Biomimetic probucol self-assembled nanoformulation, method of preparation and use thereof

Abstract

The application discloses a kind of bionic probucol self-assembly nano preparation and its preparation method and application.The nano preparation includes the nanometer core formed by the self-assembly of uncovalently modified probucol, the DSPE-PEG-OH modified component distributed on its surface and the macrophage-derived microvesicle membrane layer coated on the outer surface.The preparation method includes preparing the self-assembly nano particle of probucol modified by DSPE-PEG-OH, the dehydration treatment and rehydration treatment of RAW264.7 macrophage are carried out and centrifugal purification is carried out to obtain macrophage microvesicle, and the nano particle is mixed with the macrophage microvesicle and is treated by vortex and ultrasonic, to form the core-shell structure nano preparation coated with microvesicle membrane layer.The nano preparation has dispersion stability in the tested aqueous medium, and exhibits cell compatibility and biological safety under the tested in-vivo and in-vitro conditions, and can be used for preparing the drug for treating atherosclerosis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a biomimetic probucol self-assembled nanoformulation, its preparation method, and its application in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a group of chronic vascular diseases characterized by lipid deposition, endothelial dysfunction, inflammatory cell infiltration, increased oxidative stress, and plaque formation and progression. Endothelial cell damage and macrophage foaming are crucial steps in the development of AS. Oxidative stress, inflammatory responses, apoptosis, and increased uptake of oxidized low-density lipoprotein can all promote plaque progression and affect plaque stability. Current treatments for AS typically include interventions such as lipid regulation, anti-inflammation, antiplatelet therapy, and improvement of endothelial function. Probucol, a small molecule drug with antioxidant, anti-inflammatory, lipid-regulating, and endothelial-protective effects, has been used to treat AS-related diseases. However, probucol is highly hydrophobic, and conventional administration methods result in poor water dispersibility, insufficient effective accumulation at lesion sites, and limited cellular uptake, affecting its efficacy in atherosclerotic lesions.

[0003] Nanoparticle drug delivery technology can improve the circulation, distribution, and cellular uptake of drugs in vivo through nanoscale particles, and is therefore used to enhance the delivery efficiency of hydrophobic drugs. Existing nanoparticle drug delivery systems typically use liposomes, polymer micelles, inorganic nanomaterials, or other carrier materials to load drugs, which can improve drug solubility and in vivo distribution to some extent. However, these carrier-based nanomedicines usually require the introduction of a large amount of exogenous carrier material, making the preparation process relatively complex, and may suffer from problems such as limited drug loading, difficulty in batch-to-batch stability control, and increased burden on carrier metabolism or safety evaluation. Therefore, for hydrophobic small molecule drugs such as probucol, there is still a need to develop nanoparticle delivery systems with relatively simple preparation processes, high drug loading ratios, and good dispersion stability.

[0004] Furthermore, atherosclerotic lesions exhibit a distinct inflammatory microenvironment, with vascular endothelial cells and macrophages at the lesion site serving as crucial target cells for drug intervention. To enhance the accumulation of nanomedicines in inflammatory lesions, existing techniques have explored surface modification of nanoparticles using cell membranes, extracellular vesicles, or other biomimetic membrane structures to improve biocompatibility, immune evasion capabilities, and lesion targeting. However, current biomimetic membrane-coated nanomedicines may still suffer from challenges such as complex preparation steps, cumbersome membrane source processing, insufficient matching between the membrane coating structure and the drug nanocore, and the need to improve their ability to target inflammatory plaques and key pathological cells. For nanoformulations with probucol as the primary drug component, a simpler and more effective technical solution remains to be found that balances high drug loading, formulation stability, biocompatibility, and accumulation capacity in inflammatory lesions.

[0005] Therefore, existing technologies still require a probucol nanoformulation suitable for the treatment of atherosclerosis and its preparation method, which can improve the aqueous dispersion, dispersion stability, atherosclerosis-related distribution properties, and cellular uptake properties of the nanoformulation while reducing dependence on exogenous carriers as the main structure of the nano-core and improving the effective delivery efficiency of probucol. This would provide a new formulation for the drug delivery of probucol in atherosclerosis-related lesions. Summary of the Invention

[0006] In view of the shortcomings of the above-mentioned related technologies, the purpose of this application is to provide a biomimetic probucol self-assembled nano-formulation, its preparation method and its application in the preparation of drugs for treating atherosclerosis, so as to at least improve the problem that it is difficult to simultaneously achieve probucol nano-delivery system with probucol nano-sizing, dispersion stability, biocompatibility and atherosclerotic lesion-related distribution and related cellular uptake performance.

[0007] To achieve the above and other related objectives, the first aspect of this application provides a biomimetic probucol self-assembled nano-formulation, comprising a probucol self-assembled nanocore, a DSPE-PEG-OH modified component, and a macrophage-derived microvesicle membrane layer; wherein: the probucol self-assembled nanocore is formed by the self-assembly of uncovalently modified probucol molecules in an aqueous phase through non-covalent interactions, the non-covalent interactions including hydrophobic interactions, and at least one of hydrogen bonding and π-π stacking interactions; the DSPE-PEG-OH modified component is distributed on the surface of the probucol self-assembled nanocore, and the mass ratio of probucol to DSPE-PEG-OH is (8~12):1; the macrophage-derived microvesicle membrane layer is provided by macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration treatment, and coats the outer surface of the probucol self-assembled nanocore modified by the DSPE-PEG-OH modified component, thereby forming a core-shell structure in the nano-formulation.

[0008] A second aspect of this application provides a method for preparing a biomimetic probucol self-assembled nanoparticle, comprising the following steps: preparing probucol self-assembled nanoparticles modified with DSPE-PEG-OH: dissolving uncovalently modified probucol and DSPE-PEG-OH in an organic solvent to form an organic phase; adding the organic phase dropwise to an aqueous phase and stirring to form probucol self-assembled nanoparticles modified with DSPE-PEG-OH; dialyzing the resulting mixture to obtain a dispersion containing the probucol self-assembled nanoparticles modified with DSPE-PEG-OH; wherein the probucol self-assembled nanoparticles modified with DSPE-PEG-OH comprise a probucol self-assembled nanocore formed by the non-covalent self-assembly of uncovalently modified probucol molecules in an aqueous phase, and the DSPE-PEG-OH modifying component is distributed within the probucol self-assembled nanocore. On the surface, the mass ratio of probucol to DSPE-PEG-OH is (8~12):1; Preparation of macrophage microvesicles: RAW264.7 macrophages are cultured until the cell confluence reaches 80%~90%, the culture medium is removed and the cells are washed, the washed cells are dehydrated, and then the dehydrated cells are rehydrated. The supernatant after rehydration is collected and centrifuged to purify, obtaining a dispersion containing macrophage microvesicles; Preparation of biomimetic probucol self-assembled nanoparticles: The dispersion containing the DSPE-PEG-OH modified probucol self-assembled nanoparticles is mixed with the dispersion containing the macrophage microvesicles, and vortex treatment and sonication treatment are performed in sequence, so that the macrophage microvesicles are coated on the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanoparticles, obtaining a biomimetic probucol self-assembled nanoparticle with a core-shell structure.

[0009] The third aspect of this application provides the use of a biomimetic probucol self-assembled nanoformulation as described in any embodiment of the first aspect of this application, or a biomimetic probucol self-assembled nanoformulation prepared by the preparation method described in any embodiment of the second aspect of this application, in the preparation of a drug for treating atherosclerosis.

[0010] In summary, the biomimetic probucol self-assembled nanoparticles, their preparation method, and their application in the preparation of drugs for treating atherosclerosis provided in this application involve the non-covalent self-assembly of uncovalently modified probucol molecules in an aqueous phase to form a probucol self-assembled nanocore. A DSPE-PEG-OH modification component is then applied to the surface of the probucol self-assembled nanocore. Furthermore, macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration provide a membrane material, which forms on the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanoparticles, thereby obtaining a biomimetic nanoparticle with a core-shell structure.

[0011] Probucol itself, as a drug molecule forming the nanocore, helps reduce the space occupied by traditional carrier materials in the nanocore; the DSPE-PEG-OH modification component helps maintain the self-assembled probucol nanoparticles in an aqueous dispersion state; the macrophage-derived microvesicle membrane layer enables the resulting nanoformation to form a cell-derived membrane outer surface. The results of the examples show that the obtained PPBNPs and MPPBNPs generally maintained a nanoscale dispersion state in the tested aqueous media; MPPBNPs exhibited high cellular uptake levels in inflammatory-stimulated HUVECs and RAW264.7 macrophages and were able to distribute to areas related to atherosclerotic aortic lesions. Further in vivo and in vitro experiments showed that MPPBNPs could reduce oxidative stress-related indicators, inhibit lipid uptake or foam cell formation, and in ApoE... - / - The nanoformulation reduced aortic plaque burden in a mouse model of atherosclerosis. Therefore, the nanoformulation can be used to prepare drugs for treating atherosclerosis. Attached Figure Description

[0012] The specific features involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0013] Figure 1 The figure shown is a diagram illustrating the physicochemical characterization results of PPBNPs, MPPBNPs, and MMVs in one embodiment of this application.

[0014] Figure 2The figure shown is a graph illustrating the screening and self-assembly mechanism verification results of Probucol self-assembled nanoparticles in one embodiment of this application.

[0015] Figure 3 The image shows the stability characterization results of PPBNPs and MPPBNPs in different media in one embodiment of this application.

[0016] Figure 4 The figure shown is a characterization result of the potential and fluorescence properties of DiD-labeled PPBNPs and MPPBNPs in one embodiment of this application.

[0017] Figure 5 The diagram shows the cell compatibility evaluation results of PPBNPs and MPPBNPs in one embodiment of this application.

[0018] Figure 6 The figure shown is an evaluation of the uptake of DiD-labeled PPBNPs and MPPBNPs by HUVECs and RAW264.7 macrophages under normal or inflammatory stimulation conditions in one embodiment of this application.

[0019] Figure 7 This diagram shows the evaluation results of the cellular uptake pathway and intracellular distribution of PPBNPs and MPPBNPs in one embodiment of this application.

[0020] Figure 8 The figure shown is a graph illustrating the in vitro effect evaluation results of PPBNPs and MPPBNPs on HUVECs in one embodiment of this application.

[0021] Figure 9 The figure shown is an evaluation result of the in vitro effects of PPBNPs and MPPBNPs on RAW264.7 macrophages in one embodiment of this application.

[0022] Figure 10 Shown as DiD-tagged PPBNPs and MPPBNPs in one embodiment of this application in ApoE - / - The evaluation results of the distribution and blood retention in the mouse body are shown in the figure.

[0023] Figure 11 The figure shown is a graph illustrating the in vivo therapeutic effects of different dosage forms of probucol in atherosclerotic mice in one embodiment of this application.

[0024] Figure 12 The figure shown is a graph illustrating the in vivo safety and blood compatibility evaluation results of different dosage forms of probucol in atherosclerotic mice in one embodiment of this application. Detailed Implementation

[0025] The following specific embodiments illustrate the implementation of this application. Those skilled in the art can easily understand the advantages and technical effects of this application from the content disclosed in this specification. In the following description, some embodiments may be referenced to the accompanying drawings. It should be understood that other embodiments without accompanying drawings may also be used, and those skilled in the art can make reasonable adjustments to the raw material specifications, preparation methods, process conditions, testing methods, and experimental operations without departing from the technical concept of this application. The following detailed description should not be considered limiting, and the scope of the embodiments of this application is limited only to the claims published in this application. The terminology used herein is for describing specific embodiments only and is not intended to limit this application.

[0026] Unless otherwise specified, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It will also be understood that terms used herein shall be interpreted as having the meaning consistent with their meaning in the context of this specification and the relevant field, and shall not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0027] In view of the technical problems mentioned in the background art, the present application provides a biomimetic probucol self-assembled nano-formulation, its preparation method, and its application in the preparation of drugs for treating atherosclerosis. This involves the non-covalent self-assembly of unmodified probucol molecules in an aqueous phase to form a probucol self-assembled nanocore. A DSPE-PEG-OH modification component is then applied to the surface of the probucol self-assembled nanocore. Furthermore, macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration treatment provide a microvesicle membrane layer that coats the outer surface of the probucol self-assembled nanocore modified with the DSPE-PEG-OH modification component, resulting in a core-shell structure for the obtained nano-formulation. Therefore, on the one hand, probucol, as the main drug component forming the nanocore, allows probucol itself to act as a drug molecule forming the nanocore, which helps reduce the space occupied by traditional carrier materials in the nanocore and the space occupied by carrier materials in the drug loading system. On the other hand, the DSPE-PEG-OH modified component helps improve the water dispersibility and dispersion stability of probucol self-assembled nanoparticles, and the macrophage-derived microvesicle membrane layer helps to form a cell-derived biomimetic surface and improve the uptake level of nanoparticles by vascular endothelial cells and macrophages in the inflammatory stimulation cell model. The in vivo distribution results show that both PPBNPs and MPPBNPs can be distributed to the aortic lesion-related areas of atherosclerosis.

[0028] In this application, "atherosclerosis" (AS) refers to a chronic vascular disease characterized by lipid deposition, endothelial dysfunction, inflammatory response, oxidative stress, macrophage foaming, and the formation or progression of arterial plaques. Treatment of atherosclerosis in this application includes, but is not limited to, slowing down, inhibiting, or improving the progression of atherosclerotic lesions, reducing plaque area or lipid deposition within plaques, reducing the formation of necrotic cores within plaques, improving plaque stability, or alleviating oxidative stress, apoptotic damage, lipid uptake, or foam cell formation associated with atherosclerosis.

[0029] In this application, "biomimetic probucol self-assembled nanoformulation" refers to a nanoformulation comprising a probucol self-assembled nanocore, a DSPE-PEG-OH modified component, and a macrophage-derived microvesicle membrane layer. The probucol self-assembled nanocore serves as the core structure of the drug. The DSPE-PEG-OH modified component is distributed on the surface of the probucol self-assembled nanocore. The macrophage-derived microvesicle membrane layer coats the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanocore, thus forming a core-shell structure in the resulting nanoformulation. The term "biomimetic" primarily refers to the use of membrane materials provided by macrophage-derived microvesicles to form a coating structure on the outer surface of nanoparticles, indicating the cell origin of this outer membrane structure and its relative position on the outer surface of the nanoparticles.

[0030] In this application, "Probucol (PB)" refers to a hydrophobic small molecule drug used in this application as the drug molecule forming the self-assembled nanocore of probucol. Unless otherwise stated, probucol as used in this application refers to the probucol parent molecule that is not covalently linked to other drug molecules, polymer segments, or responsive linker groups.

[0031] In this application, "uncovalently modified probucol molecule" refers to a probucol molecule that is not covalently linked to other drug molecules, polymer segments, targeting groups, responsive linking groups, or prodrug linking groups. This term is used to distinguish it from probucol prodrugs, probucol polymeric prodrugs, and composite prodrugs formed by covalently linking probucol with other drugs. This term does not preclude non-covalent interactions between probucol molecules and water molecules, DSPE-PEG-OH modified components, or microvesicle membranes during nanoparticle formation.

[0032] In this application, "probucol self-assembled nanocore" refers to a nanoscale drug core structure formed by the aggregation and self-assembly of uncovalently modified probucol molecules in an aqueous phase through noncovalent interactions. This probucol self-assembled nanocore uses probucol molecules as the drug molecule forming the nanocore, which differs from drug-loaded structures formed by simply encapsulating probucol through liposomes, polymer micelles, inorganic particles, or other carrier materials.

[0033] In this application, "single-drug self-assembled nanocore" refers to a self-assembled nanocore in which the drug molecule forming the nanocore is probucol. This term is used to indicate that the probucol self-assembled nanocore is not a co-assembled drug core formed by probucol and other therapeutically active small molecule drugs. The term "single-drug self-assembled nanocore" does not exclude the presence of DSPE-PEG-OH as a surface modification component, nor does it exclude the presence of a macrophage-derived microvesicle membrane layer as an outer coating structure.

[0034] In this application, "non-covalent interaction" refers to an interaction that promotes or maintains intermolecular aggregation, self-assembly, or structural stability without being linked by covalent bonds. For the purposes of this application, the non-covalent interaction includes hydrophobic interactions, and includes at least one of hydrogen bonding and π-π stacking interactions. Specifically, hydrophobic interaction refers to the interaction in which hydrophobic molecules or hydrophobic structural segments tend to move closer together in an aqueous environment to reduce their contact area with water molecules; hydrogen bonding refers to the non-covalent interaction formed between atoms or groups containing hydrogen bond donors and hydrogen bond acceptors; and π-π stacking interaction refers to the non-covalent stacking interaction between structures containing aromatic rings or other π-electron systems through π-electron interactions.

[0035] The term "DSPE-PEG-OH" refers to hydroxyl-terminated polyethylene glycol-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG-OH, which can be described as a hydroxyl-terminated distearoyl-phosphatidylethanolamine-polyethylene glycol coupling compound). Here, DSPE represents 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG represents polyethylene glycol; and OH indicates hydroxyl-terminated. Unless otherwise stated, the molecular weight of the PEG segment described in this application refers to the average molecular weight as stated by the supplier, not the absolute molecular weight of each PEG molecule.

[0036] In this application, "DSPE-PEG-OH modified component" refers to the surface modification portion formed by DSPE-PEG-OH and distributed on the surface of the probucol self-assembled nanocore. The DSPE-PEG-OH modified component is used to improve the dispersibility and stability of the probucol self-assembled nanoparticles in aqueous phase and to provide a suitable particle surface state for the coating of macrophage-derived microvesicle membranes. The use of the DSPE-PEG-OH modified component does not imply that a covalent bond must exist between DSPE-PEG-OH and probucol.

[0037] In this application, "RAW264.7 macrophages" refers to the mouse-derived macrophage-like cell line RAW264.7 (RAW264.7 murine macrophage-like cell line). In this application, RAW264.7 macrophages are used to release macrophage microvesicles after dehydration and rehydration treatment.

[0038] In this application, "macrophage-derived microvesicles" (MMVs) refer to membranous vesicle particles obtained from RAW264.7 macrophages after dehydration and rehydration, followed by centrifugation purification. In this application, the membranous vesicle particles obtained according to the above-described cell source and preparation process, and characterized by particle size, potential, and / or transmission electron microscopy, are referred to as macrophage microvesicles.

[0039] In this application, "macrophage-derived microvesicle membrane layer" refers to a membrane-like coating structure formed on the outer surface of the nanoparticles by mixing the aforementioned macrophage microvesicles with probucol self-assembled nanoparticles modified with DSPE-PEG-OH, followed by vortexing and sonication treatment. The term "source" refers to the fact that the membrane material forming this coating structure is provided by macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration treatment.

