Micromolecule prodrug co-assembled nano-drug for targeted inhibition of pyroptosis of macrophages and preparation method and application thereof

Abstract

The invention discloses a micromolecule prodrug co-assembled nano-drug for targeted inhibition of pyroptosis of macrophages as well as a preparation method and application of the micromolecule prodrug co-assembled nano-drug. The preparation method of the micromolecular prodrug co-assembled nano-drug for targeted inhibition of pyroptosis of macrophages comprises the following steps: S1, preparing a Pu-ID prodrug; s2, preparation of MPID NPs: weighing a Pu-ID prodrug, disulfiram, DSPE-PEG-Mannose and DSPE-PEG-COOH, fully dissolving the weighed materials in an organic solvent B, and dropwise adding the obtained mixed solution into pure water in a stirring state to form an MPID NPs solution; then, the obtained MPID NPs solution is transferred into a dialysis bag for dialysis; and after dialysis is finished, collecting the MPID NPs solution in the dialysis bag, so as to obtain the micromolecule prodrug co-assembled nano-drug. The nano-drug provided by the invention can be applied to preparation of drugs for treating atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule prodrug co-assembled nanomedicine targeted to inhibit macrophage pyroptosis, and a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory vascular disease. AS has become a major risk factor for cardiovascular disease and a leading cause of death worldwide. The course of AS can progress through lipid streaks, fibrous plaques, atheromatous plaques, intraplaque hemorrhage, rupture, and thrombosis. Research has shown that the formation and progression of AS are influenced by numerous factors and involve complex mechanisms. In recent years, researchers at home and abroad have explored new mechanisms of AS formation and progression, including pyroptosis, ferroptosis, autophagy, copper death, and intestinal microbiota. Pyroptosis is a form of programmed cell death that triggers the release of a range of inflammatory substances, thereby exacerbating the inflammatory response. Pyroptosis itself is inflammatory, characterized by rapid plasma membrane rupture and the release of proinflammatory cytokines. Studies have shown that pyroptosis-induced inflammation persists throughout the development and progression of AS. Furthermore, studies have confirmed that in advanced AS lesions, macrophage pyroptosis may be a key factor in promoting the formation of a necrotic core within AS lesions and plaque instability. Inhibiting macrophage pyroptosis with anti-pyroptotic drugs can alleviate the development of AS lesions. Therefore, targeted inhibition of macrophage pyroptosis in AS lesions provides an ideal strategy for delaying the progression of AS.

[0003] Gasdermins (GSDMD) is a mediator of inflammatory cell death triggered by cytoplasmic sensing of invasive infection and danger signals. Humans have a total of six GSDM genes: GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB59. Among them, GasderminsD (GSDMD) is one of the final effector proteins in pyroptosis. Its regulatory mechanism mainly focuses on upstream factors that affect its early lysis, including multi-effect pyroptosis-related complexes that exert pro-inflammatory or anti-inflammatory effects. It is involved in the pathogenesis of many inflammasome-related diseases and tissue damage. The GSDMD protein has three types of domains: the N-terminal domain (GSDMD-N), the linker domain, and the C-terminal domain (GSDMD-C). Further studies have found that GSDMD-N can bind to phospholipids in the cytoplasmic membrane, transfer to the cell membrane after binding, and then insert into the cell membrane to form cell membrane pores. The cell membrane pores formed by GSDMD-N disrupt the integrity of the cell membrane, leading to the release of intracellular substances such as inflammatory cytokines (IL-1β, IL-18) into the extracellular space, causing inflammatory cell death. This inflammatory cell death mode is called pyroptosis. Therefore, by inhibiting the formation of GSDMD-N pores on the cell membrane surface, the release of inflammatory cytokines can be effectively blocked, thereby effectively preventing the occurrence of pyroptosis.

[0004] Existing studies on the inhibition of GSDMD-N pore formation have shown that disulfiram (DSF), the first drug approved by the US Food and Drug Administration for the treatment of alcohol dependence in 1951, has been used clinically without significant side effects. Professor Xing Liu of Boston Children's Hospital and others published an article in Nature Immunology (2020) demonstrating that DSF effectively inhibits pyroptosis in humans and mice. DSF covalently modifies Cys191 (human) / Cys192 (mouse) in GSDMD, thereby preventing GSDMD-N pore formation. Furthermore, studies have shown that reactive oxygen species (ROS) can promote GSDMD cleavage and thus GSDMD-N pore formation by affecting the inflammasome pathway. This phenomenon suggests that the plasma membrane pores of GSDMD-N are positively regulated by ROS, and that intracellular oxidative stress further promotes GSDMD-N pore formation. Therefore, combining antioxidant therapy with GSDMD-N pore inhibition can more effectively inhibit GSDMD-N pore formation.

