Experimental Methods

[0164] 1. Construction of a mouse model of carotid artery ligation

[0165] ① Preparation of experimental animals: Select healthy SPF-grade WT mice (8 weeks old), maintain the temperature of the breeding environment at 22~25℃ and the humidity at 50%~60%, with a circadian rhythm of 12 h / 12 ​​h, and allow free access to food and water.

[0166] ② Preoperative preparation: Fasting for 12 hours and water restriction for 4 hours before surgery; weighing the mice and anesthetizing them by intraperitoneal injection of sodium pentobarbital at a dose of 5 mg / kg; after anesthesia, fix the mice supine on the operating table, remove hair from the neck, disinfect the neck skin with 75% ethanol solution, and cover with a sterile drape.

[0167] ③ Surgical procedure: Make a 1-1.5 cm longitudinal incision along the midline of the neck, and dissect the skin, subcutaneous tissue and platysma muscle layer by layer to expose the left common carotid artery; carefully dissect the connective tissue and nerves around the common carotid artery to avoid damaging the blood vessels and nerves; clamp the proximal and distal ends of the common carotid artery with non-invasive vascular clips, and double ligate the common carotid artery between the two clips with 9-0 silk suture. After confirming that the ligation is secure, slowly release the vascular clips and observe whether there is any bleeding and whether the blood flow is completely blocked.

[0168] ④ Postoperative care: Rinse the surgical wound with saline, suture the platysma muscle and skin layer by layer, and disinfect the suture site with povidone-iodine; place the mouse in a warm environment to recover, and after recovery, return it to the breeding cage and feed it as usual. Observe the mouse's mental state, diet and wound healing status daily for 3 days after surgery to avoid infection.

[0169] 2. Carotid artery enrichment

[0170] ① Sample preparation: Prepare a DiD-labeled VPNPs solution (labeling efficiency > 90%), dilute it with PBS to an experimental concentration of 1 mg / mL, and use it immediately; at the same time, prepare an equal volume of PBS + DiD solution as a negative control.

[0171] ② Experimental grouping and administration: WT mice with successfully ligated carotid artery were randomly divided into a VPNPs group and a PBS+DiD control group, with 5 mice in each group. The dosage was calculated based on body weight, and 200 µL of the corresponding sample was slowly injected via the tail vein.

[0172] ③Incubation and tissue collection: After administration, the mice were fed in a routine manner and anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 5 mg / kg at 12 h. The mice were then quickly dissected, the carotid artery tissue was separated and collected, the surface residual blood was rinsed with physiological saline, and the moisture was absorbed with filter paper.

[0173] ④ Fluorescence imaging: The collected tissues were placed in black culture dishes, and fluorescence images were captured using a fluorescence imaging system. Uniform exposure parameters were set, and the distribution of DiD fluorescence signals was recorded.

[0174] II. Results Analysis

[0175] This invention establishes a carotid artery ligation mouse model, inducing endothelial inflammation by creating turbulence in the left cervical region of mice. The fluorescence intensity maps of DiD-VPNPs in the carotid arteries of mice in different treatment groups are shown below. Figure 8 As shown in the fluorescence imaging, DiD-containing nanoparticles are enriched in the inflamed left cervical fossa, further demonstrating the sustained-release effect of the nanoparticles and their ability to target inflammatory sites effectively in in vivo experiments.

Claims

1. A vitexin-probucol self-assembled nanoparticle, characterized in that, The nanoparticles are self-assembled from vitexin and probucol, with the mass ratio of vitexin to probucol being 2:4~8.

2. The nanoparticles according to claim 1, characterized in that, The mass ratio of vitexin to probucol is 2:

5.

3. The nanoparticles according to claim 1, characterized in that, The driving force for self-assembly includes any one or more of hydrogen bonding, hydrophobic interaction, and π-π conjugation.

4. A method for preparing vitexin-probucol self-assembled nanoparticles, characterized in that, Includes the following steps: (1) Mix vitexin, probucol and DSPE-PEG-OH, dissolve and disperse them in a good solvent to obtain a mixed solution; (2) Add the mixed solution obtained in step (1) to a poor solvent and use the solvent displacement effect to promote the self-assembly of vitexin and probucol to form nanoparticles.

5. The method according to claim 4, characterized in that, The good solvent includes any one or two of DMF and DMSO; the bad solvent includes any one or two of dd H2O and PBS.

6. The method according to claim 4, characterized in that, The mass ratio of vitexin, probucol, and DSPE-PEG-OH is 2:4~8:0.5-1.

7. The method according to claim 4, characterized in that, After the solvent replacement reaction in step (2) is completed, the resulting reaction solution is dialyzed to obtain vitexin-probucol self-assembled nanoparticles; the dialysis time is 4~12 h, and the molecular weight cutoff of the dialysis bag is 3000-4000 Da.

8. A drug for treating atherosclerosis, characterized in that, The active ingredient of the drug includes the nanoparticles as described in any one of claims 1-3.

9. The use of the vitexin-probucol self-assembled nanoparticles according to any one of claims 1-3 in the preparation of a medicament for treating atherosclerosis.

10. The use of the vitexin-probucol self-assembled nanoparticles according to any one of claims 1-3 in the preparation of a medicament for inhibiting endothelial cell uptake of ox-LDL and / or endothelial cell apoptosis during the development of atherosclerosis.

Images