[0040] In this application, "dehydration treatment" refers to a process in which, after removing the culture medium from RAW264.7 macrophages and washing the cells, the washing buffer is removed, and the cells are placed in an open environment in a sterile setting for a certain period of time, thus subjecting the cells to a short-term state of dehydration or fluid deficiency. In some embodiments, the dehydration treatment includes: after removing the washing DPBS, opening the culture container lid, and placing the RAW264.7 macrophages in a sterile laminar flow hood for 15-25 minutes, for example, about 20 minutes.

[0041] In this application, "rehydration treatment" refers to the process of adding buffer solution back to RAW264.7 macrophages after dehydration and allowing them to stand for a certain period of time to promote the release of macrophage microvesicles. In some embodiments, the rehydration treatment includes: adding DPBS to the dehydrated RAW264.7 macrophages and allowing them to stand for 20-40 minutes, for example, about 30 minutes; then gently tapping the edge of the culture container to promote the release of macrophage microvesicles and collecting the supernatant containing the macrophage microvesicles.

[0042] In this application, "core-shell structure" refers to a nanoparticle structure comprising self-assembled probucol nanoparticles modified with DSPE-PEG-OH internally and a membrane-coated structure provided by macrophage microvesicles externally. The thickness of the external membrane-coated structure may vary. Transmission electron microscopy can be used to observe the membrane boundary or outer layer structure located outside the internal nanoparticles, which can be used to characterize the core-shell structure; changes in hydration kinetic diameter and zeta potential before and after coating can serve as auxiliary characterization results.

[0043] In this application, "PBNPs" (short for probucol self-assembled nanoparticles) refers to nanoparticles formed by the self-assembly of unmodified probucol molecules in an aqueous phase via nanoprecipitation without the addition of DSPE-PEG-OH and macrophage microvesicles. PBNPs are mainly used for the self-assembly behavior of probucol, dosage screening, and verification of the self-assembly mechanism.

[0044] In this application, "PPBNPs" refers to DSPE-PEG-OH-modified probucol self-assembled nanoparticles. In this application, PPBNPs can be used as nanocore particles or intermediates for the preparation of MPPBNPs.

[0045] In this application, "MMVs" (short for macrophage microvesicles or macrophage-derived microvesicles) refers to macrophage microvesicles obtained by releasing RAW264.7 macrophages after dehydration and rehydration treatment and then purifying them by centrifugation.

[0046] In this application, "MPPBNPs" refers to macrophage microvesicle-coated DSPE-PEG-OH-modified probucol self-assembled nanoparticles. In this application, MPPBNPs are a specific embodiment of a biomimetic probucol self-assembled nanoformulation.

[0047] In this application, "DPBS" is short for Dulbecco's phosphate-buffered saline. In this application, DPBS can be used for washing RAW264.7 macrophages, rehydration, and resuspending macrophage microvesicles.

[0048] In this application, "DMF" is an abbreviation for N,N-dimethylformamide. In this application, DMF can be used as an organic solvent to dissolve probucol and DSPE-PEG-OH to form an organic phase, and to prepare DSPE-PEG-OH modified probucol self-assembled nanoparticles via a nanoprecipitation method.

[0049] In this application, "MWCO" is short for molecular weight cut-off, which refers to the nominal molecular weight limit that a dialysis membrane or ultrafiltration membrane allows small molecules to pass through while retaining larger molecules or particles. For example, MWCO3500Da indicates that the nominal molecular weight cut-off of the dialysis membrane is 3500Da. In this application, MWCO can be used to define the molecular weight cut-off of a dialysis bag.

[0050] In this application, "overnight dialysis" refers to a continuous dialysis treatment for a relatively long period of time, typically 8 to 16 hours, for example, about 12 hours. The overnight dialysis can be used to remove residual organic solvents and free small molecule components from the nanoprecipitation process and to obtain a dispersion of probucol self-assembled nanoparticles modified with DSPE-PEG-OH.

[0051] In this application, "aqueous dispersion for intravenous administration" refers to an aqueous dispersion system containing the biomimetic probucol self-assembled nanoformulation suitable for intravenous administration. The aqueous dispersion system may contain a pharmaceutically acceptable aqueous medium, such as sterile saline, injectable buffer solution, or other pH- and osmotically adjusted aqueous medium. This term is used to describe the administration form of the nanoformulation and does not require that the aqueous dispersion necessarily contain all the pharmaceutical excipients required in the final clinical formulation.

[0052] Unless otherwise stated, when this application refers to the drug concentration and dosage of PB, PPBNPs, or MPPBNPs, all values ​​are expressed in probucol equivalents. When referring to MMV concentrations, they may be characterized using one of the following: total protein concentration, phospholipid concentration, or particle number.

[0053] In some embodiments, this application provides a biomimetic probucol self-assembled nanoformulation comprising a probucol self-assembled nanocore, a DSPE-PEG-OH modified component, and a macrophage-derived microvesicle membrane layer. The probucol self-assembled nanocore constitutes the drug core of the nanoformulation, the DSPE-PEG-OH modified component is distributed on the surface of the probucol self-assembled nanocore, and the macrophage-derived microvesicle membrane layer coats the outer surface of the probucol self-assembled nanocore modified by the DSPE-PEG-OH modified component, thereby forming a biomimetic nanoformulation with a core-shell structure.

[0054] Specifically, the probucol self-assembled nanocore is formed by the self-assembly of uncovalently modified probucol molecules in an aqueous environment through non-covalent interactions. These non-covalent interactions include hydrophobic interactions and at least one of hydrogen bonding and π-π stacking interactions. Due to the strong hydrophobicity of probucol molecules themselves, they can aggregate through hydrophobic interactions in an aqueous environment and further form nanoscale drug core structures with the participation of hydrogen bonding and / or π-π stacking interactions. Thus, probucol molecules can serve as drug molecules forming nanocores, self-assembling to form nanoscale drug core structures without the need for covalent modification to link probucol to other drug molecules, polymer segments, or prodrug linker groups.

[0055] Furthermore, the DSPE-PEG-OH modifying component is distributed on the surface of the probucol self-assembled nanocore to form DSPE-PEG-OH modified probucol self-assembled nanoparticles, i.e., PPBNPs. In one implementation, the PPBNPs are obtained by dissolving probucol and DSPE-PEG-OH together in an organic solvent, adding the resulting organic phase dropwise to an aqueous phase, and then dialyzing. In one example, the DSPE-PEG-OH modifying component participates in the formation of PPBNPs as a surface modifying component.

[0056] In one embodiment, the macrophage-derived microvesicle membrane layer is provided by macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration. After mixing, vortexing, and sonication with DSPE-PEG-OH modified probucol self-assembled nanoparticles, the macrophage microvesicles can coat or fuse onto the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanoparticles, thereby forming a membrane-like coating structure derived from macrophage microvesicles on the outside of the nanoparticles. Thus, the biomimetic probucol self-assembled nanoformulation combines the drug-loading characteristics of a probucol self-assembled nanocore with the biomimetic surface characteristics of a macrophage-derived microvesicle membrane layer.

[0057] Please see Figure 1 The image shows the physicochemical characterization results of PPBNPs, MPPBNPs, and MMVs in one embodiment of this application. Figure 1 A shows the hydration dynamic diameter characterization results for PPBNPs, MPPBNPs, and MMVs; Figure 1 B shows the dispersion coefficient characterization results for PPBNPs, MPPBNPs, and MMVs; Figure 1 C shows the potential characterization results for PPBNPs, MPPBNPs, and MMVs; Figure 1 D shows the transmission electron microscopy observations of PPBNPs, MPPBNPs, and MMVs.

[0058] In this application, the "polydispersity index" (PDI) is used to characterize the uniformity of particle size distribution. Combined with... Figure 1 A to Figure 1 As can be seen from C, the particle size and potential of the nanoparticles changed before and after coating with macrophage microvesicles, indicating that macrophage microvesicles can alter the surface characteristics of probucol self-assembled nanoparticles modified with DSPE-PEG-OH. Combined with... Figure 1 As can be seen from D, PPBNPs exhibit a nanoparticle morphology, MMVs exhibit a membrane-bound vesicle morphology, while MPPBNPs, after coating macrophage microvesicles, exhibit a core-shell structure. Figure 1 A to Figure 1 C shows that after coating with macrophage microvesicles, the resulting MPPBNPs exhibited changes in hydration kinetic diameter and zeta potential compared to PPBNPs, indicating alterations in particle size and surface charge after treatment. Combined with... Figure 1As can be seen from D, PPBNPs exhibit a nanoparticle morphology, MMVs exhibit a membrane vesicle morphology, and MPPBNPs show a membrane boundary or outer layer structure on the outer side of the internal particles. Considering the RAW264.7 cell origin, dehydration-rehydration, and centrifugation purification process of MMVs, as well as the mixing, vortexing, and sonication treatment processes of MPPBNPs, the above results collectively support the formation of membrane material provided by MMVs on the outer surface of PPBNPs, resulting in MPPBNPs exhibiting a core-shell structure.

[0059] In this application, the core-shell structure does not require that the macrophage-derived microvesicle membrane layer form a geometrically continuous and completely closed shell of absolutely uniform thickness around all particles. The thickness of the macrophage-derived microvesicle membrane layer is not required to be absolutely uniform, but the resulting MPPBNPs should exhibit morphological characteristics of the combination of internal nanoparticles and the membrane boundary or outer structure located on their outer side. The outer membrane structure can be characterized by transmission electron microscopy, and changes in hydration dynamics diameter and Zeta potential before and after coating can serve as auxiliary characterization results.

[0060] In one specific embodiment, the biomimetic probucol self-assembled nanoformulation consists of DSPE-PEG-OH modified probucol self-assembled nanoparticles, i.e., MPPBNPs, coated with macrophage microvesicles. The MPPBNPs use inner PPBNPs as the drug core particles and an outer macrophage-derived microvesicle membrane layer as the biomimetic coating layer. Through this structural configuration, on the one hand, probucol can exist in the form of a self-assembled nanocore, which is beneficial for improving its dispersion in aqueous systems; on the other hand, the macrophage-derived microvesicle membrane layer can provide a cell-derived membrane surface structure and exhibits the effect of increasing cellular uptake levels in the inflammatory cell model of this application; the resulting nanoformulation demonstrates the ability to distribute to aortic lesion-related areas in animal experiments.

[0061] Please see Figure 2 The image shows the screening and self-assembly mechanism verification results of Probucol self-assembled nanoparticles in one embodiment of this application. Figure 2 A shows the hydration kinetic diameter characterization results of PBNPs obtained with different dosing masses from Probucol; Figure 2 B shows the dispersion coefficient characterization results of PBNPs obtained from different dosages of probucol; Figure 2 C represents the hydration kinetic diameter characterization results of nanoparticles obtained after co-treatment with NaCl, Urea, or SDS during the preparation of PBNPs. Figure 2 D represents the dispersion coefficient characterization results of nanoparticles obtained after co-treatment with NaCl, Urea, or SDS during the preparation of PBNPs. Figure 2E shows the Fourier transform infrared spectral characterization results of PBNPs; Figure 2 F shows a schematic diagram of the self-assembly of PBNPs and snapshots of the self-assembly process of probucol in aqueous dispersion at different simulation times; Figure 2 G to Figure 2 H represents the analysis results of hydrogen bonding and Leonard-Jones interaction between Probucol and water; Figure 2 I arrive Figure 2 J shows the results of solvent-accessible surface area and total potential energy analysis during the Probucol self-assembly process; Figure 2 K to Figure 2 L shows the root mean square deviation and radius of gyration analysis results for Probucol aggregates; Figure 2 M shows the free energy surface analysis results for different structural states of small molecule aggregates, determined based on root mean square deviation and radius of gyration.

[0062] In one embodiment, the probucol self-assembled nanocore is formed by the self-assembly of uncovalently modified probucol molecules in an aqueous phase. Specifically, probucol can be dissolved in an organic solvent to form an organic phase using a nanoprecipitation method, and then the organic phase is added dropwise to the aqueous phase, causing the probucol molecules to aggregate in the aqueous environment and form a nanoscale particle structure. The probucol self-assembled nanocore uses probucol as the drug molecule for forming the nanocore, and it is not necessary to pre-covalently link probucol to other drug molecules, polymer segments, targeting groups, or responsive linking groups.

[0063] In one embodiment, the probucol self-assembled nanocore is a single-drug self-assembled nanocore formed by probucol as the drug molecule. That is, the drug molecule forming the nanocore is probucol, rather than a co-assembled drug core formed by probucol and other therapeutically active small molecule drugs. It should be understood that the single-drug self-assembled nanocore is used to define the type of drug molecule participating in the self-assembly nucleation within the nanocore. Further application of a DSPE-PEG-OH modification component to the surface of the probucol self-assembled nanocore is not excluded, nor is further coating of the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanoparticles with a macrophage-derived microvesicle membrane layer.

[0064] In one specific embodiment, the probucol dosage was set to 5 mg, 10 mg, and 20 mg, respectively, and PBNPs were prepared using the same preparation system. Combined with Figure 2 A and Figure 2As can be seen from B, the hydration kinetic diameter and dispersion coefficient of PBNPs obtained under different drug dosage masses differ, indicating that the dosage of probucol affects the particle size distribution and dispersion state of the obtained nanoparticles. In this embodiment, there is a drug dosage range that can form stable nanoparticles; under the premise of ensuring nanoparticle stability, 10 mg of probucol was selected as the preferred dosage mass. The PBNPs prepared under these conditions have suitable hydration kinetic diameter and dispersion coefficient, and no significant drug loss was observed in this embodiment.

[0065] In one embodiment, to analyze the intermolecular interactions that may be involved in the self-assembly of probucol, NaCl, Urea, or SDS were added to the aqueous phase during the preparation of PBNPs, and the hydration kinetic diameter and dispersion coefficient of the resulting nanoparticles were measured after dialysis. Figure 2 C and Figure 2 As can be seen from D, compared with the control group, the hydration kinetic diameter and dispersion coefficient of the NaCl-treated group changed significantly, while the Urea-treated group and the SDS-treated group did not show the same degree of change. These results indicate that the non-covalent interactions between probucol molecules and the interactions between probucol and the aqueous environment are related to the self-assembly behavior of probucol.

[0066] Furthermore, combined Figure 2 The Fourier transform infrared spectroscopy results shown in E indicate that the characteristic absorption peaks change after probucol self-assembles into PBNPs. Specifically, at 3000 cm⁻¹... -1 Up to 3500cm -1 The broadening, intensity changes, or shifts of OH-related peaks within the range indicate potential changes in hydrogen bonding after self-assembly; at 1200 cm⁻¹... -1 Up to 1300cm -1 The shift in the CO or SO stretching vibration-related peaks within the range indicates that structural changes related to hydrophobic interactions or π-π stacking may occur after self-assembly. Therefore, in one embodiment, the probucol self-assembled nanocore can be formed from probucol molecules through non-covalent interactions including hydrophobic interactions, and these non-covalent interactions may further include at least one of hydrogen bonding and π-π stacking interactions.

[0067] In one embodiment, molecular dynamics simulations were used to further observe the aggregation process of probucol molecules in an aqueous environment. Figure 2 As can be seen from F, probucol molecules can aggregate in the aqueous dispersion during the simulation. Combined with... Figure 2 G to Figure 2 H shows that there are hydrogen bonds and Leonard-Jones interactions between Probucol and water; combined Figure 2 I arrive Figure 2 J shows that the solvent-accessible surface area and the total potential energy of the system change with time during the simulation, with the total potential energy of the system entering a relatively stable fluctuating state after changing in the initial stage of the simulation; combined with Figure 2 K to Figure 2 L shows that the root mean square deviation and radius of gyration of the Probucol aggregates change with simulation time, indicating that the aggregate size and structure are not constant; combined with Figure 2 As can be seen from M, probucol aggregates can correspond to multiple structural states. The simulation results above indicate that probucol molecules can form aggregates in the aqueous phase, and the resulting aggregates can be in a state of dynamic structural change.

[0068] Based on the above results, in this application, the probucol self-assembled nanocore can be understood as a drug core structure formed by the aggregation of uncovalently modified probucol molecules in an aqueous environment through noncovalent interactions. This drug core structure differs from drug-loaded structures formed by simply encapsulating probucol with exogenous carrier materials, and also differs from probucol prodrug nanoparticles obtained through covalent predrugation. By forming the probucol self-assembled nanocore, probucol can exist in nanoparticle form, providing a particle basis for subsequent DSPE-PEG-OH surface modification and macrophage-derived microvesicle membrane coating.

[0069] In one embodiment, the DSPE-PEG-OH modifying component is provided by DSPE-PEG-OH and distributed on the surface of the probucol self-assembled nanocore to form DSPE-PEG-OH modified probucol self-assembled nanoparticles, i.e., PPBNPs. The DSPE-PEG-OH modifying component participates in the formation of PPBNPs as a surface modifying component, but a covalent bond between the DSPE-PEG-OH and the probucol molecules is not required. During nanoprecipitation, the hydrophobic lipid segments of DSPE-PEG-OH can non-covalently associate with the hydrophobic aggregation regions of probucol, and the hydrophilic PEG segments can be distributed towards the aqueous phase, thus allowing DSPE-PEG-OH to participate in the formation of PPBNPs as a surface modifying component. This description is intended to explain possible structural arrangements and does not indicate that the specific orientation of each DSPE-PEG-OH molecule has been determined through surface chemical analysis.

[0070] In one embodiment, the molecular weight of the PEG segment in the DSPE-PEG-OH is between 3000 Da and 4000 Da. For example, it can be approximately 3000 Da, 3100 Da, 3200 Da, 3300 Da, 3400 Da, 3500 Da, 3600 Da, 3700 Da, 3800 Da, 3900 Da, or 4000 Da. By using DSPE-PEG-OH with the above molecular weight range, DSPE-PEG-OH can be co-prepared with probucol to obtain DSPE-PEG-OH modified probucol self-assembled nanoparticles. In one implementation, probucol and DSPE-PEG-OH can be co-dissolved in an organic solvent to form an organic phase, which is then added dropwise to an aqueous phase and dialyzed to obtain a dispersion containing PPBNPs.

[0071] In one embodiment, the mass ratio of probucol to DSPE-PEG-OH is (8~12):1. For example, it can be approximately 8:1, 9:1, 10:1, 11:1, or 12:1. Preferably, the mass ratio of probucol to DSPE-PEG-OH is 10:1. In a specific implementation, 10 mg of probucol and 1 mg of DSPE-PEG-OH are dissolved together in 1 mL of N,N-dimethylformamide, and then the resulting organic phase is added dropwise to 4 mL of aqueous phase, and after dialyzing, a PPBNPs dispersion is obtained. Thus, PPBNPs can be used as core particles or intermediates for subsequent macrophage microvesicle coating. Within the above mass ratio range, whether the obtained formulation is suitable for subsequent microvesicle coating can be confirmed by hydration kinetic diameter, dispersion coefficient, and transmission electron microscopy morphology; when the obtained particles maintain a nanoscale dispersion and do not show obvious precipitation or irreversible aggregation, they can be used as PPBNPs for subsequent coating.