[0005] In recent years, nanomedicines based on nanotechnology have become an innovative form of pharmaceutical formulation. This involves the design and application of drugs at the nanoscale, namely, using nanofabrication technology to process the original drug into nanoscale particles, or using appropriate carrier materials to load the original drug to synthesize nanoscale particles and ultimately prepare a pharmaceutical formulation. Among them, small-molecule prodrugs (SMPs) are a new prodrug design strategy that involves linking small molecule drugs to themselves or other small molecules through cleavable active bonds to form prodrugs. Synthesized small molecule prodrugs can self-assemble to form small molecule nanoprodrugs. These self-assembled small molecule nanoprodrugs exhibit high drug loading (drug loading greater than 50%). At the same time, these small molecule nanoprodrugs have unique pharmacokinetic characteristics and may have more significant advantages in regulating drug release rate and addressing multidrug resistance.

[0006] Probucol (Pu) is a compound containing a bisphenolic hydroxyl group that exhibits antioxidant and anti-inflammatory properties and can prevent plaque formation during the progression of atherosclerosis. Idebenone (ID) was developed and marketed in Japan in 1986. Previous literature indicates that idebenone possesses strong ROS scavenging and antioxidant capabilities, as well as anti-endothelial cell apoptosis, enhanced mitochondrial function, and reduced inflammatory responses. Furthermore, related research suggests that idebenone can attenuate the production of pro-inflammatory NF-κB and MAPK mechanisms, both of which are involved in the molecular mechanisms that promote atherosclerosis. Most importantly, idebenone has a favorable safety and tolerability profile. Clinical trial results have shown that IDE is highly tolerable and has minimal side effects. Therefore, if idebenone can be used as a mitochondrial-targeted antioxidant to prevent and treat early vascular atherosclerosis plaque formation, it holds great promise.

[0007] Mannose is a naturally occurring monosaccharide belonging to the hexose family. The hydroxyl groups in its molecular structure make it electrically neutral under physiological conditions. In atherosclerosis (AS), mannose can exert its effects by targeting CD206 (mannose receptor, MRC1). CD206 is a C-type lectin receptor highly expressed on the surface of macrophages and activated endothelial cells. It specifically recognizes and binds mannose, mediating endocytosis. Within atherosclerotic plaques, infiltrating inflammatory macrophages (such as M1) and damaged endothelial cells significantly upregulate CD206 expression. Studies have demonstrated that PEGylated mannose liposomes have a 2.3-fold longer half-life in mouse blood than unmodified particles. Furthermore, while mannose-modified nanoparticles are taken up by CD206, their local retention (>72 hours) in plaques is prolonged due to the EPR effect (enhanced permeability retention) and receptor enrichment at lesion sites, while maintaining systemic clearance. Summary of the Invention

[0008] The present invention uses PEGylated mannose to modify the surface of nanoparticles to achieve delivery of nanoparticles to macrophages. Experimental studies have confirmed that this approach can prolong the circulation time of nanomedicines in the body while specifically targeting the drug to AS lesions. Based on this, the present invention protects the following technical solutions:

[0009] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a method for preparing a small molecule prodrug co-assembled nanodrug for targeted inhibition of macrophage pyroptosis, comprising the following steps:

[0011] S1. Preparation of Pu-ID prodrug:

[0012] S1.1. Add idebenone to organic solvent A to obtain an idebenone solution, then add oxalyl chloride dropwise to the idebenone solution and allow to react at 0-4°C. After the reaction is complete, drain the remaining reaction liquid to obtain an intermediate product, which is then dissolved in organic solvent A to obtain an intermediate product solution.

[0013] S1.2. Dissolve 4-dimethylaminopyridine (DMAP), triethylamine, and probucol in organic solvent A to obtain a mixed solution. Add the mixed solution dropwise to the intermediate product solution at 0-4°C. After addition, move the reaction flask to 20-25°C for full reaction. After the reaction is complete, add a quencher to quench the reaction. Extract the mixture with organic solvent A and elute the extract with an eluent to obtain the Pu-ID prodrug.

[0014] S2. Preparation of MPIDNPs:

[0015] Pu-ID prodrug, disulfiram, DSPE-PEG-Mannose and DSPE-PEG-COOH are weighed and fully dissolved in organic solvent B, and the obtained mixed solution is dropped into pure water under stirring to form an MPIDNPs solution; the obtained MPIDNPs solution is then transferred to a dialysis bag for dialysis; after the dialysis is completed, the MPIDNPs solution in the dialysis bag is collected to obtain the small molecule prodrug co-assembled nanodrug.