[0072] Please continue reading. Figure 1 , combined Figure 1 A to Figure 1 As can be seen from C, PPBNPs possess detectable nanoscale particle size, dispersion index, and surface potential; combined with Figure 1 As can be seen from D, PPBNPs exhibit a nanoparticle morphology. These results demonstrate that PPBNPs with a nanoparticle morphology can be obtained after modification with the DSPE-PEG-OH modifier, and can be further used to prepare macrophage-derived microvesicle membrane-coated MPPBNPs.

[0073] In one embodiment, the macrophage-derived microvesicle membrane layer is provided by macrophage microvesicles released from RAW264.7 macrophages. Specifically, the RAW264.7 macrophages release macrophage microvesicles after dehydration and rehydration treatment. These macrophage microvesicles, after centrifugation purification, can be used to coat probucol self-assembled nanoparticles modified with DSPE-PEG-OH, i.e., PPBNPs.

[0074] In one embodiment, the macrophage microvesicles are obtained as follows: RAW264.7 macrophages are cultured until the cell confluence reaches 80% to 90%, the culture medium is removed and the cells are washed; subsequently, the washed RAW264.7 macrophages are dehydrated, and then the dehydrated RAW264.7 macrophages are rehydrated to allow the RAW264.7 macrophages to release macrophage microvesicles; the supernatant after rehydration is collected and purified by centrifugation to obtain a dispersion containing the macrophage microvesicles. The macrophage microvesicles can be mixed with PPBNPs in subsequent steps and coated onto the outer surface of the PPBNPs by vortexing and sonication.

[0075] In one specific embodiment, the dehydration treatment includes: after removing the washing buffer, placing RAW264.7 macrophages in an open environment in a sterile laminar flow hood for 15 to 25 minutes, for example, about 20 minutes. The rehydration treatment includes: adding buffer to the dehydrated RAW264.7 macrophages and allowing them to stand for 20 to 40 minutes, for example, about 30 minutes. The centrifugal purification includes: centrifuging at 4000-6000×g for 3-8 minutes (e.g., 5000×g for 5 minutes) to remove detached cells, and centrifuging at 12000-16000×g for 5-15 minutes (e.g., 14000×g for 10 minutes) to enrich the macrophage microvesicles. The macrophage microvesicles obtained thereby can be used as source material for forming the macrophage-derived microvesicle membrane layer.

[0076] Furthermore, after mixing the macrophage microvesicles with PPBNPs, they can coat or fuse onto the outer surface of the PPBNPs under external force. In one embodiment, the external force includes vortex treatment and ultrasonic treatment. After the above treatment, the macrophage microvesicles can form a membrane-like coating structure on the outside of the PPBNPs, thereby obtaining macrophage microvesicle-coated DSPE-PEG-OH modified probucol self-assembled nanoparticles, i.e., MPPBNPs. The macrophage-derived microvesicle membrane layer in the MPPBNPs serves as the outer membrane structure, coating the outer surface of the PPBNPs, thus giving the obtained nano-formulation a surface structure distinct from that of uncoated PPBNPs. In one implementation, the MPPBNPs nano-formulation is obtained by mixing a dispersion containing a probucol self-assembled nanocore modified with the DSPE-PEG-OH modified component and a dispersion containing the macrophage microvesicles at a volume ratio of (7~11):1, followed by vortex treatment and ultrasonic treatment. For example, a dispersion containing PPBNPs can be mixed with a dispersion containing MMVs at a volume ratio of approximately 7:1, 8:1, 9:1, 10:1, or 11:1, and then subjected to vortexing and ultrasonic treatment to obtain MPPBNPs.

[0077] In one embodiment, the PPBNP dispersion and the macrophage microvesicle dispersion can be mixed in a specific ratio to achieve a suitable coating amount of macrophage microvesicles relative to PPBNPs. After mixing, intermittent vortexing can promote sufficient contact and dispersion between the two; further ultrasonic treatment in a water bath can promote the coating or fusion of macrophage microvesicles with PPBNPs, thereby distributing the membrane structure provided by the macrophage microvesicles on the outer surface of PPBNPs. In one specific embodiment, the volume ratio of the dispersion containing the probucol self-assembled nanocore modified by the DSPE-PEG-OH modified component to the dispersion containing macrophage microvesicles is 9:1. The vortexing treatment is performed once every 7-12 minutes (e.g., once every 10 minutes), for a total of 2-3 times (e.g., 3 times). The ultrasonic treatment is water bath ultrasonication, and the ultrasonic conditions are 40-44 kHz (e.g., 42 kHz), 90-110 W (e.g., 100 W), and 2.5-4 minutes (e.g., 3 minutes).

[0078] Please continue reading. Figure 1 , combined Figure 1 A to Figure 1 As can be seen from C, compared to uncoated PPBNPs, the hydration dynamics diameter and potential of MPPBNPs changed, indicating that macrophage microvesicles participated in the formation of PPBNP surface structures. Combined with... Figure 1As can be seen from D, MMVs exhibit a membrane-bound vesicle morphology, while MPPBNPs display a core-shell structure distinct from PPBNPs. These results demonstrate that macrophage microvesicles can serve as membrane-derived materials to coat the outer surface of PPBNPs, forming the macrophage-derived microvesicle membrane layer. It should be understood that the macrophage-derived microvesicle membrane layer can coat, cover, or fuse to at least a portion of the outer surface of PPBNPs, without requiring it to form a perfectly uniform geometric shell of absolute thickness around each nanoparticle.

[0079] In one embodiment, the biomimetic probucol self-assembled nanoformulation is an aqueous dispersion for intravenous administration. The aqueous dispersion for intravenous administration comprises an aqueous dispersion medium and MPPBNPs dispersed in the aqueous dispersion medium. In other words, the MPPBNPs are dispersed in the aqueous dispersion medium as nanoparticles, rather than as free probucol crystals or organic solvent solutions.

[0080] In one embodiment, the aqueous dispersion medium may be water, a buffer solution, or a physiologically relevant aqueous medium. For example, the aqueous dispersion medium in the intravenous administration aqueous dispersion may be a pharmaceutically acceptable aqueous medium, such as sterile saline, injectable phosphate buffer, or other aqueous media adjusted for pH and osmotic pressure. It should be understood that the aqueous dispersion medium is used to maintain the MPPBNPs in a dispersed state; prior to specific administration, the concentration, osmotic pressure, pH, and sterility of the aqueous dispersion may be adjusted according to animal experiments or pharmaceutical formulation requirements.

[0081] In one embodiment, the aqueous dispersion for intravenous administration can be obtained directly from the prepared MPPBNPs, or it can be obtained by diluting a concentrated dispersion containing MPPBNPs with an aqueous dispersion medium. In a specific example, during the preparation of PPBNPs, probucol and DSPE-PEG-OH are added dropwise to the aqueous phase via an organic phase and dialyzed to form a PPBNPs dispersion; further, PPBNPs and macrophage microvesicles are mixed, vortexed, and sonicated to form an MPPBNPs dispersion. Thus, the obtained MPPBNPs can exist in aqueous dispersion form and can be used for subsequent in vitro characterization, in vivo distribution evaluation, or animal intravenous administration experiments.

[0082] Please see Figure 3 The image shows the stability characterization results of PPBNPs and MPPBNPs in different media in one embodiment of this application. Figure 3 A to Figure 3D shows the changes in the hydration kinetic diameter of PPBNPs and MPPBNPs over time in water, 0.9% NaCl solution, PBS, and culture medium containing 10% fetal bovine serum, respectively. In one specific embodiment, aqueous dispersions of PPBNPs or MPPBNPs were added to the above different media, and the hydration kinetic diameter was measured daily from 1 to 7 days. Figure 3 A to Figure 3 As can be seen from D, PPBNPs and MPPBNPs can maintain detectable nanoscale particle sizes in various aqueous media. These results indicate that MPPBNPs can exist in aqueous dispersion form and provide a basis for further evaluation as an aqueous dispersion for intravenous administration.

[0083] In one embodiment, the aqueous dispersion of the intravenously administered drug can be used for in vivo distribution evaluation in animals. Specifically, fluorescently labeled PPBNPs or MPPBNPs can be prepared into an aqueous dispersion, administered via tail vein injection, and blood, aorta, and major organs can be collected at a predetermined time after administration for fluorescence imaging analysis. Through this method, the distribution of nanoparticles in blood, aortic lesions, and major organs can be evaluated.

[0084] In one embodiment, the intravenous administration water dispersion can also be used for therapeutic evaluation of animal models of atherosclerosis. Specifically, PB, PPBNPs, or MPPBNPs can be formulated into administration systems for tail vein injection and administered to atherosclerosis model animals according to predetermined doses and frequencies. In one specific embodiment, ApoE induced by a high-fat diet... - / - Mice with atherosclerosis were administered PB, PPBNPs, or MPPBNPs via tail vein injection at a dose of 6 mg / kg, with a volume of 200 μL per administration, twice weekly for 8 weeks. After administration, the status of atherosclerotic plaques was evaluated by aortic Oil Red O staining and aortic root section staining.

[0085] In this application, the aqueous dispersion for intravenous administration is used to define the delivery form of MPPBNPs. That is, the MPPBNPs can be evaluated for intravenous administration as nanoparticles dispersed in an aqueous medium. This form differs from the direct administration of poorly soluble free probucol and also from administration forms using organic solvents as the main continuous phase. By preparing MPPBNPs as an aqueous dispersion for intravenous administration, a delivery basis can be provided for evaluating their in vivo distribution and therapeutic efficacy in atherosclerosis models.

[0086] This application provides a method for preparing a biomimetic probucol self-assembled nanoparticle in one embodiment. The method is used to prepare DSPE-PEG-OH modified probucol self-assembled nanoparticles, i.e., MPPBNPs, coated with macrophage microvesicles. The preparation method includes steps S110, S120, and S130.

[0087] Step S110 involves preparing probucol self-assembled nanoparticles (PPBNPs) modified with DSPE-PEG-OH. In this step, uncovalently modified probucol serves as the drug molecule forming the self-assembled nanocore of probucol, while DSPE-PEG-OH acts as a surface modification component participating in the formation of PPBNPs. By dissolving uncovalently modified probucol and DSPE-PEG-OH together in an organic solvent to form an organic phase, and then adding the organic phase dropwise to an aqueous phase while stirring, probucol can form self-assembled nanoparticles in the aqueous environment. Simultaneously, DSPE-PEG-OH participates in the formation of these self-assembled nanoparticles, thereby obtaining DSPE-PEG-OH modified probucol self-assembled nanoparticles.

[0088] Specifically, step S110 includes: dissolving uncovalently modified probucol and DSPE-PEG-OH in an organic solvent to form an organic phase; adding the organic phase dropwise to an aqueous phase and stirring to form DSPE-PEG-OH modified probucol self-assembled nanoparticles; dialyzing the resulting mixture to obtain a dispersion containing the DSPE-PEG-OH modified probucol self-assembled nanoparticles; wherein the DSPE-PEG-OH modified probucol self-assembled nanoparticles include a probucol self-assembled nanocore formed by the non-covalent self-assembly of uncovalently modified probucol molecules in an aqueous phase, the DSPE-PEG-OH modified component being distributed on the surface of the probucol self-assembled nanocore, and the mass ratio of probucol to DSPE-PEG-OH being (8~12):1.

[0089] In step S110, the amount of uncovalently modified probucol is 5-20 mg, the amount of DSPE-PEG-OH is 0.5-2 mg, and the mass ratio of probucol to DSPE-PEG-OH is (8-12):1. For example, in some examples, the amount of uncovalently modified probucol may be about 5 mg, about 8 mg, about 10 mg, about 12 mg, about 15 mg, or about 20 mg, and the amount of DSPE-PEG-OH may be about 0.5 mg, about 0.8 mg, about 1 mg, about 1.2 mg, about 1.5 mg, or about 2 mg. The uncovalently modified probucol and the DSPE-PEG-OH can be used in combination to meet the above-mentioned mass ratio range. For example, the dosage of the two can be about 8 mg and about 1 mg, about 9 mg and about 1 mg, about 10 mg and about 1 mg, about 11 mg and about 1 mg, or about 12 mg and about 1 mg, respectively. In this case, the mass ratio of the uncovalently modified probucol to the DSPE-PEG-OH is about 8:1, about 9:1, about 10:1, about 11:1, or about 12:1.

[0090] In one embodiment, the volume ratio of the organic solvent to the aqueous phase is 1:(3~5). Specifically, the organic solvent is used to dissolve uncovalently modified probucol and DSPE-PEG-OH to form an organic phase containing probucol and DSPE-PEG-OH; the aqueous phase is used to receive the dropwise addition of the organic phase, allowing probucol and DSPE-PEG-OH to form a nanoparticle dispersion in an aqueous environment. The aqueous phase can be pure water or ultrapure water. In some examples, the volume ratio of the organic solvent to the aqueous phase can be approximately 1:3, 1:4, or 1:5.

[0091] In one specific embodiment, in step S110, the amount of uncovalently modified probucol is 10 mg, the amount of DSPE-PEG-OH is 1 mg, the organic solvent is N,N-dimethylformamide, the volume of the organic solvent is 1 mL, and the aqueous phase is pure water or ultrapure water, the volume of the aqueous phase is 4 mL. That is, 10 mg of uncovalently modified probucol and 1 mg of DSPE-PEG-OH are dissolved together in 1 mL of N,N-dimethylformamide, and mixed to form an organic phase; the organic phase and 4 mL of pure water or ultrapure water constitute a granulation system with a volume ratio of 1:4.

[0092] In one embodiment, in step S110, the molecular weight of the PEG segment in the DSPE-PEG-OH is 3000~4000 Da, for example, approximately 3000 Da, 3100 Da, 3200 Da, 3300 Da, 3400 Da, 3500 Da, 3600 Da, 3700 Da, 3800 Da, 3900 Da, or 4000 Da. By using DSPE-PEG-OH within the above molecular weight range, DSPE-PEG-OH can participate in the formation of PPBNPs as a surface modification component.

[0093] Further, in step S110, the organic phase is vortex-mixed for 15-30 seconds and then added dropwise to an aqueous phase under stirring at a speed of 400-600 rpm. By adding the organic phase dropwise to the aqueous phase under stirring conditions, the organic phase can be dispersed in the aqueous phase, and probucol and DSPE-PEG-OH can form an initial nanoparticle dispersion containing PPBNPs in the aqueous environment. In a specific example, the organic phase can be vortex-mixed for 20 seconds and then added dropwise uniformly, slowly, and incrementally to an aqueous phase under stirring at a speed of 500 rpm. After the addition is complete, a mixture containing probucol self-assembled nanoparticles modified with DSPE-PEG-OH is obtained. The mixture contains the initially formed PPBNPs and small molecule components such as organic solvents that need to be further removed.

[0094] Further, in step S110, the obtained mixture is transferred to a dialysis bag with a molecular weight cutoff of 3000-4000 Da, and dialysis is performed under stirring conditions of the dialysis fluid. The dialysis treatment removes organic solvents and small molecule components, ensuring that the PPBNPs exist as an aqueous dispersion. After dialysis, the liquid in the dialysis bag is collected, yielding the PPBNPs dispersion.

[0095] In one specific embodiment, the stirring speed of the aqueous phase is 500 rpm, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed of the dialysis fluid is 200 rpm, and the dialysis is overnight dialysis. After dialysis, the liquid in the dialysis bag is collected to obtain an aqueous dispersion of probucol self-assembled nanoparticles (PPBNPs) modified with DSPE-PEG-OH. The obtained PPBNPs dispersion can be used as the core particle dispersion for macrophage microvesicle coating in subsequent step S130, and can also be used for particle size, potential, morphology, or stability characterization.

[0096] Step S120 involves preparing macrophage microvesicles (MMVs). In this step, the macrophage microvesicles are derived from RAW264.7 macrophages. Specifically, RAW264.7 macrophages are first cultured to a predetermined cell state, then dehydrated and rehydrated to release macrophage microvesicles, and finally purified by centrifugation to obtain the macrophage microvesicles (MMVs).

[0097] Specifically, step S120 includes culturing RAW264.7 macrophages until the cell confluence reaches 80%~90%, removing the culture medium and washing the cells, dehydrating the washed cells, rehydrating the dehydrated cells, collecting the supernatant after rehydration and centrifuging to purify it, and obtaining a dispersion containing macrophage microvesicles.

[0098] In step S120, after removing the culture medium, the cells are washed twice with DPBS. After removing the DPBS, the culture container lid is opened, and the RAW264.7 macrophages are placed in a sterile laminar flow hood with the opening exposed for 15-25 minutes (e.g., 20 minutes) to perform the dehydration process. This washing process removes the culture medium and free impurities, providing a relatively clean cell system for subsequent dehydration and rehydration. The short period of exposure keeps the RAW264.7 macrophages in a dehydrated state, facilitating the release of macrophage microvesicles during the subsequent rehydration process.

[0099] Further, in the rehydration treatment, DPBS is added to the dehydrated RAW264.7 macrophages and allowed to stand for 20-40 minutes. Then, the edge of the culture container is gently tapped to promote the release of macrophage microvesicles, and the supernatant containing the macrophage microvesicles is collected. In one specific embodiment, DPBS is added to the dehydrated RAW264.7 macrophages and allowed to stand for approximately 30 minutes, after which the supernatant is collected. Optionally, the culture container can be gently tapped before collecting the supernatant to promote the entry of macrophage microvesicles into the supernatant.

[0100] In the centrifugal purification process, the supernatant containing the macrophage microvesicles is centrifuged at 4000-6000×g for 3-8 min to remove detached cells. The supernatant is then centrifuged at 12000-16000×g for 5-15 min to enrich the macrophage microvesicles, and the enriched macrophage microvesicles are resuspended in DPBS. In a specific example, the supernatant containing the macrophage microvesicles is centrifuged at 5000×g for 5 min to remove detached cells; the precipitate is discarded and the supernatant is transferred, then centrifuged at 14000×g for 10 min to enrich the macrophage microvesicles; after centrifugation, the precipitate is collected and resuspended in DPBS to obtain a macrophage microvesicle (MMV) dispersion. The obtained MMV dispersion can be used as a membrane source material for coating PPBNPs in the subsequent step S130.

[0101] Step S130 involves preparing biomimetic probucol self-assembled nanoparticles (MPPBNPs). In this step, a dispersion containing the DSPE-PEG-OH modified probucol self-assembled nanoparticles (i.e., PPBNPs dispersion) is mixed with a dispersion containing the macrophage microvesicles (i.e., MMVs dispersion), and then subjected to vortexing and sonication treatments sequentially. This allows the macrophage microvesicles to coat the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanoparticles, resulting in a biomimetic probucol self-assembled nanoparticle formulation (i.e., MPPBNPs) with a core-shell structure.

[0102] Before mixing, the concentration of the PPBNPs dispersion was 1.42 mg / mL based on probucol equivalents. The MMVs dispersion was obtained from RAW264.7 macrophages in a 100 mm culture dish after dehydration and rehydration treatment, resuspended in 5 mL DPBS, centrifuged at 5000 rpm, and the supernatant was collected and centrifuged again at 14000 rpm. The precipitate was collected and resuspended in 200 μL ddH2O. In a specific embodiment, in step S130, the dispersion containing the DSPE-PEG-OH modified probucol self-assembled nanoparticles was mixed with the dispersion containing the macrophage microvesicles at a volume ratio of 9:1. The mixture was vortexed once every 10 min for a total of 3 vortexes, each vortex lasting 20 s. A vortex mixer (4000 rpm) was used for vortexing. Subsequently, the mixture was sonicated in a water bath at 42 kHz and 100 W for 3 min to obtain the biomimetic probucol self-assembled nanoformulation. In this embodiment, the volume ratio of PPBNPs dispersion to MMVs dispersion is configured as 9:1, which allows macrophage microvesicles to fully contact the PPBNPs as a membrane-derived material, facilitating the subsequent formation of a macrophage-derived microvesicle membrane layer on the outer surface of the PPBNPs. Intermittent vortexing promotes thorough mixing and contact between the PPBNPs and macrophage microvesicles in the dispersion system, reducing local aggregation or insufficient contact, and providing a relatively uniform mixing system for subsequent water bath sonication. Through water bath sonication, macrophage microvesicles can coat or fuse onto the outer surface of the PPBNPs, thereby forming a macrophage-derived microvesicle membrane layer located on the outer side of the PPBNPs, resulting in the biomimetic probucol self-assembled nanoformulation MPPBNPs.

[0103] In one embodiment, the MPPBNPs are obtained in the form of a dispersion. The MPPBNPs comprise a probucol self-assembled nanocore, a DSPE-PEG-OH modified component distributed on the surface of the probucol self-assembled nanocore, and a macrophage-derived microvesicle membrane layer coating the outer surface of the MPPBNPs. The resulting MPPBNP dispersion can be used for subsequent characterization of particle size, potential, morphology, and stability, and can also be further used as an aqueous dispersion for intravenous administration to evaluate in vivo distribution or therapeutic efficacy in animal models of atherosclerosis. In a preferred embodiment, 10 mg of uncovalently modified probucol and 1 mg of DSPE-PEG-OH were dissolved in 1 mL of DMF. The resulting organic phase was added dropwise to 4 mL of aqueous phase and dialyzed overnight using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain a PPBNPs dispersion. RAW264.7 macrophages were dehydrated for 20 min and rehydrated for 30 min, and then centrifuged at 5000×g for 5 min and 14000×g for 10 min, respectively, to obtain an MMVs dispersion. The PPBNPs dispersion and the MMVs dispersion were mixed at a volume ratio of 9:1, vortexed once every 10 min for a total of 3 times, and then sonicated in a water bath at 42 kHz and 100 W for 3 min to obtain an MPPBNPs dispersion.

[0104] It should be noted that in step S130, the macrophage microvesicles coating or fusing onto the outer surface of PPBNPs does not require that each PPBNP particle form a continuous shell of absolutely uniform thickness and perfectly regular geometry. As long as the membrane structure provided by the macrophage microvesicles can be distributed on at least a portion of the outer surface of the PPBNPs, and the resulting particles exhibit a surface structure distinct from uncoated PPBNPs, the macrophage-derived microvesicle membrane layer can be considered to have been formed.

[0105] In one embodiment, this application also provides the application of the aforementioned biomimetic probucol self-assembled nanoformulations in the preparation of drugs for treating atherosclerosis. The biomimetic probucol self-assembled nanoformulations can be MPPBNPs as described in any of the foregoing embodiments, or MPPBNPs prepared by the preparation methods described in any of the foregoing embodiments.

[0106] In one embodiment, the treatment of atherosclerosis includes slowing, inhibiting, or improving the progression of atherosclerotic lesions, reducing aortic plaque area, reducing intraplaque lipid deposition, reducing intraplaque necrotic core formation, improving plaque-related histological features, or alleviating oxidative stress, apoptotic damage, lipid uptake, or foam cell formation associated with atherosclerosis.

[0107] In one embodiment, the drug may include the biomimetic probucol self-assembled nanoformulation and pharmaceutically acceptable excipients, carriers, or dispersion media. The drug may be an aqueous dispersion suitable for intravenous administration. In a specific embodiment, the aqueous dispersion includes an aqueous dispersion medium and MPPBNPs dispersed in the aqueous dispersion medium, wherein the aqueous dispersion medium may include at least one of water, buffer solution, physiological saline, and DPBS.

[0108] In one embodiment, when the biomimetic probucol self-assembled nano-formulation is used to prepare a drug for treating atherosclerosis, it can exert probucol-related effects such as anti-oxidative stress, anti-apoptotic damage, inhibition of lipid uptake, or inhibition of foam cell formation through the probucol self-assembled nano-core; and the macrophage-derived microvesicle membrane layer can provide the nano-formulation with a cell-derived membrane surface structure, enabling the nano-formulation to have cell uptake and in vivo distribution characteristics that are different from those of uncoated PPBNPs.

[0109] In one embodiment, when the biomimetic probucol self-assembled nanoformulation is used to prepare a drug for treating atherosclerosis, it can be used to reduce the aortic plaque burden in atherosclerosis model animals, reduce lipid deposition in aortic root plaques, improve the histological characteristics related to aortic root plaques, or alleviate oxidative stress damage, apoptosis damage, lipid uptake, or foam cell formation in atherosclerosis-related cell models.

[0110] In one specific embodiment, the biomimetic probucol self-assembled nanoformulation is used to prepare an intravenously administered drug for treating atherosclerosis. The intravenously administered drug can be used to treat ApoE induced by a high-fat diet. - / - The drug was administered to mice with atherosclerosis, and its therapeutic effect on atherosclerotic plaques could be evaluated by whole aortic Oil Red O staining, aortic root Oil Red O staining, toluidine blue staining, Masson staining, and HE staining.

[0111] To verify the formation, structural characteristics, dispersion stability, and effects of the biomimetic probucol self-assembled nanoformulations described in this application in atherosclerosis-related models, the following experimental examples characterize and evaluate the performance of PPBNPs, MMVs, and MPPBNPs. Unless otherwise stated, the PPBNPs, MMVs, and MPPBNPs used in the following examples can be prepared according to the methods described in steps S110 to S130 above.

[0112] In one embodiment, the particle size distribution, surface charge, and morphological structure of PPBNPs, MMVs, and MPPBNPs can be characterized by hydration kinetic diameter, dispersion coefficient, potential, and transmission electron microscopy observations to confirm the nanoparticle morphology of PPBNPs, the membrane vesicle morphology of MMVs, and the core-shell structure of MPPBNPs. Furthermore, the dispersion stability of PPBNPs and MPPBNPs can be evaluated by measuring particle size variations in different media, thereby demonstrating their feasibility as aqueous dispersions for subsequent in vitro and in vivo evaluations.

[0113] In one embodiment, the cell compatibility, cellular uptake capacity, and effects on atherosclerosis-related cell damage processes of the nanoformulation can also be evaluated through cell experiments. Specifically, vascular endothelial cells and macrophages can be used as in vitro evaluation subjects, and atherosclerosis-related cell models can be constructed through inflammatory stimulation, oxidative stress stimulation, or lipid loading stimulation to evaluate the effects of PPBNPs and MPPBNPs on cellular uptake, anti-oxidative stress, anti-apoptotic damage, inhibition of lipid uptake, or inhibition of foam cell formation.

[0114] In one embodiment, the in vivo distribution, lesion-site-related distribution or enrichment, and therapeutic efficacy of the nano-formulation can also be evaluated through animal experiments. Specifically, ApoE can be used. - / - A mouse model of atherosclerosis was established, and the distribution characteristics, anti-atherosclerotic effects, and preliminary safety of MPPBNPs after in vivo administration were comprehensively analyzed by fluorescence tracing, whole aorta Oil Red O staining, aortic root histological staining, and biosafety evaluation.

[0115] It should be noted that the DiD labeling involved in the following experimental examples is mainly used for fluorescence tracing, cellular uptake observation, or in vivo distribution evaluation. DiD labeling does not constitute an essential component of therapeutic PPBNPs or MPPBNPs, nor is it used to limit the therapeutic active ingredient of the biomimetic probucol self-assembled nanoformulation of this application.

[0116] Example 1: Formation and mechanism verification of probucol self-assembled nanoparticles (PBNPs)

[0117] This embodiment is used to verify the ability of uncovalently modified probucol molecules to form nanoparticles without exogenous support materials and without chemical modification, and to preliminarily verify its self-assembly mechanism. For ease of description, the probucol self-assembled nanoparticles formed by the individual self-assembly of probucol molecules in this embodiment are denoted as PBNPs.

[0118] (1) Set up three groups of probucol dosages of 5 mg, 10 mg, and 20 mg. Take three 2 mL EP tubes and number them 1-3 respectively. Add solutions containing 5 mg, 10 mg, and 20 mg of probucol to each EP tube respectively, and add N,N-dimethylformamide to each tube until the total volume is 1 mL. In a specific example, add 125 μL, 250 μL, and 500 μL of 40 mg / mL probucol DMF stock solution to the three EP tubes respectively, and add 875 μL, 750 μL, and 500 μL of N,N-dimethylformamide to each tube until the total volume is 1 mL. Then, vortex the resulting 1 mL mixed organic phase solution for 20 s.

[0119] (2) Take a 10mL glass bottle containing a rotor, rinse it with water, add 4mL of water phase to the glass bottle, and place the glass bottle on a magnetic stirrer and adjust the speed to 500rpm.

[0120] (3) The organic phase solution obtained in step (1) is added dropwise to the glass bottle containing the aqueous phase at a uniform rate, slowly, so that the probucol molecules self-assemble in the aqueous environment and form nanoparticles. After the addition is complete, the magnetic stirrer is turned off, the glass bottle is removed, and the obtained system can be observed by laser irradiation to see if it has the Tyndall effect.

[0121] (4) Transfer the nanoparticle solution obtained in step (3) into a dialysis bag with a molecular weight cutoff of 3500 Da. After clamping both ends of the dialysis bag with clamps, place it in a beaker containing 1 L of double-distilled water and a rotor. Then, place the beaker on a magnetic stirrer, set the speed to 200 rpm, and dialyze for 4 h.

[0122] (5) After dialysis, the liquid in the dialysis bag was collected, and the hydration dynamic diameter and dispersion coefficient of the obtained PBNPs were determined by dynamic light scattering method.

[0123] The results are as follows Figure 2 A and Figure 2 As shown in Figure B, the hydration kinetic diameter and dispersion coefficient of PBNPs obtained with different probucol dosages differ. Experimental results indicate that, under the same preparation system, the probucol dosage affects the self-assembly state of the PBNPs. While ensuring nanoparticle stability, PBNPs corresponding to 10 mg of probucol exhibit suitable hydration kinetic diameter and dispersion coefficient; therefore, 10 mg is selected as the preferred dosage for subsequent PPBNP preparation. Thus, 10 mg of probucol can be considered the preferred dosage for the subsequent preparation of DSPE-PEG-OH modified probucol self-assembled nanoparticles.

[0124] To further analyze the intermolecular interactions that may be involved in the formation of PBNPs, intermolecular force perturbation experiments were conducted.

[0125] (6) Weigh 10 mg of probucol and dissolve it in 1 mL of N,N-dimethylformamide to obtain an organic phase solution.

[0126] (7) The 1 mL organic phase solution obtained in step (6) was added dropwise at a uniform rate to 4 mL of ultrapure water containing 50 mM Urea, 50 mM NaCl or 50 mM SDS, and nanoparticle dispersions were formed by nanoprecipitation. An ultrapure water group without Urea, NaCl or SDS was set up as a control group. The other preparation and dialysis conditions were the same as those of each treatment group.

[0127] (8) Dialyze the system obtained in step (7) in distilled water for 12 hours. After dialysis, collect the resulting aqueous dispersion and measure its hydration kinetic diameter and dispersion coefficient.

[0128] The results are as follows Figure 2 C and Figure 2 As shown in Figure D, compared to the control group, the hydration kinetic diameter and dispersion coefficient of the nanoparticles obtained in the NaCl-treated group changed significantly, while the Urea-treated and SDS-treated groups did not show the same degree of change as the NaCl-treated group. These results indicate that the formation or stable state of PBNPs is related to changes in the aqueous environment, and suggest that the non-covalent interactions between probucol molecules and the interactions between probucol and the aqueous environment participate in or influence its self-assembly behavior.

[0129] Furthermore, the probucol powder and PBNPs lyophilized powder were characterized by Fourier transform infrared spectroscopy.

[0130] (9) Take appropriate amounts of probucol powder and PBNPs freeze-dried powder respectively, and use Fourier transform infrared spectrometer to detect them. Record the absorption peak characteristics of each sample in the infrared wavenumber range, and analyze the changes of relevant characteristic absorption peaks before and after probucol self-assembly by comparing the infrared spectra of probucol powder and PBNPs freeze-dried powder.

[0131] The results are as follows Figure 2 As shown in E, after Probucol self-assembles into PBNPs, 3000 cm⁻¹ -1 Up to 3500cm -1 The broadening, intensity changes, or shifts of OH-related peaks within the range indicate that the hydrogen bonding environment changes before and after probucol self-assembly; 1200 cm⁻¹ -1 Up to 1300cm -1The shift in the CO or SO stretching vibration-related peaks within the range indicates a change in the local chemical environment of the molecule after probucol self-assembly. Combined with the aforementioned intermolecular force perturbation experimental results, these results support the correlation between the formation of PBNPs and non-covalent interactions, involving at least hydrophobic interactions and changes in the hydrogen bonding environment, and possibly accompanied by interactions between π-electron systems.

[0132] To further observe the aggregation process of probucol molecules in an aqueous environment, molecular dynamics simulations were performed.

[0133] (10) Molecular dynamics simulations were performed using Gromacs 2022.3 software. The steepest descent method was first used to minimize the energy in the simulation system; then isothermal and isochoric ensemble equilibrations were performed respectively; after equilibration, free molecular dynamics simulations were performed. After the simulation, the simulation trajectory was analyzed using the software's built-in tools, and the root mean square deviation, radius of gyration, hydrogen bonds, free energy morphology diagram, and interaction data between Probucol and water were calculated for the small molecule's trajectory.

[0134] The results are as follows Figure 2 F to Figure 2 As shown in Figure M, probucol molecules can aggregate in aqueous dispersions. Hydrogen bonds and Leonard-Jones interactions exist between probucol and water. The short-range Leonard-Jones energy between probucol and water is strongly positive, indicating a repulsive tendency between probucol and water molecules in close contact. During the simulation, the solvent-accessible surface area and the total potential energy of the system change over time, and the total potential energy enters a relatively stable fluctuating state after the initial changes in the simulation. Simultaneously, the root-mean-square deviation and radius of gyration of the probucol aggregates change with simulation time, and multiple low free energy regions exist in the free energy surface diagram. These results indicate that the aggregates formed by probucol molecules in the aqueous phase are not a single static conformation, but rather a dynamic state that can exist between multiple structural states.

[0135] In summary, this embodiment demonstrates that uncovalently modified probucol molecules can self-assemble into probucol self-assembled nanoparticles (PBNPs) in an aqueous environment. The formation of PBNPs is related to the non-covalent interactions between probucol molecules and the interaction between probucol and the aqueous environment. This result provides an experimental basis for subsequent steps such as introducing DSPE-PEG-OH into PBNPs to form PPBNPs, and further encapsulating macrophage microvesicles to form MPPBNPs.

[0136] Example 2: Preparation and characterization of probucol self-assembled nanoparticles (PPBNPs) modified with DSPE-PEG-OH

[0137] This embodiment illustrates the preparation process of probucol self-assembled nanoparticles (PPBNPs) modified with DSPE-PEG-OH, and characterizes the particle size, potential, and morphology of the obtained PPBNPs.

[0138] (1) Weigh 10 mg of probucol and 1 mg of DSPE-PEG-OH, add them to 1 mL of N,N-dimethylformamide, and mix thoroughly to obtain an organic phase containing probucol and DSPE-PEG-OH. The molecular weight of the PEG segment in the DSPE-PEG-OH is 3000 Da to 4000 Da.

[0139] (2) Place the 1 mL organic phase obtained in step (1) into a vortex shaker and shake for 20 s to fully mix probucol and DSPE-PEG-OH in the organic phase.

[0140] (3) Prepare 4 mL of pure water as the aqueous phase and place the aqueous phase on a magnetic stirrer, setting the stirring speed to 500 rpm. Then, the organic phase obtained in step (2) is added dropwise at a uniform speed to the aqueous phase under stirring, so that probucol and DSPE-PEG-OH form a nanoparticle dispersion in the aqueous environment.

[0141] (4) The nanoparticle dispersion obtained in step (3) was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. The stirring speed of the dialysis external liquid was set to 200 rpm, and dialysis was performed overnight. After dialysis, the liquid in the dialysis bag was collected to obtain a dispersion of probucol self-assembled nanoparticles (PPBNPs) modified with DSPE-PEG-OH.

[0142] (5) The hydration kinetic diameter and dispersion coefficient of PPBNPs in the aqueous dispersion were detected by a laser particle size analyzer, and the surface potential of PPBNPs was detected by a potential detection method. During the test, the samples were tested at room temperature, and each group of samples was measured three times.

[0143] (6) The microstructure of PPBNPs was observed using a transmission electron microscope. Specifically, one drop of PPBNPs aqueous dispersion was added to a 200-mesh carbon film copper mesh, and the mesh was allowed to dry naturally at room temperature. Then, one drop of 0.2% phosphotungstic acid solution was added for negative staining, which was carried out for 10 minutes. Subsequently, the residual phosphotungstic acid staining solution on the surface of the copper mesh was removed, and the mesh was rinsed twice with deionized water and allowed to air dry again. Finally, the treated sample was placed under a transmission electron microscope, and an appropriate field of view was selected for observation.