[0016] Preferably, in step S1.1, the reaction is carried out at 0-4° C. for 3-6 hours, the molar ratio of idebenone to oxalyl chloride is 1:4-6, and the idebenone solution is preferably prepared according to a ratio of idebenone to organic solvent A of 1 mmol:8-12 ml; preferably, the intermediate product prepared by 1 mmol of idebenone is dissolved in 8-12 ml of organic solvent A to prepare an intermediate product solution;

[0017] The organic solvent A is selected from dichloromethane.

[0018] Preferably, in step S1.2, the temperature is moved to 20-25° C. and the reaction is performed for 10-16 hours, the quencher is pure water, and the eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 14-16:1;

[0019] The molar ratio of DMAP, triethylamine and probucol is 0.15-0.25:1.0-1.2:1, and the usage ratio of probucol to organic solvent A is 1 mmol:4-6 ml.

[0020] Preferably, in step S2, the mass ratio of the Pu-ID prodrug, disulfiram, DSPE-PEG-Mannose, and DSPE-PEG-COOH is (4-6):1:(220-280):(220-280), and the organic solvent B is DMF or DMSO; the molecular weight of PEG of DSPE-PEG-Mannose and DSPE-PEG-COOH is 1000-3000, preferably 1500-2500;

[0021] The mass volume ratio of the disulfiram to the organic solvent B is (1-2):(0.8-1.2).

[0022] Preferably, the molecular weight cut-off (MWCO) of the dialysis bag is 3000-4000 Da, preferably MWCO 3500 Da.

[0023] Preferably, in step S2, the obtained mixed solution is added dropwise into pure water under stirring, and the volume ratio of the mixed solution to pure water is 1:(3-5); the obtained MPIDNPs solution is transferred to a dialysis bag and dialyzed in ultrapure water for 4-14 hours.

[0024] The second aspect of the present invention provides a small molecule prodrug co-assembled nanomedicine targeted to inhibit macrophage pyroptosis, which is prepared by any of the methods described above.

[0025] The third aspect of the present invention provides the use of the above-mentioned small molecule prodrug co-assembled nanomedicine that targets and inhibits macrophage pyroptosis in the preparation of a drug for treating atherosclerosis.

[0026] In the application technology solution, the small molecule prodrugs are co-assembled into nanomedicines to target and inhibit macrophage pyroptosis in atherosclerotic lesions.

[0027] In the application technology scheme, the small molecule prodrug co-assembled nanodrug accumulates in the atherosclerotic lesion site and is taken up by the inflammatory macrophages in the area. Under high intracellular ROS levels, Pu, ID and disulfiram drugs are released. Pu and ID can effectively inhibit the generation of ROS, and synergistically inhibit GSDMD-NT pore formation together with DSF, inhibit the occurrence of macrophage pyroptosis, and inhibit the occurrence and development of atherosclerosis.

[0028] The beneficial effects of the present invention are:

[0029] The present invention first couples probucol (Pu) and idibenquinone (ID) via an oxalate bond with ROS-responsive cleavage to synthesize a ROS-responsive heterodimeric small molecule nanoprodrug. This system can form a nanomedicine through self-assembly. When in an environment with relatively low ROS concentrations, the pharmacophore of Pu-ID is coupled to the hydroxyl group in the lactone ring, thereby demonstrating the pharmacological inertness of the system and ensuring the stability of the prodrug. When the plaque inflammation microenvironment with a higher ROS concentration is reached, the oxalate bond in Pu-ID breaks, allowing the system to release the two drugs to exert their efficacy. This can achieve "intelligent" drug release while improving the oxidative stress microenvironment in the plaque. In addition, the synthesized Pu-ID can be used as a carrier to load hydrophobic drugs to form nanomedicines. Therefore, the present invention uses Pu-ID loaded with DSF to co-assemble into small molecule nanoprodrugs (PIDNPs), thereby achieving efficient co-loading of antioxidant functional drugs and DSF. In order to achieve the function of targeting macrophages in AS lesions, DSPE-PEG-mannose was used to functionally modify PIDNPs, and the prepared nanomedicine (MPIDNPs) can efficiently target and aggregate to the AS lesions and be taken up by inflammatory macrophages in the area. MPIDNPs entering the inflammatory macrophages release Pu, ID and DSF drugs under high intracellular ROS levels. Pu and ID can effectively inhibit the production of ROS, thereby synergistically inhibiting GSDMD-NT pore formation together with DSF and inhibiting the occurrence of macrophage pyroptosis. In short, the nanomedicine can achieve efficient targeted inhibition of the occurrence of macrophage pyroptosis in AS lesions, thereby effectively inhibiting the occurrence and development of AS. The present invention provides a new drug for the efficient targeted treatment of AS and provides a new idea and treatment strategy for the treatment of AS. The present invention achieves efficient targeting and safe treatment of AS by constructing a new small molecule prodrug co-assembled nanomedicine that targets and inhibits macrophage pyroptosis in AS lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The synthesis and characterization of the biomimetic small molecule prodrug Pu-ID and its in vitro safety evaluation; (a) the synthetic route of the small molecule prodrug Pu-ID, and (b) the in vitro cytotoxicity evaluation of probucol, (c) idibenzoquinone and (d) Pu-ID prodrugs on RAW264.7 cells at different concentrations; (n=3, mean±SD).