[0144] The results are as follows Figure 1 A to Figure 1As shown in Figure C, PPBNPs exhibit detectable hydration kinetic diameter, dispersion coefficient, and surface potential, indicating that a PPBNPs dispersion with an aqueous continuous phase can be prepared using the aforementioned nanoprecipitation method. The results are as follows... Figure 1 As shown in Figure D, PPBNPs exhibit a nanoparticle morphology. These results indicate that DSPE-PEG-OH-modified probucol self-assembled nanoparticles (PPBNPs) can be prepared by dissolving probucol and DSPE-PEG-OH together in an organic phase and adding them dropwise to an aqueous phase, followed by dialysis. The obtained PPBNPs can be used as core particles for subsequent macrophage microvesicle coating.

[0145] Example 3: Preparation and characterization of macrophage microvesicles (MMVs)

[0146] This embodiment illustrates the preparation process of macrophage microvesicles (MMVs) and characterizes the particle size, potential, and morphology of the obtained MMVs.

[0147] (1) Culture RAW264.7 macrophages until the cell confluence reaches 80%~90%, remove the culture medium, and wash the cells twice with DPBS. In a specific example, 5 mL of DPBS can be used to wash the cells each time to remove residual culture medium and free impurities. The RAW264.7 macrophages are cultured in RAW264.7 cell-specific culture medium (Pronosai CM-0190), which contains 10% fetal bovine serum, 1% penicillin and antibiotics, and RAW264.7 cell-specific additives. The culture conditions are 37℃ and 5% CO2. The culture container is a 10 cm diameter culture dish, and 12 culture dishes are used per batch.

[0148] (2) After removing the washing DPBS, open the culture container lid and place the RAW264.7 macrophages in an open position inside a sterile laminar flow hood to dehydrate the RAW264.7 macrophages. In one specific example, the dehydration time is 20 min.

[0149] (3) After dehydration, add DPBS to the culture container to rehydrate the RAW264.7 macrophages. In one specific example, add 5 mL of DPBS to the culture container and let it stand for 30 min. After rehydration, gently tap the edge of the culture container to allow the macrophage microvesicles released by the RAW264.7 macrophages to enter the supernatant, and then collect the supernatant containing macrophage microvesicles.

[0150] (4) The supernatant collected in step (3) is centrifuged to remove detached cells and enrich macrophage microvesicles. In a specific example, the supernatant is first centrifuged at 5000×g for 5 min to remove detached cells; after centrifugation, the precipitate is discarded and the supernatant is transferred, and then the resulting supernatant is centrifuged at 14000×g for 10 min to enrich macrophage microvesicles. After centrifugation, the precipitate is collected and resuspended in DPBS to obtain a macrophage microvesicle (MMV) dispersion. After centrifugation, the precipitate is collected and resuspended in 200 μL ddH2O.

[0151] (5) The hydration kinetic diameter and dispersion coefficient of MMVs in aqueous dispersion were detected by dynamic light scattering method, and the surface potential of MMVs was detected by potential detection method. During the test, the samples were tested at room temperature, and each group of samples was measured three times.

[0152] (6) The microstructure of MMVs was observed using a transmission electron microscope. Specifically, one drop of MMV dispersion was added to a 200-mesh carbon film copper mesh, and the mesh was allowed to dry naturally at room temperature. Then, one drop of 0.2% phosphotungstic acid solution was added for negative staining, which was carried out for 10 minutes. Subsequently, the residual phosphotungstic acid staining solution on the surface of the copper mesh was removed, and the mesh was rinsed twice with deionized water and allowed to air dry again. Finally, the treated sample was placed under a transmission electron microscope, and a suitable field of view was selected for observation.

[0153] The results are as follows Figure 1 A to Figure 1 As shown in Figure C, the MMVs possess detectable hydration kinetic diameter, dispersion coefficient, and surface potential, indicating that macrophage microvesicles in aqueous dispersion form can be obtained through the above dehydration and rehydration treatments. The results are as follows... Figure 1 As shown in Figure D, MMVs exhibit a membrane-like vesicle morphology. The results are as follows: Figure 1 A to Figure 1 As shown in C, the particulate components obtained by the above-described dehydration, rehydration, and centrifugal purification steps have detectable hydration kinetic diameter, dispersion coefficient, and Zeta potential. Figure 1 D shows that the particulate component exhibits a membrane-bound vesicle morphology. Based on the RAW264.7 cell origin, preparation process, and membrane-bound vesicle morphology of the particulate component, this application refers to the particulate component as macrophage microvesicles (MMVs) and uses it as the source material for the subsequent formation of the outer membrane structure of MPPBNPs.

[0154] Example 4: Preparation and characterization of MPPBNPs

[0155] This embodiment illustrates the preparation process of macrophage microvesicle-coated PPBNPs, i.e., MPPBNPs, and characterizes the particle size, potential, and morphology of the obtained MPPBNPs.

[0156] (1) Take the PPBNPs dispersion prepared in Example 2 and the MMVs dispersion prepared in Example 3, and mix the PPBNPs dispersion and the MMVs dispersion at a volume ratio of 9:1 to obtain a mixture.

[0157] (2) The mixture obtained in step (1) is subjected to intermittent vortex treatment. Specifically, the mixture is vortexed once every 10 minutes for a total of three times, so as to fully mix PPBNPs and MMVs and promote contact between MMVs and PPBNPs.

[0158] (3) After the vortex treatment, the mixture obtained in step (2) was placed in an ultrasonic water bath for ultrasonic treatment. The ultrasonic conditions were 42 kHz, 100 W, and the ultrasonic time was 3 min. After ultrasonic treatment, macrophage microvesicle-coated PPBNPs, i.e., MPPBNPs, were obtained.

[0159] (4) The hydration kinetic diameter and dispersion coefficient of MPPBNPs in the aqueous dispersion were detected by a laser particle size analyzer, and the surface potential of MPPBNPs was detected by a potential detection method. During the test, the samples were tested at room temperature, and each group of samples was measured three times.

[0160] (5) The microstructure of MPPBNPs was observed using a transmission electron microscope. Specifically, one drop of MPPBNPs aqueous dispersion was added to a 200-mesh carbon film copper mesh, and the mesh was allowed to dry naturally at room temperature. Then, one drop of 0.2% phosphotungstic acid solution was added for negative staining, which was carried out for 10 minutes. Subsequently, the residual phosphotungstic acid staining solution on the surface of the copper mesh was removed, and the mesh was rinsed twice with deionized water and then allowed to air dry again. Finally, the treated sample was placed under a transmission electron microscope, and a suitable field of view was selected for observation.

[0161] The results are as follows Figure 1 A to Figure 1 As shown in Figure C, compared to PPBNPs, MPPBNPs obtained after MMVs mixing, vortexing, and water bath ultrasonic treatment showed changes in hydration dynamic diameter and Zeta potential, indicating that the particle size and surface electrical properties changed after treatment. Figure 1 D shows that MPPBNPs exhibit a membrane boundary or outer layer structure on the outside of the internal nanoparticles. Combined with the source and preparation process of MMVs and the preparation steps of MPPBNPs, the above results collectively support the formation of membrane material provided by MMVs on the outer surface of PPBNPs, thus obtaining macrophage microvesicle-coated PPBNPs, i.e., MPPBNPs.

[0162] Example 5: Preparation and fluorescence characterization of DiD-labeled PPBNPs and MPPBNPs

[0163] This embodiment illustrates the preparation process of DiD-labeled PPBNPs and MPPBNPs, and verifies whether the nanoparticles obtained after DiD labeling retain their corresponding electrical and fluorescent properties. The DiD-labeled PPBNPs and MPPBNPs can be used for subsequent cellular uptake, in vivo distribution, and fluorescence tracing experiments.

[0164] (1) PPBNPs were labeled using the near-infrared fluorescent probe DiD. Specifically, DiD-labeled PPBNPs were prepared according to the preparation method of PPBNPs in Example 2, except that a DiD solution with a concentration of 5 mg / mL was further added to the organic phase of the PPBNPs so that DiD was introduced into the resulting nanoparticles during the formation of PPBNPs. After dialysis, the liquid in the dialysis bag was collected to obtain DiD-labeled PPBNPs, which were denoted as DiD-PPBNPs. Specifically, the amount of DiD added to the DiD-PPBNPs prepared for the cell uptake experiment was 2 μL (5 mg / mL stock solution), and the concentration after dialysis was 0.0014 mg / mL; the amount of DiD added to the DiD-PPBNPs prepared for the animal distribution experiment was 10 μL (5 mg / mL stock solution), and the DiD concentration after dialysis was 0.007 mg / mL; the DiD equivalent was the same in each group of examples, and the final solvent of the Free-DiD group was PBS with a concentration of 0.007 mg / mL. It should be noted that "Free-DiD" or "Free DiD" mentioned in the foregoing and subsequent embodiments of this application refer to the experimental group that does not contain DiD.

[0165] (2) MPPBNPs were labeled using the near-infrared fluorescent probe DiD. Specifically, following the preparation method of MPPBNPs in Example 4, the DiD-PPBNPs prepared in step (1) were mixed with the MMVs dispersion at a volume ratio of 9:1, and vortexed once every 10 minutes for a total of three vortexes; then, the mixture was subjected to ultrasonic treatment in an ultrasonic water bath at 42 kHz, 100 W, and for 3 minutes. After ultrasonic treatment, DiD-labeled MPPBNPs were obtained, denoted as DiD-MPPBNPs.

[0166] (3) The surface potentials of DiD-PPBNPs and DiD-MPPBNPs were detected by a potential detection method to evaluate whether the DiD labeling significantly affected the charged properties of PPBNPs and MPPBNPs. During the detection, each group of samples was measured three times.

[0167] (4) The fluorescence intensity of free DiD, DiD-PPBNPs and DiD-MPPBNPs under the same conditions was detected by a fluorescence microplate reader to evaluate whether the fluorescence signal intensity of DiD-PPBNPs and DiD-MPPBNPs is comparable.

[0168] (5) The fluorescence spectra of free DiD, DiD-PPBNPs and DiD-MPPBNPs were detected by a fluorescence spectrophotometer to evaluate whether the excitation and emission wavelengths of DiD changed significantly after being introduced into PPBNPs and MPPBNPs.

[0169] Please see Figure 4 The figure shows the characterization results of the potential and fluorescence properties of DiD-labeled PPBNPs and MPPBNPs in one embodiment of this application. Figure 4 A shows the potential characterization results of PPBNPs before and after DiD labeling; Figure 4 B shows the fluorescence intensity detection results for Free DiD, DiD-PPBNPs, and DiD-MPPBNPs; Figure 4 C shows the excitation and emission fluorescence spectra of Free-DiD, DiD-PPBNPs, and DiD-MPPBNPs.

[0170] exist Figure 4 In A, the horizontal axis represents the mass of probucol added (5 mg and 10 mg), respectively, and + indicates the addition of DiD for labeling. The results are as follows: Figure 4 As shown in Figure A, the surface potential of PPBNPs did not change significantly after DiD labeling, indicating that DiD labeling did not significantly affect the charged properties of PPBNPs. The results are as follows... Figure 4 As shown in Figure B, under the same concentration conditions, there was no significant difference in fluorescence intensity between DiD-PPBNPs and DiD-MPPBNPs, indicating that they can be used for comparative analysis in subsequent fluorescence tracer experiments. The results are as follows... Figure 4 As shown in Figure C, both DiD-PPBNPs and DiD-MPPBNPs exhibit the same or essentially the same excitation and emission wavelengths as free DiD, indicating that the fluorescence properties of DiD did not change significantly after being introduced into PPBNPs and MPPBNPs.

[0171] The above results indicate that after labeling PPBNPs and MPPBNPs with DiD, the resulting DiD-PPBNPs and DiD-MPPBNPs retain their corresponding surface charge and fluorescence characteristics, and can be used as fluorescent tracer samples in subsequent in vitro cell uptake experiments and in vivo animal distribution experiments. It should be noted that DiD is mainly used for fluorescence tracer and imaging observation, and does not constitute a necessary component of therapeutic PPBNPs or MPPBNPs, nor is it used to limit the therapeutic active ingredient of the biomimetic probucol self-assembled nanoformulation of this application.

[0172] Example 6: Stability evaluation of PPBNPs and MPPBNPs

[0173] This embodiment is used to evaluate the dispersion stability of PPBNPs and MPPBNPs in different aqueous media, in order to verify whether PPBNPs and MPPBNPs can maintain a relatively stable nano-dispersion state in the aqueous environment involved in subsequent in vitro experiments or in vivo drug delivery evaluations.

[0174] (1) The PPBNPs aqueous dispersion prepared in Example 2 and the MPPBNPs aqueous dispersion prepared in Example 4 were taken as test samples, respectively. In a specific example, the concentration of the PPBNPs aqueous dispersion and the MPPBNPs aqueous dispersion was 1.42 mg / mL.

[0175] (2) Take 500 μL of the PPBNPs aqueous dispersion and add it to 1.5 mL ddH2O, 1.5 mL PBS aqueous solution, 1.5 mL 0.9% NaCl solution or 1.5 mL culture medium containing 10% fetal bovine serum. Mix well to obtain the stability test samples of PPBNPs in different media.

[0176] (3) Take 500 μL of the MPPBNPs aqueous dispersion and add it to 1.5 mL ddH2O, 1.5 mL PBS aqueous solution, 1.5 mL 0.9% NaCl solution or 1.5 mL culture medium containing 10% fetal bovine serum. Mix well to obtain MPPBNPs stability test samples in different media.

[0177] (4) Place the samples obtained in steps (2) and (3) under the same conditions, and detect the hydration dynamic diameter of nanoparticles in each group of samples daily from day 1 to day 7 to evaluate the particle size variation of PPBNPs and MPPBNPs in different media. Three parallel groups are set up for each group of samples.

[0178] (5) While detecting the hydration dynamic diameter, observe the dispersion state of each group of samples to determine whether PPBNPs and MPPBNPs exhibit obvious precipitation, aggregation or stratification in different aqueous media.

[0179] The results are as follows Figure 3 A to Figure 3 As shown in D, after 7 days in ddH2O, 0.9% NaCl solution, PBS aqueous solution and culture medium containing 10% fetal bovine serum, PPBNPs and MPPBNPs generally maintained a nanoscale dispersion in the above media, but some particle size fluctuations were observed in some media and time points. No continuous precipitation, obvious stratification or irreversible macroscopic aggregation was observed, indicating that PPBNPs and MPPBNPs can maintain a relatively stable nanoscale dispersion in the above different aqueous media.

[0180] The above results demonstrate that PPBNPs and MPPBNPs exhibit good in vitro dispersion stability. Specifically, MPPBNPs, even after being coated with macrophage microvesicles, maintained stable dispersion in various aqueous media, indicating that the microvesicle coating process did not compromise their aqueous dispersion stability as nanoparticles. These results provide an experimental basis for the use of PPBNPs and MPPBNPs in subsequent cell experiments, in vivo distribution evaluation, and animal drug administration experiments.

[0181] Example 7: Evaluation of the cytocompatibility of PPBNPs and MPPBNPs

[0182] This embodiment is used to evaluate the cytocompatibility of PPBNPs and MPPBNPs with human umbilical vein endothelial cells (HUVECs) and RAW264.7 macrophages.

[0183] (1) Human umbilical vein endothelial cells (HUVECs) and RAW264.7 macrophages in the logarithmic growth phase were taken, washed with sterile 1×PBS, and digested with 0.05% trypsin at room temperature for 3 min. Subsequently, the digestion was terminated by adding the corresponding complete culture medium, and the cells were resuspended to obtain cell suspension.

[0184] (2) Take a 96-well plate and add 100 μL of the cell suspension obtained in step (1) to each well, so that the number of cells seeded in each well is approximately 1 × 10⁻⁶. 4 The 96-well plates were then pre-cultured at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere and grow.

[0185] (3) After the pre-incubation, discard the culture medium in each well and add complete culture medium containing different concentrations of the test samples to each well. The test samples include PPBNPs and MPPBNPs. In one specific example, the concentrations of the test samples were 0, 10, 50, 100, 200, and 300 μM. After adding the drugs, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 h. All concentrations are expressed in probucol equivalents.

[0186] (4) After incubation, aspirate the culture medium from the culture plate and rinse the cells with 1×PBS. Then, prepare CCK-8 dilution buffer according to the number of wells in the culture plate. In one specific example, CCK-8 reagent and basal culture medium were prepared at a volume ratio of 1:10. 100 μL of the CCK-8 dilution buffer was added to each well, and the culture plate was placed in a 37°C, 5% CO2 incubator for 30 min.

[0187] (5) After incubation, remove the culture plate, remove the air bubbles in the wells, and use an ELISA reader to detect the absorbance value of each well at a wavelength of 450 nm.

[0188] (6) Calculate cell viability based on the absorbance values ​​of each well. Specifically, subtract the background OD value from the OD value of each test well, and calculate the mean ± SD of the OD values ​​of each replicate well. Cell viability is expressed as T / C%, where T is the OD value of the drug-treated cells and C is the OD value of the control cells. The calculation formula is as follows:

[0189] Cell viability % = OD value of drug-treated cells / OD value of control cells × 100%.

[0190] Please see Figure 5 The image shows the cell compatibility evaluation results of PPBNPs and MPPBNPs in one embodiment of this application. Figure 5 A shows the cell viability assay results of HUVECs after treatment with different concentrations of PPBNPs; Figure 5 B shows the cell viability assay results of HUVECs after treatment with different concentrations of MPPBNPs; Figure 5 C shows the cell viability assay results of RAW264.7 macrophages after treatment with different concentrations of PPBNPs; Figure 5 D shows the cell viability assay results of RAW264.7 macrophages after treatment with different concentrations of MPPBNPs.

[0191] The results are as follows Figure 5 A to Figure 5As shown in Figure D, after co-incubating HUVECs and RAW264.7 macrophages for 24 h at the above concentration ranges, PPBNPs and MPPBNPs did not significantly inhibit the activity of HUVECs and RAW264.7 macrophages at lower and medium concentrations. At some higher concentrations, HUVECs cell activity decreased to some extent, but overall, detectable cell viability was maintained. Therefore, subsequent in vitro experiments should select concentrations with acceptable cell compatibility. These results indicate that PPBNPs and MPPBNPs have good in vitro cell compatibility with HUVECs and RAW264.7 macrophages within the tested concentration ranges and can be used for subsequent cell uptake experiments and functional evaluation in atherosclerosis-related cell models.

[0192] Example 8: Evaluation of cellular uptake of PPBNPs and MPPBNPs under inflammatory stimulation conditions

[0193] This embodiment is used to evaluate the uptake of DiD-labeled PPBNPs and MPPBNPs by HUVECs and RAW264.7 macrophages under normal or inflammatory stimulation conditions.

[0194] (1) Human umbilical vein endothelial cells (HUVECs) cultured to approximately 90% confluence were digested with trypsin and resuspended to obtain a HUVECs cell suspension. In one specific example, HUVECs were cultured at 2 × 10⁻⁶ cells / year. 5 Cells were seeded at a concentration of 10 cells / mL into 12-well plates containing a climbing sheet. 500 μL of cell suspension was added to each well, and the plates were incubated at 37°C and 5% CO2 for 12 h.

[0195] (2) After the HUVECs have completely adhered to the well, a portion of the HUVECs are subjected to inflammatory stimulation. In one specific example, 500 μL of LPS at a concentration of 1 μg / mL was added to the corresponding well, and induction was continued for 12 h to obtain LPS-activated HUVECs; the normal group of HUVECs were not treated with LPS.

[0196] (3) After the inflammatory stimulation treatment, DiD-PPBNPs or DiD-MPPBNPs were added to the normal group and LPS-activated group HUVECs, respectively. In one specific example, DiD-PPBNPs or DiD-MPPBNPs were added to each well to a final concentration of 100 μg / mL, and the nanoparticles were gently blown to distribute evenly in the culture medium, followed by incubation for 12 h. The concentrations of DiD-PPBNPs and DiD-MPPBNPs were calculated using the same probucol equivalent and the same DiD equivalent.

[0197] (4) After incubation, remove the culture medium and wash the cells with PBS. For samples observed by laser confocal microscopy, fix with 4% paraformaldehyde for 30 min, wash with PBS, add 0.1% Triton X-100 and let stand, wash with PBS again, add a mixture of 1% BSA and phalloidin for cytoskeleton staining, and then stain the cell nuclei with DAPI staining solution. After staining, remove the slides, mount them, store them in the dark, and observe the uptake of DiD-PPBNPs or DiD-MPPBNPs by HUVECs using laser confocal microscopy.

[0198] (5) For samples to be detected by flow cytometry, after incubation of DiD-PPBNPs or DiD-MPPBNPs with HUVECs, the cells were washed with PBS and the cells were collected to form a cell suspension. Then, the intracellular DiD fluorescence intensity was detected by flow cytometry to quantitatively analyze the uptake level of DiD-PPBNPs or DiD-MPPBNPs by HUVECs.

[0199] (6) Take RAW264.7 macrophages cultured to approximately 90% confluence, gently pipette and resuspend to obtain a RAW264.7 cell suspension. In one specific example, RAW264.7 macrophages were cultured at a concentration of 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of 1 cell / mL in 12-well plates, with 1 mL of cell suspension added to each well, and incubated at 37°C in a 5% CO2 incubator for 12 h.

[0200] (7) After the RAW264.7 macrophages adhered to the well, a portion of the RAW264.7 macrophages were subjected to inflammatory stimulation. In one specific example, LPS at a concentration of 1 μg / mL was added to the corresponding well and induced for 8 h to obtain LPS-activated RAW264.7 macrophages; the normal group of RAW264.7 macrophages were not treated with LPS.

[0201] (8) After the inflammatory stimulation treatment, DiD-PPBNPs or DiD-MPPBNPs were added to RAW264.7 macrophages in the normal group and LPS-activated group, respectively. In one specific example, DiD-PPBNPs or DiD-MPPBNPs were added to a final concentration of 100 μg / mL, and the nanoparticles were gently pipetted to distribute them evenly in the culture medium, followed by incubation for 4 h. The concentrations of DiD-PPBNPs and DiD-MPPBNPs were calculated using the same probucol equivalents and the same DiD equivalents.

[0202] (9) For flow cytometry samples, the culture medium was removed, and the cells were washed with PBS. The cells were collected and a cell suspension was formed. The fluorescence intensity of DiD in RAW264.7 macrophages was detected by flow cytometry to quantitatively analyze the uptake level of DiD-PPBNPs or DiD-MPPBNPs by RAW264.7 macrophages.

[0203] (10) For samples observed by laser confocal microscopy, after incubation of RAW264.7 macrophages with DiD-PPBNPs or DiD-MPPBNPs, the culture medium was removed and the cells were washed with PBS. Subsequently, 4% paraformaldehyde was added for fixation for 30 min, followed by PBS washing and then 0.1% Triton X-100 treatment. After washing with PBS again, a mixture of 1% BSA and phalloidin was added for cytoskeleton staining, followed by DAPI staining for cell nuclei. After staining, the slides were removed, mounted, and stored in the dark. The uptake of DiD-PPBNPs or DiD-MPPBNPs by RAW264.7 macrophages was observed using laser confocal microscopy.

[0204] Please see Figure 6 The image shows the evaluation results of the uptake of DiD-labeled PPBNPs and MPPBNPs by HUVECs and RAW264.7 macrophages under normal or inflammatory stimulation conditions in one embodiment of this application. Figure 6 A shows the results of laser confocal microscopy observations of DiD-PPBNPs and DiD-MPPBNPs taken up by normal or LPS-activated HUVECs. Figure 6 B to Figure 6 C shows the results of quantitative analysis of HUVECs uptake fluorescence by flow cytometry; Figure 6 D shows the results of laser confocal microscopy observation of the uptake of DiD-PPBNPs and DiD-MPPBNPs by RAW264.7 macrophages, which are either normal or LPS-activated. Figure 6 E to Figure 6 F shows the results of quantitative analysis of fluorescence uptake by RAW264.7 macrophages using flow cytometry.

[0205] The results are as follows Figure 6 A to Figure 6As shown in Figure C, HUVECs can take up DiD-PPBNPs and DiD-MPPBNPs. After LPS activation, the DiD-related fluorescence signal in HUVECs is enhanced, indicating that inflammatory stimulation can improve the uptake level of the nanoparticles by HUVECs. Compared with the DiD-PPBNPs group, the DiD-MPPBNPs group showed a stronger intracellular fluorescence signal in HUVECs, indicating that macrophage microvesicle coating is beneficial to improving the ability of HUVECs to take up MPPBNPs after inflammatory stimulation.

[0206] The results are as follows Figure 6 D to Figure 6 As shown in Figure F, RAW264.7 macrophages were also able to take up DiD-PPBNPs and DiD-MPPBNPs. After LPS activation, the DiD-related fluorescence signal in RAW264.7 macrophages was enhanced, indicating that inflammatory stimulation can improve the uptake level of the nanoparticles by RAW264.7 macrophages. Compared with the DiD-PPBNPs group, the DiD-MPPBNPs group showed a stronger intracellular fluorescence signal in RAW264.7 macrophages, indicating that macrophage microvesicle coating is beneficial to improving the ability of RAW264.7 macrophages to take up MPPBNPs after inflammatory stimulation.

[0207] The above results indicate that both PPBNPs and MPPBNPs can be taken up by HUVECs and RAW264.7 macrophages; under LPS-induced inflammatory stimulation, the uptake level of nanoparticles by both cell types is further enhanced; at the same time, MPPBNPs show a higher cellular uptake level than PPBNPs, indicating that macrophage microvesicle coating can improve the uptake performance of nanoparticles in atherosclerosis-related inflammatory cell models.

[0208] Example 9: Evaluation of cellular uptake pathways and intracellular distribution of PPBNPs and MPPBNPs

[0209] This embodiment is used to evaluate the uptake pathways involved when PPBNPs and MPPBNPs are taken up by cells, and to further observe the intracellular distribution of PPBNPs and MPPBNPs after they enter the cells.

[0210] (1) Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were seeded in a culture plate and cultured in a 37°C, 5% CO2 incubator to allow the cells to adhere and grow.

[0211] (2) After HUVECs adhered to the cell wall, they were pretreated with different endocytosis pathway inhibitors or low-temperature conditions. The low-temperature condition could be 4°C treatment to inhibit energy-dependent endocytosis; the endocytosis pathway inhibitors could be chlorpromazine, genistein, methyl-β-cyclodextrin, vormeningocele, and cytochalasin D, which were used to intervene in different endocytosis or cytoskeleton-related processes. Cells without nanoparticles were used as a blank group. In addition, a control group was set up where neither low-temperature treatment nor endocytosis pathway inhibitor treatment was performed, but only DiD-PPBNPs or DiD-MPPBNPs were added. Specifically, the control group was not treated with 4°C low-temperature treatment, nor was any of the endocytosis pathway inhibitors, chlorpromazine, genistein, methyl-β-cyclodextrin, vormeningocele, or cytochalasin D added, but was directly incubated with the corresponding DiD-PPBNPs or DiD-MPPBNPs at 37°C.

[0212] (3) After pretreatment, DiD-PPBNPs or DiD-MPPBNPs were added to each group of HUVECs and incubated for another 6 hours. In one specific example, DiD-PPBNPs or DiD-MPPBNPs were co-incubated with HUVECs for 6 hours.

[0213] (4) After incubation, the culture medium was removed and the cells were washed with 1×PBS to remove nanoparticles that were not taken up by the cells. The cells were then collected and resuspended, and the intracellular DiD-related fluorescence intensity was detected by flow cytometry to evaluate the effect of different pretreatment conditions on the uptake of DiD-PPBNPs or DiD-MPPBNPs by HUVECs.

[0214] (5) To further observe the intracellular distribution of PPBNPs and MPPBNPs after entering the cells, HUVECs with a growth density of about 80% were digested with 0.25% trypsin, resuspended after digestion was terminated, and seeded in confocal culture dishes and incubated at 37°C and 5% CO2 for 24 hours.

[0215] (6) After incubation for 24 hours, remove the original culture medium and replace it with fresh culture medium containing DiD-PPBNPs or DiD-MPPBNPs, so that HUVECs are co-incubated with DiD-PPBNPs or DiD-MPPBNPs. In a specific example, the concentration of DiD-PPBNPs or DiD-MPPBNPs is 100 μg / mL, and the co-incubation time is 6 hours.

[0216] (7) After incubation, discard the culture medium containing nanoparticles and wash the cells with 1×PBS. Subsequently, organelles were stained using organelle fluorescent tracer probes. In a specific example, the endoplasmic reticulum was stained with ER-Tracker dye and the Golgi apparatus was stained with Golgi dye; the concentration of ER-Tracker dye could be 1 μM and the concentration of Golgi dye could be 5 μM, and the staining conditions were incubation at 37°C in the dark for 30 min.

[0217] (8) After organelle staining, the cells were washed with 1×PBS and the nuclei were stained with Hoechst 33342. In one specific example, the Hoechst 33342 staining time was 1 min. After staining, the cells were washed again with 1×PBS and images were taken using a laser confocal microscope system.

[0218] (9) Further, lysosomal fluorescent probes were used to stain nanoparticles at 2h and 6h of incubation to compare the colocalization of nanoparticles and lysosomes at different incubation times, so as to evaluate the lysosomal-related transport process after PPBNPs and MPPBNPs enter the cells.

[0219] Please see Figure 7 The image shows the evaluation results of the cellular uptake pathways and intracellular distribution of PPBNPs and MPPBNPs in one embodiment of this application. Figure 7 A and Figure 7 C shows the flow cytometry results of HUVECs after co-incubation with DiD-PPBNPs for 6 h following pretreatment with different endocytosis inhibitors or low temperature conditions. Figure 7 B and Figure 7 D shows the flow cytometry results of HUVECs after co-incubation with DiD-MPPBNPs for 6 h following pretreatment with different endocytosis inhibitors or low temperature conditions. Figure 7 E shows the colocalization images of PPBNPs and MPPBNPs with the endoplasmic reticulum after 6 hours of co-culture with HUVECs; Figure 7 F is displayed as Figure 7 E corresponds to the colocation coefficient; Figure 7 G shows the colocalization images of PPBNPs and MPPBNPs with the Golgi apparatus after 6 hours of co-culture with HUVECs; Figure 7 H is displayed as Figure 7 The colocation coefficient corresponding to G; Figure 7 Image I shows the colocalization of PPBNPs and MPPBNPs with lysosomes after co-culturing with HUVECs; Figure 7J shows the colocalization of PPBNPs and MPPBNPs with lysosomes after co-culturing with RAW264.7 macrophages.

[0220] The results are as follows Figure 7 A to Figure 7 As shown in Figure D, the blank group without added nanoparticles showed only weak background fluorescence. The control group without inhibition treatment showed that HUVECs were able to take up DiD-PPBNPs and DiD-MPPBNPs, and the intracellular fluorescence intensity of the DiD-MPPBNPs group was higher than that of the DiD-PPBNPs group, indicating that macrophage microvesicle coating can improve the internalization level of nanoparticles by cells. Furthermore, both 4°C low-temperature treatment and chlorpromazine treatment reduced the uptake levels of DiD-PPBNPs and DiD-MPPBNPs by HUVECs, indicating that the cellular uptake of PPBNPs and MPPBNPs is at least partially dependent on energy-dependent endocytosis and is related to the clathrin-mediated endocytosis pathway.

[0221] The results are as follows Figure 7 E to Figure 7 As shown in Figure H, after DiD-PPBNPs and DiD-MPPBNPs enter HUVECs, co-localization signals with the endoplasmic reticulum and Golgi apparatus can be observed, indicating that PPBNPs and MPPBNPs can be distributed in intracellular structures such as the endoplasmic reticulum and Golgi apparatus after being taken up by cells. The results are as follows... Figure 7 I arrive Figure 7 As shown in J, DiD-PPBNPs and DiD-MPPBNPs can also colocalize with lysosome-associated fluorescence signals, indicating that PPBNPs and MPPBNPs undergo lysosome-associated transport after entering the cell.

[0222] These results indicate that PPBNPs and MPPBNPs can be taken up by HUVECs, with MPPBNPs exhibiting a higher cellular uptake level compared to PPBNPs. The cellular uptake process of PPBNPs and MPPBNPs is associated with energy-dependent endocytosis and clathrin-mediated endocytosis. After entering the cell, PPBNPs and MPPBNPs can be distributed in intracellular structures such as lysosomes, endoplasmic reticulum, and Golgi apparatus, providing a basis for their intracellular pharmacological effects related to probucol.

[0223] Example 10: Effects of PPBNPs and MPPBNPs on oxidative stress, apoptosis, and lipid uptake in HUVECs

[0224] This embodiment is used to evaluate the effects of PPBNPs and MPPBNPs on reactive oxygen species levels, oxidative stress-related apoptosis, and lipid uptake in human umbilical vein endothelial cells (HUVECs).

[0225] (1) Take HUVECs in the logarithmic growth phase, inoculate them in a culture plate, and incubate them in a 37°C, 5% CO2 incubator to allow the cells to adhere and grow.

[0226] (2) To evaluate the effects of PPBNPs and MPPBNPs on intracellular reactive oxygen species levels in HUVECs, LPS stimulation was used to construct an inflammation-related oxidative stress model. After LPS stimulation, cells were washed with 1×PBS and treated with free probucol PB, PPBNPs, or MPPBNPs, respectively. In one specific example, after LPS stimulation, HUVECs were treated with PB, PPBNPs, or MPPBNPs, respectively, and incubated at 37°C in a 5% CO2 incubator for 4 hours.

[0227] (3) After drug treatment, remove the culture medium and wash the cells with 1×PBS. Then, dilute DCFH-DA to a final concentration of 10 μM with serum-free culture medium, load the probe onto HUVECs, ensure that the DCFH-DA working solution fully covers the cells, and incubate at 37°C for 30 min. During incubation, gently shake the cells every 5 min and observe them under a microscope.

[0228] (4) After DCFH-DA incubation, the culture medium was removed and the cells were washed with serum-free fresh basal medium to remove the DCFH-DA fluorescent probes that had not entered the cells. Subsequently, some samples were used for fluorescence microscopy to observe and collect fluorescence images, while the other samples were used to collect cells and then flow cytometry to detect the intracellular DCFH-related fluorescence intensity to evaluate the reactive oxygen species levels in HUVECs of each treatment group.

[0229] (5) To evaluate the effects of PPBNPs and MPPBNPs on oxidative stress-related apoptosis damage in HUVECs, HUVECs were diluted to 1×10⁻⁶. 5 Cells were seeded at 1 mL / well in a 6-well plate and incubated at 37°C for 12 h in a 5% CO2 incubator.

[0230] (6) When HUVECs reached 70%–80% confluence, the original culture medium was removed, and fresh culture medium containing PB, PPBNPs, or MPPBNPs was added for pretreatment. In one specific example, the concentrations of PB, PPBNPs, and MPPBNPs were all 100 μg / mL, and the pretreatment time was 12 h. Subsequently, tBHP was added to induce oxidative stress-related apoptosis in HUVECs. In one specific example, the concentration of tBHP was 5 mM, and the induction time was 2 h.

[0231] (7) After tBHP induction, the survival and death status of cells were observed by Calcein-AM / PI live and dead cell staining, and the apoptosis detection reagents used in actual flow cytometry were used to quantitatively detect the cells in each group.

[0232] (8) To evaluate the effects of PPBNPs and MPPBNPs on lipid uptake in HUVECs, HUVECs were seeded in culture plates and cultured to a suitable state. They were then pretreated with PB, PPBNPs, or MPPBNPs, respectively, followed by the addition of oxidized low-density lipoprotein (ox-LDL) to induce lipid uptake in HUVECs. In one specific example, PB, PPBNPs, and MPPBNPs were treated with the same probucol equivalents, followed by co-culture with ox-LDL.

[0233] (9) After ox-LDL treatment, Oil Red O staining was used to stain intracellular lipid deposits, and optical microscopy was used to observe lipid uptake or lipid droplet deposition in HUVECs of different treatment groups. Furthermore, ImageJ software was used to perform quantitative analysis of the Oil Red O staining results.

[0234] Please see Figure 8 The image shows the in vitro efficacy evaluation results of PPBNPs and MPPBNPs on HUVECs in one embodiment of this application. Figure 8 A shows representative fluorescence microscopy images of HUVECs from different treatment groups after LPS induction and DCFH-DA staining; Figure 8 B to Figure 8 C shows the quantitative results of DCFH-related fluorescence intensity analysis in HUVECs by flow cytometry. Figure 8 D shows representative fluorescence images of the apoptosis or survival status of HUVECs in different treatment groups; Figure 8 E shows the apoptosis status of HUVECs obtained by flow cytometry in different treatment groups; Figure 8 F shows the statistical results of HUVECs cell survival rate; Figure 8 G shows representative optical microscopic images of HUVECs uptake of ox-LDL after treatment in different treatment groups; Figure 8 H is displayed as a result of ImageJ. Figure 8 The results of quantitative analysis of lipid uptake or lipid droplet deposition in G.