[0031] Figure 2The optimization of PidNPs and the characterization of PIDNPs / MPIDNPs are shown: (a) PidNPs aqueous solution was prepared by nanoprecipitation method at different ratios of Pu-ID and DSF; (b) Tyndall effect of PidNPs aqueous solution with different ratios after irradiation with laser pen; particle size (c), surface potential (d) and PDI (e) of PidNPs prepared at different ratios of Pu-ID and DSF (n=3, average ± SD); changes in particle size (f) and PDI (g) of PidNPs prepared at different ratios of Pu-ID and DSF within nine days at room temperature (n=3, average ± SD), and precipitation after nine days at room temperature (h); TEM images of PID NPs (i) and MPID NPs (j) prepared under Pu-ID:DSF 5:1 conditions (i: scale bar = 200 nm; j: scale bar = 100 nm); PID was prepared under Pu-ID:DSF 5:1 conditions. Particle size (k), surface potential (l) and PDI (m) measurement results of NPs (n=3, mean ± SD); changes in particle size and PDI of MPID NPs (n)(o) and PID NPs (p)(q) in water, PBS and culture medium containing 10% FBS within 9 days were measured (n=3, mean ± SD).

[0032] Figure 3 In vitro safety evaluation of PID NPs and MPID NPs: (a) In vitro cytotoxicity evaluation of PID NPs and (b) MPID NPs at different concentrations on RAW264.7 cells; (n=4, mean±SD); in vitro cytotoxicity evaluation of PID NPs (c) and MPID NPs (d) at different concentrations on HUVEC cells (n=3, mean±SD); in vitro cytotoxicity evaluation of PID, PID NPs and MPID NPs at a concentration of 100ug / mL on RAW 264.7 (e) and HUVEC (f) using CCK-8 method (n=3, mean±SD); blood compatibility of PID NPs / MPID NPs: (g) hemolysis test images of PID NPs and MPID NPs, and the absorbance of each group of solutions (h) (n=3, mean±SD).

[0033] Figure 4The results of the PID NPs / MPID NPs HUVECs cell phagocytosis experiment are shown: CLSM image (a) (scale bar = 10 μm), fluorescence quantitative analysis statistical graph (b) and flow cytometer analysis graph (c) of HUVECs cells phagocytosing 200 μg / mL PID NPs / MPID NPs after different treatments (n = 3, mean ± SD); MPID NPs Transwell schematic diagram (d); fluorescence intensity (644 nm) OD value in RAW 264.7 cell culture medium in the Transwell lower chamber (e) (n = 3, mean ± SD); fluorescence quantitative analysis statistical results (f), CLSM image (g) (scale bar 10 μm) and fluorescence quantitative analysis (h) (n = 3, mean ± SD) of MPID NPs phagocytosis by RAW264.7 cells in the Transwell lower chamber.

[0034] Figure 5 Shown is the lysosomal escape of PID NPs / MPID NPs in HUVECs cells: CLSM images of PID NPs / MPID NPs being phagocytosed by HUVECs cells at different times (scale bar = 10 μm).

[0035] Figure 6 The experimental results of the ROS scavenging ability of PID NPs at different concentrations and PID / PID NPs / MPID NPs at the same concentration in HUVECs cells are shown: fluorescence image (a) (scale bar = 100 μm), fluorescence quantitative statistical result image (b) and flow cytometer analysis image (c) (n = 3, mean ± SD) of the inhibition of ROS production by PID NPs at different concentrations; fluorescence image (d) (scale bar = 100 μm), fluorescence quantitative statistical result image (e) and flow cytometer analysis image (f) (n = 3, mean ± SD) of the inhibition of ROS production by HUVECs cells by different dosing groups (200 μg / mL).