[0235] The results are as follows Figure 8 A to Figure 8As shown in Figure C, LPS stimulation enhanced the DCFH-related fluorescence signal within HUVECs, indicating an increase in intracellular reactive oxygen species (ROS) levels. Treatment with PB, PPBNPs, or MPPBNPs reduced the DCFH-related fluorescence signal within HUVECs. Compared to free PB, the reduction in fluorescence signal within HUVECs after treatment with PPBNPs and MPPBNPs was more significant, suggesting that preparing probucol as nanoparticles can enhance its effect in reducing intracellular ROS levels in HUVECs.

[0236] The results are as follows Figure 8 D to Figure 8 As shown in Figure F, tBHP treatment induced significant oxidative stress-related apoptosis or cell death in HUVECs. Pretreatment with PB, PPBNPs, or MPPBNPs reduced the degree of HUVEC apoptosis or cell death and improved cell survival. Compared with free PB, PPBNPs and MPPBNPs showed a more significant protective effect against tBHP-induced HUVEC damage, indicating that PPBNPs and MPPBNPs can alleviate oxidative stress-mediated apoptosis in HUVECs.

[0237] The results are as follows Figure 8 G to Figure 8 As shown in Figure H, ox-LDL treatment increased lipid uptake or lipid droplet deposition in HUVECs; treatment with PB, PPBNPs, or MPPBNPs reduced Oil Red O positive signal in HUVECs. Compared to free PB, PPBNPs and MPPBNPs more significantly reduced the uptake or lipid deposition levels of ox-LDL in HUVECs, indicating that PPBNPs and MPPBNPs can enhance the effect of probucol in inhibiting lipid uptake in HUVECs.

[0238] The above results indicate that PPBNPs and MPPBNPs can reduce intracellular reactive oxygen species levels, alleviate oxidative stress-related apoptosis damage, and inhibit ox-LDL-induced lipid uptake or lipid deposition in the HUVECs model, suggesting that the nano-formulations can intervene in the process of atherosclerosis-related endothelial cell injury.

[0239] Example 11: Effects of PPBNPs and MPPBNPs on oxidative stress, apoptosis, and foam cell formation in RAW264.7 macrophages

[0240] This embodiment was used to evaluate the effects of PPBNPs and MPPBNPs on reactive oxygen species levels, oxidative stress-related apoptosis, and foam cell formation in RAW264.7 macrophages.

[0241] (1) Take RAW264.7 macrophages in the logarithmic growth phase, seed them in a culture plate, and incubate them in a 37°C, 5% CO2 incubator to allow the cells to adhere and grow.

[0242] (2) To evaluate the effects of PPBNPs and MPPBNPs on the intracellular reactive oxygen species level of RAW264.7 macrophages, RAW264.7 macrophages were stimulated with LPS to construct an inflammation-related oxidative stress model. After LPS stimulation, the cells were washed with 1×PBS and treated with free probucol PB, PPBNPs or MPPBNPs, respectively.

[0243] (3) After drug treatment, the culture medium was removed, and the cells were rinsed with 1×PBS. Subsequently, DCFH-DA was diluted with serum-free culture medium and used to load probes onto RAW264.7 macrophages, ensuring that the DCFH-DA working solution fully covered the cells. The cells were then incubated at 37°C in the dark. After incubation, the DCFH-DA working solution was removed, and the cells were washed with serum-free culture medium or 1×PBS to remove any DCFH-DA fluorescent probes that had not entered the cells.

[0244] (4) Perform fluorescence imaging on the RAW264.7 macrophages after step (3), and further collect cells and use flow cytometry to detect the intracellular DCFH-related fluorescence intensity to evaluate the reactive oxygen species level in each treatment group of RAW264.7 macrophages.

[0245] (5) To evaluate the effects of PPBNPs and MPPBNPs on oxidative stress-related apoptosis damage in RAW264.7 macrophages, RAW264.7 macrophages were treated with tert-butyl hydroperoxide (tBHP) to construct a macrophage oxidative damage apoptosis model.

[0246] (6) Before or during tBHP induction, RAW264.7 macrophages were treated with PB, PPBNPs, or MPPBNPs, respectively. After treatment, apoptosis or survival of RAW264.7 macrophages in different treatment groups were detected by apoptosis detection methods or live / dead cell staining methods, and flow cytometry was used to further quantify apoptosis.

[0247] (7) To evaluate the effects of PPBNPs and MPPBNPs on foam formation of RAW264.7 macrophages, RAW264.7 macrophages were seeded in culture plates. After the cells adhered and reached a suitable state, they were pretreated with PB, PPBNPs or MPPBNPs respectively. Then, oxidized low-density lipoprotein ox-LDL was added to induce RAW264.7 macrophages to take up lipids and form foam cells.

[0248] (8) After ox-LDL treatment, oil red O staining was used to stain lipid deposits in RAW264.7 macrophages, and the lipid uptake or foam cell formation in RAW264.7 macrophages of different treatment groups was observed using an optical microscope. Furthermore, the Oil Red O staining results can be quantitatively analyzed using ImageJ software.

[0249] Please see Figure 9 The image shows the in vitro efficacy evaluation results of PPBNPs and MPPBNPs on RAW264.7 macrophages in one embodiment of this application. Figure 9 Image A shows representative fluorescence microscopy images of RAW264.7 macrophages from different treatment groups after LPS induction and DCFH-DA staining; Figure 9 B to Figure 9 C shows the quantitative results of DCFH-related fluorescence intensity analysis in RAW264.7 macrophages by flow cytometry. Figure 9 D shows representative fluorescent images of apoptosis or survival status of RAW264.7 macrophages in different treatment groups; Figure 9 E shows the apoptosis status of RAW264.7 macrophages obtained by flow cytometry analysis in different treatment groups; Figure 9 F shows the statistical results of RAW264.7 macrophage survival rate; Figure 9 G shows representative optical microscopic images of RAW264.7 macrophages uptake ox-LDL after treatment in different treatment groups; Figure 9 H is displayed as a result of ImageJ. Figure 9 The results of quantitative analysis of lipid uptake or foam cell formation in G.

[0250] The results are as follows Figure 9 A to Figure 9 As shown in Figure C, LPS stimulation enhanced DCFH-related fluorescence signals in RAW264.7 macrophages, indicating an increase in intracellular reactive oxygen species (ROS) levels. Treatment with PB, PPBNPs, or MPPBNPs decreased DCFH-related fluorescence signals in RAW264.7 macrophages. Compared to the model group, PPBNPs and MPPBNPs treatments reduced ROS levels in RAW264.7 macrophages, suggesting that PPBNPs and MPPBNPs can alleviate LPS-induced oxidative stress in macrophages.

[0251] The results are as follows Figure 9 D to Figure 9As shown in Figure F, tBHP treatment induced significant oxidative stress-related apoptosis or cell death in RAW264.7 macrophages. Treatment with PB, PPBNPs, or MPPBNPs reduced the degree of apoptosis or cell death in RAW264.7 macrophages, improving cell viability. In one specific result, tBHP treatment reduced the survival rate of RAW264.7 macrophages to 8.95%, while MPPBNP pretreatment increased the survival rate to 89.64%. These results indicate that MPPBNPs can effectively alleviate tBHP-induced oxidative damage to RAW264.7 macrophages and improve cell viability.

[0252] The results are as follows Figure 9 G to Figure 9 As shown in Figure H, ox-LDL treatment increased lipid uptake and lipid droplet deposition in RAW264.7 macrophages, indicating that RAW264.7 macrophages underwent foam cell transformation. Treatment with PB, PPBNPs, or MPPBNPs reduced Oil Red O positive signal in RAW264.7 macrophages. Compared to the model group, MPPBNPs reduced ox-LDL uptake and lipid deposition levels in RAW264.7 macrophages, indicating that MPPBNPs inhibited ox-LDL-induced foam cell formation in RAW264.7 macrophages.

[0253] These results indicate that PPBNPs and MPPBNPs can reduce intracellular reactive oxygen species levels, alleviate oxidative stress-related apoptosis, and inhibit ox-LDL-induced lipid uptake or foam cell formation in the RAW264.7 macrophage model. These results demonstrate that the nanoparticles can intervene in atherosclerosis-related macrophage damage and foaming processes.

[0254] Example 12: DiD-labeled PPBNPs and MPPBNPs in ApoE - / - Distribution evaluation in mice

[0255] This embodiment is used to evaluate the blood retention, aortic-related distribution, and major organ distribution of DiD-labeled PPBNPs and MPPBNPs in atherosclerotic model mice.

[0256] (1) Select ApoE cells that have been induced by a high-fat diet for 8 weeks - / - Mice were used as an animal model of atherosclerosis, and the ApoE was used... - / - Mice were randomly grouped. In one specific example, the ApoE... - / -Mice were randomly divided into three groups: FreeDiD group, DiD-PPBNPs group, and DiD-MPPBNPs group.

[0257] (2) Free DiD, DiD-PPBNPs and DiD-MPPBNPs were formulated into drug delivery systems suitable for tail vein injection, and ApoE was administered to the corresponding groups via tail vein injection. - / - Mice. The DiD-PPBNPs and DiD-MPPBNPs were prepared according to the method described in Example 5. Specifically, the administration volume for each group was 200 μL, the DiD equivalent was 0.0014 mg, and the probucol equivalent of DiD-PPBNPs and DiD-MPPBNPs was 0.284 mg.

[0258] (3) To evaluate the retention of different drug delivery systems in the blood, mouse blood samples were collected at predetermined time points after drug administration, and fluorescence imaging or fluorescence signal detection was performed. In a specific example, blood samples could be collected 24 hours after drug administration, and the DiD-related fluorescence signals in the blood samples could be imaged and quantitatively analyzed. Furthermore, blood samples could also be collected at different time points after drug administration by tail clipping, and the DiD-related fluorescence signals in the blood samples could be detected to evaluate the changes in fluorescence signals of different drug delivery systems in the blood.

[0259] (4) To evaluate the distribution of different drug administration systems in the aorta, mice were sacrificed 24 hours after drug administration and aortic tissue was removed. Fluorescence imaging was performed on the isolated aortic tissue, and the DiD-related fluorescence signal in the aortic tissue was quantitatively analyzed.

[0260] (5) To evaluate the distribution of different drug delivery systems in major organs, major organs of mice were removed 24 hours after drug administration and subjected to in vitro fluorescence imaging. In a specific example, the major organs included the heart, liver, spleen, lungs, and kidneys. Furthermore, the DiD-related fluorescence signals in each major organ could be quantitatively analyzed.

[0261] Please see Figure 10 The image shows DiD-tagged PPBNPs and MPPBNPs in ApoE in one embodiment of this application. - / - The evaluation results of the distribution and blood retention in mice are shown in the figure. Figure 10 A shows that Free DiD, DiD-PPBNPs, and DiD-MPPBNPs were injected into ApoE. - / - Fluorescence imaging results of blood in model mice 24 hours later; Figure 10 B is displayed as Figure 10Quantitative analysis results of fluorescence signals in blood sample A; Figure 10 C shows that Free DiD, DiD-PPBNPs, and DiD-MPPBNPs were injected into ApoE. - / - Fluorescence imaging results of isolated aortic tissue from model mice 24 hours after in vivo in vivo; Figure 10 D is displayed as Figure 10 Quantitative analysis results of fluorescence signals in the aortic tissue of C; Figure 10 E shows that Free DiD, DiD-PPBNPs, and DiD-MPPBNPs were injected into ApoE. - / - After 24 hours in vivo in the model mice, one mouse from each of the three drug-treated groups was selected to obtain the in vitro fluorescence imaging results of the major organs (heart, liver, spleen, lung and kidney); Figure 10 F shows the results obtained by quantifying the DiD-related fluorescence signals in the major organs of three mice in each drug administration group and calculating the mean within each group. Figure 10 G shows the fluorescence imaging and quantitative analysis results of blood samples taken by tail clipping at different time points after drug administration.

[0262] It should be noted that, Figure 10 E is only used to display in vitro fluorescence images of major organs of individual mice in each drug administration group. Figure 10 F is not only about Figure 10 The quantification shown in E is based on a single mouse, but rather on intragroup statistical analysis of the fluorescence quantitative results of corresponding organs from all three mice in each treatment group. Therefore, Figure 10 The organ fluorescence image of a single mouse shown in E is compared with... Figure 10 The average quantitative results within group F do not constitute a one-to-one correspondence. The two may differ in relative signal strength due to individual differences among animals within the group.

[0263] also, Figure 10 E is a pseudo-color fluorescence image, mainly used to visually display the spatial distribution of DiD-related fluorescence signals; Figure 10 F represents the statistical results obtained after quantitative fluorescence analysis of the corresponding organ's region of interest using a unified analytical method. Therefore, quantitative comparisons of fluorescence signals between groups and between organs of major organs are based on... Figure 10 The statistical results shown in F shall prevail.

[0264] The results are as follows Figure 10 A to Figure 10 B and Figure 10As shown in Figure G, the semi-quantitative analysis of blood fluorescence revealed that, compared to the FreeDiD group, the DiD-PPBNPs and DiD-MPPBNPs groups maintained DiD-related fluorescence signals in post-drug administration blood samples for a longer period, and this trend persisted up to 48 hours post-drug administration. These results indicate that, compared to free DiD, the fluorescence signals corresponding to DiD-labeled PPBNPs and MPPBNPs have a prolonged retention time in blood, suggesting that nano-formulations possess a longer blood circulation retention characteristic.

[0265] The results are as follows Figure 10 C to Figure 10 As shown in Figure D, after drug administration, both the DiD-PPBNPs group and the DiD-MPPBNPs group exhibited significant DiD-related fluorescence signals in the aortic plaque region. Compared to the Free DiD group, the DiD-related fluorescence signals were more pronounced in the aortic lesion region. These results indicate that PPBNPs and MPPBNPs have significant effects on ApoE activity. - / - The nanomaterials were able to distribute to the aortic plaque-related area in mice with an atherosclerosis model, indicating that the nanomaterials have the ability to distribute to the atherosclerosis lesion-related area.

[0266] The results are as follows Figure 10 E to Figure 10 As shown in F, 24 hours after administration, varying degrees of DiD-related fluorescence signals were detected in the heart, liver, spleen, lungs, and kidneys in the Free DiD group, DiD-PPBNPs group, and DiD-MPPBNPs group. Figure 10 E is used to display the fluorescence distribution of major organs in individual mice within each group. Figure 10 F represents the within-group statistical results of the quantitative fluorescence results of the corresponding organs in the three mice of each group. Figure 10 As can be seen from F, there are certain differences in the distribution of DiD-related fluorescence signals in major organs among different drug delivery systems. The above results reflect the relative fluorescence distribution of each drug delivery system in major organs.

[0267] The above results indicate that DiD-labeled PPBNPs and MPPBNPs in ApoE - / -In mouse models of atherosclerosis, DiD-PPBNPs and DiD-MPPBNPs exhibited in vivo distribution characteristics distinct from Free DiD: firstly, they showed significant fluorescence signals in aortic plaque-related regions, indicating their ability to distribute to atherosclerotic lesion-related areas; secondly, the retention time of DiD-related fluorescence signals in the blood was prolonged, suggesting that the nanoformulated formulations have a longer blood circulation retention characteristic. Furthermore, the distribution results in major organs showed that varying degrees of DiD-related fluorescence signals could be detected in multiple major organs after administration of Free DiD, DiD-PPBNPs, and DiD-MPPBNPs.

[0268] Example 13: PPBNPs and MPPBNPs in ApoE - / - Evaluation of therapeutic effects in a mouse model of atherosclerosis

[0269] This embodiment is used to evaluate PPBNPs and MPPBNPs in ApoE. - / - In vivo therapeutic effects in a mouse model of atherosclerosis.

[0270] (1) Select ApoE - / - Mice were fed a high-fat diet to establish an atherosclerosis model. In one specific example, 48 ApoE mice were selected. - / - After feeding mice with a high-fat diet for 8 weeks, an atherosclerosis model was established. After excluding the number of dead mice, 5-6 mice were used in each group for statistical analysis.

[0271] (2) ApoE after 8 weeks of feeding with a high-fat diet - / - Atherosclerosis model mice were randomly divided into six groups. In one specific example, the model mice were randomly divided into six groups: PBS model control group, free PB group, PPBNPs group, MPPBNPs group, PPBNPs gavage group, and atorvastatin group. The PBS model control group, free PB group, PPBNPs group, and MPPBNPs group were administered the medication via tail vein injection; the PPBNPs gavage group and atorvastatin group were administered the medication via gavage.

[0272] (3) For the tail vein injection group, PB, PPBNPs or MPPBNPs were prepared into a drug delivery system suitable for tail vein injection and administered according to the same probucol equivalent. In a specific example, the dosage of the free PB group, PPBNPs group and MPPBNPs group was 6 mg / kg, the volume of each administration was 200 μL, and the administration was twice a week; the PBS model control group received 200 μL of PBS via tail vein injection each time.

[0273] (4) For the gavage administration group, the gavage administration was performed daily at the predetermined dose. In one specific example, the PPBNPs gavage group was administered 2 mg / kg once daily; the atorvastatin group served as the positive control group and was administered gavage according to the corresponding administration conditions.

[0274] (5) The treatment period for each group of mice was 8 weeks. During the treatment period, the mice in each group continued to maintain a high-fat diet to maintain the atherosclerosis model. After each tail vein administration, the weight of the mice was recorded to observe the changes in the general condition of the mice during the treatment process.

[0275] (6) After treatment, mice in each group were sacrificed and the entire aorta was isolated. After removing the adipose tissue around the aorta, the aortic tissue was rinsed with PBS to remove residual blood. Subsequently, the entire aorta was stained with Oil Red O, and overall images of the aorta were acquired to evaluate the effect of different drug treatments on the atherosclerotic plaque burden of the aorta. Furthermore, ImageJ software was used for quantitative analysis of the Oil Red O positive areas of the aorta.

[0276] (7) After treatment, mouse hearts were collected and tissue sections containing the aortic root and aortic valve regions were prepared. The aortic root tissue sections were stained with Oil Red O, Toluidine Blue, Masson staining and HE staining, and microscopic images were acquired.

[0277] (8) Among these, Oil Red O staining of the aortic root is used to observe and evaluate lipid deposition within the aortic root plaque; toluidine blue staining is used to observe the plaque-related tissue structures or necrotic core-related areas of the aortic root; Masson staining is used to observe collagen fiber-related structures in the aortic root plaque; and HE staining is used to observe the tissue morphology of the aortic root and the overall lesion status of the plaque. Furthermore, the above staining results can be quantitatively analyzed to evaluate the effects of different drug treatments on the degree of atherosclerotic lesions and plaque structural characteristics.