[0036] Figure 7The results of the PID NPs / MPID NPs RAW264.7 cell phagocytosis experiment are shown: CLSM image (a) (scale bar = 10 μm) of 100 μg / mL PID NPs / MPID NPs phagocytosis by RAW264.7 cells after different treatments, fluorescence quantitative statistical results (b) and flow cytometry analysis (c) (n = 3, mean ± SD); PID NPs / MPID NPs lysosomal escape in RAW 264.7 cells: CLSM image of PID NPs / MPID NPs phagocytized by RAW264.7 cells at different times (d) (scale bar = 10 μm); ROS scavenging ability experiment in RAW 264.7 cells: different concentrations of PID Fluorescence graph (e) (scale bar = 100 μm), fluorescence quantitative statistical results (f) and flow cytometer analysis (g) (n = 3, mean ± SD) of the inhibition of ROS production in RAW264.7 cells by NPs; fluorescence graph (h) (scale bar = 100 μm), fluorescence quantitative statistical results (i) and flow cytometer analysis (j) (n = 3, mean ± SD) of the inhibition of ROS production in RAW264.7 cells by different drug groups.

[0037] Figure 8 To evaluate the anti-RAW264.7 cell death ability of PID NPs / MPID NPs; CCK-8 was used to detect the cell viability of RAW264.7 cells treated with cholesterol (10 μg / mL) for 24 h using PID NPs (a) and MPID NPs (b) at different concentrations (n=3, mean ± SD); different concentrations of PID NPs (75, 100 and 200 μg / mL) inhibited RAW264.7 cell death (c) (n=3, mean ± SD) and fluorescence images (e) (scale bar = 100 μm); flow cytometry results of the inhibition of RAW264.7 cell death after treatment with different dosing groups (d) (n=3, mean ± SD) and fluorescence images (f) (scale bar = 100 μm); GSDMD-N (g) and GSDMD-FL (h) were labeled in RAW264.7 cells treated with ATP+LPS, and immunofluorescence was used to detect the expression of PID / PID NPs in the cells. Expression in cells after NPs / MPID NPs treatment (scale = 100 μm); CCK-8 was used to detect the anti-pyroptosis ability of RAW264.7 cells treated with ATP (5 mmol / L) + LPS (0.5 μg / mL) for 12 h after different concentrations of MPID NPs (i) (n = 3, mean ± SD); ELISA was used to detect the secretion of TNF-a (j) and IL-1β in RAW264.7 cells after treatment with ATP + LPS in vitro (k) (n = 3, mean ± SD).

[0038] Figure 9 Figure 1: (a) Animal experimental model; (b) Oil red O staining of mouse aorta; (c) Quantitative analysis of plaques in mouse aorta (n=3, mean±SD); Quantitative analysis of ORO (d), TB (e) and Masson (f) staining (scale bar=100μm) of mouse tricuspid valve sections and ORO, TB and Masson staining (g) of mouse tricuspid valve sections (n=3, mean±SD); inflammatory cytokine CD68, IL-1β and TNF-α staining of mouse tricuspid valve sections (h) (scale bar=100μm); Quantitative analysis of CD68 (i), IL-1β (j) and TNF-α (k) staining of mouse tricuspid valve sections (n=3, mean±SD); (lo) Quantitative results of mouse blood routine data (n=5, mean±SD);

[0039] Figure 10 In vivo biosafety assessment of MPID NPs; (a) H&E images of mice in different drug treatment groups after treatment (scale bar = 100 μm); (bi) Plasma levels of aspartate aminotransferase (AST), blood urea nitrogen (BUN), alanine aminotransferase (ALT), creatinine (CREA), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) after treatment (n = 5, mean ± SD). DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0042] Example 1

[0043] 1 Main reagents and sources

[0044]

[0045] 2 Methods

[0046] 2.1 Evaluation of cytotoxicity of PU, ID, and Pu-ID small molecule prodrugs in RAW264.7 cells

[0047] RAW264.7 cells were plated at 1×10 4 Cells were seeded in a 96-well plate and incubated for 24 hours at 37°C in a cell culture incubator with 5% CO2. 100 μL of a small molecule prodrug containing PU, ID, and Pu-ID was added to each well. After 24 hours of incubation, 100 μL of CCK-8 solution was added to each well and incubated for 1-2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0048] The cell viability was calculated as follows:

[0049] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0050] in:

[0051] As: absorbance of the experimental group (culture medium containing cells, CCK-8, and test drug)

[0052] Ac: absorbance of the control group (culture medium containing cells, CCK-8, and no test drug)

[0053] Ab: absorbance of blank group (culture medium without cells and test drugs, CCK-8)

[0054] 2.2 Preparation and characterization of NPs

[0055] 2.2.1 Preparation of Pid NPs:

[0056] 1. Preparation of Pu-ID prodrug

[0057] Follow these steps:

[0058] 1) Idebenone (1 mmol) was added to the reaction solvent, dichloromethane (10 ml), to obtain an idebenone solution. Oxalyl chloride (C₂Cl₂O₂, 5 mmol) was then added dropwise to the idebenone solution. The reaction was allowed to proceed at 0°C for 4 h. Due to the excess oxalyl chloride, the reaction liquid (including excess oxalyl chloride and byproduct HCl) was pumped under reduced pressure into a cold trap after completion to remove the excess oxalyl chloride and byproduct, yielding the intermediate product. After draining the remaining reaction liquid, 10 ml of dichloromethane was added to obtain the intermediate product solution.

[0059] 2) 0.2 mmol DMAP (4-dimethylaminopyridine), 1.1 mmol triethylamine, and 1 mmol probucol were dissolved in 5 ml of dichloromethane to obtain a mixed solution. This mixed solution was added dropwise to the intermediate product solution at 0°C. After addition, the reaction flask was moved to room temperature and reacted for 12 h. After 12 h, water was added to quench the reaction. The product was then extracted with dichloromethane and eluted with an eluent (petroleum ether:ethyl acetate, volume ratio 15:1) to obtain the Pu-ID prodrug.

[0060] 2. Preparation of Pid NPs

[0061] 5 mg of Pu-ID prodrug was weighed using a precision balance and mixed according to different mass ratios of Pu-ID prodrug and disulfiram (DSF) (5:1, 5:2, and 5:4), and fully dissolved in 1 mL of N,N-dimethylformamide (DMF). Then, 1 mL of the mixed solution was dropwise added to 4 mL of ddH2O under stirring (500 rpm) to obtain a Pid NPs solution. The prepared Pid NPs solutions containing different mass ratios were then transferred to a regenerated fiber bag (MWCO: 3500 Da) and dialyzed against ultrapure water overnight. After dialysis, the Pid NPs solution in the dialysis bag was collected and its hydration kinetic diameter, surface potential, and PDI were measured using a nanoparticle size potentiostat.

[0062] 2.2.2PID NPs preparation:

[0063] 5 mg of Pu-ID prodrug, 1 mg of DSF, and 500 mg of DSPE-PEG-COOH were weighed using a precision balance and thoroughly dissolved in 1 mL of DMF. The resulting mixture was then added dropwise to 4 mL of ddH2O under stirring (500 rpm) to form a PID NPs solution. The resulting PID NPs solution was then transferred to a regenerated fiber bag (MWCO: 3500 Da) and dialyzed against ultrapure water overnight. After dialysis, the PID NPs solution in the dialysis bag was collected and its hydration kinetic diameter, surface potential, and PDI were measured using a nanoparticle size potentiometry instrument.

[0064] 2.2.3MPID NPs Preparation:

[0065] 5 mg of Pu-ID prodrug, 1 mg of DSF, 250 mg of DSPE-PEG-Mannose, and 250 mg of DSPE-PEG-COOH were weighed using a precision balance and thoroughly dissolved in 1 mL of DMF (N,N-dimethylformamide). The resulting mixture was then added dropwise to 4 mL of stirred ddH2O (500 rpm for 30 seconds) to form an MPID NPs solution. The resulting MPID NPs solution was then transferred to a regenerated fiber bag (MWCO: 3500 Da) and dialyzed against ultrapure water overnight (the purpose of dialysis was to remove the organic solvent DMF). After dialysis, the MPID NPs solution in the dialysis bag was collected and its hydration kinetic diameter, surface potential, and PDI were measured using a nanoparticle size potentiometry instrument.

[0066] 2.2.4 Characterization of Particle Size, Potential, and Morphology of Pid NPs, PID NPs, and MPID NPs

[0067] The hydration kinetic diameters and surface potentials of PID NPs and MPID NPs in aqueous solution were measured at room temperature using a laser particle size analyzer. The morphological characteristics of PID NPs and MPID NPs were determined by transmission electron microscopy (TEM).

[0068] 2.2.5 Stability testing of PID NPs and MPID NPs

[0069] To evaluate the in vitro stability of PID and MPID NPs, 500 mL of a 0.85 mg / mL aqueous solution of PID and MPID NPs was added to 4.5 mL of ddH2O, PBS, and culture medium (containing 10% fetal bovine serum), respectively. The aqueous kinetic diameter and PDI of the nanoparticles were measured daily over 1-9 days, and their aqueous stability was observed. Three parallel groups were set up for each group.

[0070] 2.2.6 Calculation of PID NPs encapsulation efficiency

[0071] After dissolving DSF in HPLC-grade acetonitrile, a standard curve was established using HPLC. Pid NPs prepared using the nanoprecipitation method were freeze-dried and dissolved in a certain volume of HPLC-grade acetonitrile. The mixture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was filtered and then injected into the liquid phase to measure the drug concentration (C). Three replicates were run, and the drug loading (DL) and encapsulation efficiency (EE) were calculated according to the following formulas: DL = CV / W1, EE = CV / W2 (W2 is the total mass of the drug input).