[0278] Please see Figure 11 The image shows the in vivo therapeutic efficacy evaluation results of different dosage forms of probucol in atherosclerotic mice in one embodiment of this application. Figure 11 A is displayed as ApoE - / - Schematic diagram of mouse high-fat diet modeling and drug administration process; Figure 11 B shows the statistical results of weight changes in mice in each group during the treatment period; Figure 11 C shows the Oil Red O staining results of the entire aorta of mice in different treatment groups; Figure 11 D shows the Oil Red O staining results of aortic root tissue sections from mice in different treatment groups; Figure 11 E shows the toluidine blue staining results of aortic root tissue sections from mice in different treatment groups; Figure 11 F shows the Masson staining results of aortic root tissue sections from mice in different treatment groups; Figure 11 G shows the HE staining results of aortic root tissue sections from mice in different treatment groups; Figure 11 H is displayed as Figure 11 Quantitative analysis results of the entire aortic plaque area in C; Figure 11 I is displayed as Figure 11 Quantitative analysis results of the aortic root plaque area or lipid deposition area in D; Figure 11 J is displayed as Figure 11 Quantitative analysis results of the core-related region of aortic root necrosis in E; Figure 11 K is displayed as Figure 11 Quantitative analysis results of collagen content or collagen-positive area in aortic root plaques in F.

[0279] The results are as follows Figure 11 As shown in Figure B, during the treatment period, the overall body weight of mice in each group remained within the corresponding range of change, and no sustained significant weight loss related to the drug administration was observed. These results indicate that, under the drug administration conditions of this embodiment, mice in each drug administration group were able to complete the corresponding treatment cycle, providing a basis for further evaluation of the treatment effect.

[0280] The results are as follows Figure 11 C and Figure 11 As shown in Figure H, after treatment, the entire aorta of mice in the PBS model control group showed a more obvious Oil Red O positive plaque area; compared with the PBS model control group, the Oil Red O positive area in the aorta of mice in the MPPBNPs group was reduced, indicating that MPPBNPs can reduce ApoE. - / - Aortic plaque burden in a mouse model of atherosclerosis.

[0281] The results are as follows Figure 11 D and Figure 11As shown in Figure I, Oil Red O staining results of aortic root tissue sections revealed significant lipid deposition in the aortic root plaque region of the PBS model control mice. Treatment with PB, PPBNPs, or MPPBNPs reduced the degree of lipid deposition in the aortic root, with a more pronounced reduction in the MPPBNPs group. These results indicate that MPPBNPs can reduce lipid deposition within aortic root plaques, thereby improving atherosclerosis-related lipid load.

[0282] The results are as follows Figure 11 E and Figure 11 As shown in Figure J, toluidine blue staining results of aortic root tissue sections revealed differences in tissue structure or necrotic core-related areas in the aortic root lesion region among different treatment groups. Compared to the PBS model control group, the relevant staining results in the MPPBNPs group were improved, indicating that MPPBNPs can reduce the severity of plaque-related tissue damage or necrotic core-related lesions in the aortic root.

[0283] The results are as follows Figure 11 F and Figure 11 As shown in K, Masson staining results of aortic root tissue sections revealed differences in collagen fiber-related structures in plaque regions among different treatment groups. Compared to the PBS model control group, Masson staining results in the MPPBNPs group showed improved plaque collagen-related structures, indicating that MPPBNPs can influence plaque collagen fiber-related structures and improve plaque structural characteristics.

[0284] The results are as follows Figure 11 As shown in G, HE staining of aortic root tissue sections revealed that obvious atherosclerotic plaque lesions were visible in the aortic root of mice in the PBS model control group. After treatment with MPPBNPs, the degree of plaque-related lesions in the aortic root was reduced, and the tissue morphology was improved compared with the model control group.

[0285] The above results indicate that in ApoE - / - In a mouse model of atherosclerosis, MPPBNPs reduced plaque burden throughout the aorta, decreased lipid deposition in the aortic root, and improved plaque-related histological features in the aortic root. Combined with the aforementioned in vivo distribution evaluation results, MPPBNPs demonstrated good in vivo therapeutic effects in the atherosclerosis model and can be used to slow, inhibit, or improve the progression of atherosclerotic lesions.

[0286] Example 14: Evaluation of in vivo safety and blood compatibility of PPBNPs and MPPBNPs

[0287] This embodiment is used to evaluate PPBNPs and MPPBNPs in ApoE. - / -The in vivo safety of PPBNPs and MPPBNPs in a mouse model of atherosclerosis was investigated, and the blood compatibility of PPBNPs and MPPBNPs was further evaluated.

[0288] (1) After the treatment cycle described in Example 13 was completed, the major organs of each group of mice were collected. The major organs included the heart, liver, spleen, lungs and kidneys. The collected major organs were fixed, dehydrated, cleared, impregnated with paraffin and embedded to prepare paraffin sections, and the obtained paraffin sections were stained with HE to observe the changes in the tissue morphology of the major organs after different drug treatments.

[0289] (2) In a specific example, after routine dewaxing and rehydration of paraffin sections of major organs, hematoxylin staining was performed on the cell nuclei. After differentiation, blueing, and rinsing, eosin staining was performed on the cytoplasm. After staining, the sections were dehydrated in a gradient, cleared, and mounted. The histological morphology of each major organ was then observed using an optical microscope.

[0290] (3) After the treatment cycle described in Example 13, blood samples were collected from each group of mice, and serum biochemical tests, complete blood count tests, and blood lipid tests were performed on the blood samples. The serum biochemical tests included liver function-related indicators and kidney function-related indicators. The liver function-related indicators included alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the kidney function-related indicators included creatinine (CREA) and urea (UREA). The complete blood count tests included red blood cell count (RBC), white blood cell count (WBC), hemoglobin (HGB), and platelet count (PLT). The blood lipid tests included triglycerides (TG), cholesterol (CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL).

[0291] (4) To further evaluate the blood compatibility of PPBNPs and MPPBNPs, PPBNPs or MPPBNPs were co-incubated with anticoagulated blood samples, and centrifuged after co-incubation. The color change of the supernatant after centrifugation was observed, and the absorbance of the supernatant was measured to evaluate whether PPBNPs and MPPBNPs caused hemolytic reactions of erythrocytes. In a specific example, the detection concentration of PPBNPs and MPPBNPs was 0.8 mg / mL. An equal volume of PBS was added to the negative control group, and an equal volume of ultrapure water was added to the positive control group. Each group of samples was incubated with diluted anticoagulated blood at 37°C, and then the supernatant was collected by centrifugation and the absorbance was measured. The hemolysis rate was calculated as HR (%) = (A sample - A negative) / (A positive - A negative) × 100%.

[0292] Please see Figure 12 The figure shows the in vivo safety and blood compatibility evaluation results of different dosage forms of probucol in atherosclerotic mice in one embodiment of this application. Figure 12 A shows the ApoE levels in each group after treatment.- / - HE staining pathological analysis results of major organ sections from a mouse model of atherosclerosis; Figure 12 B shows the serum biochemical, blood routine, and blood lipid test results of mice in each group after treatment; Figure 12 C shows the hemolysis observation results and absorbance statistics of PPBNPs and MPPBNPs after co-incubation with anticoagulated blood and centrifugation.

[0293] The results are as follows Figure 12 As shown in Figure A, after treatment, HE staining of the heart, liver, spleen, lungs, and kidneys of mice in each treatment group did not show significant tissue damage or pathological abnormalities. These results indicate that, under the administration conditions of this embodiment, treatment with PB, PPBNPs, and MPPBNPs did not cause significant histological damage to major organs.

[0294] The results are as follows Figure 12 As shown in Figure B, no significant abnormalities were observed in liver and kidney function-related indicators such as ALT, AST, CREA, and UREA in the mice of each treatment group; no significant abnormalities were observed in routine blood tests such as RBC, WBC, HGB, and PLT; and no significant abnormalities were observed in lipid-related indicators such as TG, CHO, HDL, and LDL. These results indicate that, under the administration conditions of this embodiment, PPBNPs and MPPBNPs did not cause significant liver and kidney function damage, hematological abnormalities, or abnormal lipid levels.

[0295] The results are as follows Figure 12 As shown in Figure C, after co-incubation and centrifugation of anticoagulated blood samples with PPBNPs and MPPBNPs, no obvious hemolysis of erythrocytes was observed. Corresponding absorbance statistics also showed that at a concentration of 0.8 mg / mL, PPBNPs and MPPBNPs did not induce a significant hemolytic reaction. These results indicate that PPBNPs and MPPBNPs have good blood compatibility.

[0296] The above results indicate that, under the in vivo administration conditions of this embodiment, PPBNPs and MPPBNPs did not cause significant histological damage to major organs, nor did they cause significant abnormalities in blood routine tests, liver and kidney function, or blood lipid levels. Furthermore, PPBNPs and MPPBNPs did not cause significant erythrocyte hemolysis in the in vitro hemolysis experiment. This demonstrates that the biomimetic probucol self-assembled nanoformulation possesses good preliminary in vivo safety and blood compatibility.

[0297] In summary, the biomimetic probucol self-assembled nano-formulation, its preparation method, and its application in the preparation of drugs for treating atherosclerosis disclosed in this application involve the non-covalent self-assembly of unmodified probucol molecules in an aqueous phase to form a probucol self-assembled nanocore. A DSPE-PEG-OH modification component is then applied to the surface of the probucol self-assembled nanocore. Furthermore, macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration provide a microvesicle membrane layer that coats the outer surface of the probucol self-assembled nanocore, resulting in a core-shell structure for the obtained nano-formulation.

[0298] Furthermore, stability evaluation results showed that PPBNPs and MPPBNPs could maintain a nanoscale dispersion in various aqueous media; cell experiments showed that PPBNPs and MPPBNPs had good cell compatibility and could reduce reactive oxygen species levels, alleviate oxidative stress-related apoptosis, and inhibit lipid uptake or foam cell formation in atherosclerosis-related cell models; animal experiments showed that MPPBNPs in ApoE - / - In a mouse model of atherosclerosis, PPBNPs and MPPBNPs demonstrated the ability to distribute to aortic plaque-related areas, reduce aortic plaque burden, decrease intraplaque lipid deposition, and improve plaque-related histological features. Safety evaluation results showed that, under the administration conditions described in this application, PPBNPs and MPPBNPs did not cause significant histological damage to major organs or significant hemolytic reactions.

[0299] The above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Those skilled in the art, based on the disclosure of this application, can make reasonable adjustments or equivalent substitutions to the raw material specifications, dosages, process conditions, testing methods, or specific operations without departing from the technical concept of this application. All equivalent changes or substitutions made within the scope of the claims of this application shall fall within the scope of protection of this application.

Claims

1. A biomimetic probucol self-assembled nano-formulation, characterized in that, It includes a probucol self-assembled nanocore, a DSPE-PEG-OH modified component, and a macrophage-derived microvesicle membrane layer; among which: The probucol self-assembled nanocore is formed by the self-assembly of uncovalently modified probucol molecules in an aqueous phase through non-covalent interactions, including hydrophobic interactions and at least one of hydrogen bonding and π-π stacking interactions; the probucol self-assembled nanocore is a single-drug self-assembled nanocore formed by probucol as a drug molecule. The DSPE-PEG-OH modified component is distributed on the surface of the probucol self-assembled nanocore, and the mass ratio of probucol to DSPE-PEG-OH is (8~12):1; The macrophage-derived microvesicle membrane is provided by macrophage microvesicles released from RAW264.7 macrophages after dehydration and rehydration treatment, and is coated on the outer surface of the probucol self-assembled nanocore modified by the DSPE-PEG-OH modified component, so that the nanoformulation forms a core-shell structure.

2. The biomimetic probucol self-assembled nanoformulation according to claim 1, characterized in that, The molecular weight of the PEG segment in the DSPE-PEG-OH is 3000~4000 Da.

3. The biomimetic probucol self-assembled nanoformulation according to claim 1, characterized in that, The mass ratio of probucol to DSPE-PEG-OH is 10:

1.

4. The biomimetic probucol self-assembled nanoformulation according to claim 1, characterized in that, The macrophage microvesicles were obtained as follows: RAW264.7 macrophages were cultured until the cell confluence reached 80%~90%, the culture medium was removed and the cells were washed, the washed cells were dehydrated, and the dehydrated cells were rehydrated. The supernatant after rehydration was collected and purified by centrifugation to obtain the macrophage microvesicles.

5. The biomimetic probucol self-assembled nanoformulation according to claim 4, characterized in that, The dehydration process includes: after removing the washing buffer, placing the RAW264.7 macrophages (after removing the washing buffer) in a sterile laminar flow hood with the opening open for 15-25 minutes; the rehydration process includes: adding buffer to the dehydrated RAW264.7 macrophages and allowing them to stand for 20-40 minutes; the centrifugation purification process includes: centrifuging at 4000-6000×g for 3-8 minutes to remove detached cells, and centrifuging at 12000-16000×g for 5-15 minutes to enrich the macrophage microvesicles.

6. The biomimetic probucol self-assembled nanoformulation according to claim 1, characterized in that, The nano-formulation is obtained by mixing a dispersion containing a probucol self-assembled nanocore modified by the DSPE-PEG-OH modified component and a dispersion containing macrophage microvesicles at a volume ratio of (7~11):1, followed by vortexing and ultrasonic treatment.

7. The biomimetic probucol self-assembled nanoformulation according to claim 6, characterized in that, The volume ratio of the dispersion containing the probucol self-assembled nanocore modified by the DSPE-PEG-OH modified component to the dispersion containing macrophage microvesicles is 9:1; the vortexing treatment is performed once every 7-12 minutes, for a total of 2-3 times; the ultrasonic treatment is water bath ultrasonication, and the ultrasonic conditions are 40-44 kHz, 90-110 W, and 2.5-4 min.

8. The biomimetic probucol self-assembled nanoformulation according to claim 1, characterized in that, The nano-formulation is an aqueous dispersion for intravenous injection.

9. A method for preparing a biomimetic probucol self-assembled nano-formulation, characterized in that, Includes the following steps: Preparation of DSPE-PEG-OH modified probucol self-assembled nanoparticles: Uncovalently modified probucol and DSPE-PEG-OH are dissolved in an organic solvent to form an organic phase. The organic phase is added dropwise to an aqueous phase and stirred to form DSPE-PEG-OH modified probucol self-assembled nanoparticles. The resulting mixture is dialyzed to obtain a dispersion containing the DSPE-PEG-OH modified probucol self-assembled nanoparticles. The DSPE-PEG-OH modified probucol self-assembled nanoparticles include a probucol self-assembled nanocore formed by the non-covalent self-assembly of uncovalently modified probucol molecules in an aqueous phase. The DSPE-PEG-OH modification component is distributed on the surface of the probucol self-assembled nanocore. The mass ratio of probucol to DSPE-PEG-OH is (8~12):

1. Preparation of macrophage microvesicles: RAW264.7 macrophages were cultured until the cell confluence reached 80%~90%, the culture medium was removed and the cells were washed. The washed cells were dehydrated and then rehydrated. The supernatant after rehydration was collected and centrifuged to purify the solution, which contained macrophage microvesicles. Preparation of biomimetic probucol self-assembled nanoformulations: A dispersion containing the DSPE-PEG-OH modified probucol self-assembled nanoparticles was mixed with a dispersion containing the macrophage microvesicles, and then subjected to vortexing and sonication treatments in sequence, so that the macrophage microvesicles were coated on the outer surface of the DSPE-PEG-OH modified probucol self-assembled nanoparticles, thereby obtaining a biomimetic probucol self-assembled nanoformulation with a core-shell structure.

10. The preparation method according to claim 9, characterized in that, In the step of preparing probucol self-assembled nanoparticles modified with DSPE-PEG-OH, the amount of uncovalently modified probucol is 5~20 mg, the amount of DSPE-PEG-OH is 0.5~2 mg, and the mass ratio of probucol to DSPE-PEG-OH is (8~12):1; the volume ratio of organic solvent to aqueous phase is 1:(3~5).

11. The preparation method according to claim 10, characterized in that, In the step of preparing probucol self-assembled nanoparticles modified with DSPE-PEG-OH, the amount of uncovalently modified probucol is 10 mg, the amount of DSPE-PEG-OH is 1 mg, the organic solvent is N,N-dimethylformamide, the volume of the organic solvent is 1 mL, and the aqueous phase is pure water or ultrapure water, the volume of the aqueous phase is 4 mL.

12. The preparation method according to claim 9, characterized in that, In the step of preparing probucol self-assembled nanoparticles modified with DSPE-PEG-OH, the molecular weight of the PEG segment in the DSPE-PEG-OH is 3000~4000 Da.

13. The preparation method according to claim 9, characterized in that, In the step of preparing probucol self-assembled nanoparticles modified with DSPE-PEG-OH, the organic phase is vortex-mixed for 15-30 seconds and then added dropwise to the aqueous phase under stirring, the stirring speed of the aqueous phase being 400-600 rpm. The resulting mixture was transferred to a dialysis bag with a molecular weight cutoff of 3000-4000 Da, and dialysis was performed under the condition of stirring of the dialysis fluid.

14. The preparation method according to claim 13, characterized in that, The stirring speed of the aqueous phase is 500 rpm, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed of the dialysis fluid is 200 rpm, and the dialysis is overnight dialysis.

15. The preparation method according to claim 9, characterized in that, In the step of preparing macrophage microvesicles, after removing the culture medium, the cells were washed twice with DPBS. After removing the washing DPBS, the cap of the culture container was opened and the RAW264.7 macrophages were placed in a sterile laminar flow hood for 15-25 minutes to perform the dehydration treatment.

16. The preparation method according to claim 15, characterized in that, In the rehydration process, DPBS was added to the dehydrated RAW264.7 macrophages and allowed to stand for 20-40 minutes. Then, the macrophage microvesicles were released and the supernatant containing the macrophage microvesicles was collected.

17. The preparation method according to claim 16, characterized in that, In the centrifugal purification process, the supernatant containing the macrophage microvesicles is centrifuged at 4000-6000×g for 3-8 min to remove detached cells. The supernatant is then centrifuged at 12000-16000×g for 5-15 min to enrich the macrophage microvesicles. The enriched macrophage microvesicles are then resuspended in DPBS.

18. The preparation method according to claim 9, characterized in that, In the step of preparing the biomimetic probucol self-assembled nanoformulation, the dispersion containing the probucol self-assembled nanoparticles modified with DSPE-PEG-OH and the dispersion containing the macrophage microvesicles are mixed at a volume ratio of 9:1, and vortexed once every 10 min for a total of 3 times; then, the mixture is subjected to water bath sonication at 42 kHz and 100 W for 3 min to obtain the biomimetic probucol self-assembled nanoformulation.

19. The use of the biomimetic probucol self-assembled nanoformulation according to any one of claims 1 to 8 or the biomimetic probucol self-assembled nanoformulation prepared by the preparation method according to any one of claims 9 to 18 in the preparation of a drug for treating atherosclerosis.

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