[0072] 2.3PID NPs / MPID NPs hemolysis experiment

[0073] Blood was collected from the abdominal aorta of SD rats and placed in an anticoagulant tube (EDTA.2K), and then shaken up and down thoroughly.

[0074] PID, PID NPs, and MPID NPs were mixed with 10×PBS (volume ratio 9:1) respectively. The negative control group was 1×PBS; the positive control group was ddH2O. Three replicates were set up for each group. All centrifuge tubes were placed in a 37°C constant temperature shaker for incubation. After 30 minutes, anticoagulant blood diluted with 1×PBS was added to each prepared drug (anticoagulant blood was diluted with 1×PBS in a volume ratio of 4:5). Place it in a 37°C constant temperature shaker and incubate for 60 minutes again. After incubation, use a centrifuge at 5000 rpm for 5 minutes. After centrifugation, use an enzyme reader to detect the absorbance value of the supernatant of each group at a wavelength of 545 nm.

[0075] Calculate the hemolysis rate of each group. Hemolysis rate formula: HR (%) = [(AB) / (CB)] × 100%

[0076] Wherein: HR: sample hemolysis rate; A: sample absorbance; B: absorbance of negative control group; C: absorbance of positive control group.

[0077] 2.4 Toxicity evaluation

[0078] 2.4.1 Evaluation of cytotoxicity of PID, PID NPs, and MPID NPs on RAW 264.7 cells

[0079] RAW264.7 cells were cultured at a concentration of 1 × 10 5 A cell suspension of 100 μg / mL was added to a 96-well plate and incubated at 37°C in a 5% CO2 incubator. After 24 hours, PDI, PDI NPs, and MPID NPs were added to each well. After 24 hours of incubation, 100 μL of CCK-8 solution was added to each well. After 1-2 hours of incubation, the absorbance at 450 nm was measured using a microplate reader.

[0080] The cell viability calculation method was the same as before.

[0081] 2.4.2 Toxicity evaluation of PID, PID NPs, and MPID NPs on HUVECs

[0082] HUVECs were plated at 1×10 4 Cells were seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 24 hours, 100 μL of a solution containing different doses of PID, PID NPs, and MPID NPs was added to each well of the plate. After 24 hours of incubation, 100 μL of CCK-8 solution was added to the plate. After incubation for 1-2 hours in the incubator, the absorbance of each group at 450 nm was measured using a microplate reader.

[0083] The cell viability calculation method was the same as before.

[0084] Effects of 2.5PID NPs and MPID NPs on RAW 264.7 cell viability

[0085] 1. CCK-8 assay

[0086] RAW264.7 cells were plated at 1×10 4Cells were seeded in 96-well plates and incubated in a cell culture incubator at 37°C with 5% CO2 for 24 hours. Different concentrations of PID NPs / MPID NPs were then added to the culture medium and incubated for 3 hours. Cells were treated with cholesterol (final concentration of 20 μg / mL) and 24 hours later, 10 μL of CCK-8 solution was added to each well and incubated for 1-2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0087] The cell viability calculation method was the same as before.

[0088] 2. Fluorescence microscopy and flow cytometry

[0089] RAW264.7 cells were plated at 2 × 10 4 The number of cells was seeded in a 12-well plate and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. After that, different concentrations of PID NPs were added to the culture medium and incubated for 3 hours. RAW264.7 cells were treated with cholesterol (final concentration of 20 μg / mL) and stained with a live-dead cell staining reagent 24 hours later. After 30 minutes, the cells were observed and photographed using an inverted fluorescence microscope.

[0090] Flow cytometry was used to measure the effects of different concentrations of PID NPs on RAW264.7 cell viability. After staining the cells with a live-dead stain for 30 minutes, the cells were pipetted free. The cells were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0091] 2.6 Lysosomal escape

[0092] 2.6.1 Lysosomal escape of PID NPs and MPID NPs in RAW 264.7 cells

[0093] RAW264.7 cells were plated at 2 × 10 4 Cells were seeded in confocal dishes and incubated in a cell culture incubator at 37°C and 5% CO2. After 12 hours, DiD-labeled DPID NPs and DMPID NPs (100 μg / mL) were added to each well. Lysosomes and cell nuclei were stained with a lysosomal probe and Hoechst 33342, respectively, after 1 hour and 6 hours, respectively. The images were then photographed using an inverted fluorescence microscope.

[0094] 2.6.2 Lysosomal escape of PID NPs and MPID NPs in HUVECs

[0095] HUVECs were plated at 3 × 10 per well. 5The cells were seeded in a confocal dish and incubated in a cell culture incubator at 37°C and 5% CO2. After 12 hours, DiD-labeled DPID NPs and DMPID NPs (concentration 200 μg / mL) were added to each well. Lysosomes and cell nuclei were stained with lysosomal probes and Hoechst 33342 after 1 hour and 6 hours, respectively, and then photographed using a fluorescence inverted microscope.

[0096] 2.7 Ability of PID NPs to scavenge ROS in RAW 264.7 cells

[0097] RAW264.7 cells were plated at 2 × 10 4 RAW264.7 cells were seeded in 12-well plates and incubated for 24 hours at 37°C in a cell culture incubator with 5% CO2. Lipopolysaccharide (LPS) (final concentration 10 μg / mL) was then treated with PID NPs 24 hours later. After 24 hours, cells were stained with a reactive oxygen species detection kit and Hoechst 33342 staining solution, respectively, and then observed and photographed using an inverted fluorescence microscope.

[0098] 2.8MPID NPs Transwell

[0099] HUVECs were plated at 3 × 10 per well. 4 RAW264.7 cells were seeded in the chamber of a 6-well Transwell plate at a density of 3 × 10 cells per well. 4 Cells were seeded in the lower chamber of a 6-well Transwell plate containing a cell slide and incubated for 12 hours at 37°C in a cell culture incubator with 5% CO2. HUVECs in the upper chamber were treated with tumor necrosis factor TNF-α (final concentration 10 nM). 12 hours later, DiD-labeled DPID NPs and DMPID NPs (concentration 200 μg / mL) were added to each well. After 24 hours, the cells were fixed with paraformaldehyde and stained with phalloidin-FITC and DAPI for the cytoskeleton and nuclei, respectively. Images were then taken using an inverted fluorescence microscope.

[0100] Flow cytometry was performed after RAW264.7 cells in the lower chamber phagocytized DPID / DMPID NPs. After incubating RAW264.7 cells with DPID / DMPID NPs (at a concentration of 200 μg / mL) for 24 hours, the cells were dislodged using a pipette. The cells were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes, after which the supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0101] 2.9 Cellular uptake of PID NPs and MPID NPs

[0102] 2.9.1 Uptake of PID NPs and MPID NPs by HUVECs

[0103] HUVECs were plated at 2×10 4 HUVECs were seeded in 12-well plates and incubated for 12 hours at 37°C in a cell culture incubator with 5% CO2. HUVECs were treated with tumor necrosis factor TNF-α (final concentration 10 nM). DiD-labeled DPID NPs and DMPID NPs (concentration 75 μg / mL) were added to each well 12 hours later. Four hours later, the cells were fixed with paraformaldehyde and stained with phalloidin-FITC and DAPI for the cytoskeleton and nuclei, respectively. Images were then taken using an inverted fluorescence microscope.

[0104] HUVECs were incubated with DPID and DMPID NPs for 24 hours before the cells were digested and harvested in a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0105] 2.9.2 Uptake of PID NPs and MPID NPs by RAW 264.7 cells

[0106] RAW264.7 cells were plated at 2 × 10 4 Cells were seeded in 12-well plates and incubated for 24 hours in a cell culture incubator at 37°C and 5% CO2. RAW264.7 cells were treated with lipopolysaccharide (LPS) (final concentration 1 μg / mL). 24 hours later, DiD-containing nanoparticles (DPID NPs) and MPID NPs (75 μg / mL) were added to each well. Six hours later, the cells were fixed with paraformaldehyde and stained with phalloidin-FITC and DAPI for the cytoskeleton and nuclei, respectively. Images were then taken using an inverted fluorescence microscope.

[0107] RAW264.7 cells were subjected to flow cytometry after uptake of DPID and DMPID NPs. After incubation of DPID and DMPID NPs with RAW264.7 cells for 6 hours, the cells were dislodged using a pipette. The cells were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0108] 2.10 ROS removal capability experiment

[0109] 2.10.1 Experiment on the ability of PID NPs to scavenge ROS in HUVECs

[0110] HUVECs were plated at 2 × 10 per well. 4 Cells were seeded in 12-well plates and incubated for 12 hours at 37°C in a cell culture incubator with 5% CO2. HUVECs were treated with lipopolysaccharide (LPS) (final concentration 1 μg / mL) for 4 hours. PID NPs at different concentrations were added to each well and incubated for 24 hours. Cells and nuclei were stained using a reactive oxygen species detection kit and Hoechst 33342 staining solution, respectively, and then photographed using an inverted fluorescence microscope.

[0111] HUVECs were incubated with PID NPs for 24 hours before flow cytometry analysis. After 24 hours of incubation, the cells were digested and collected in a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0112] 2.10.2 Experimental study on the ability of PID, PID NPs and MPID NPs to scavenge ROS in HUVECs

[0113] HUVECs were plated at 2 × 10 per well. 4 HUVECs were seeded in 12-well plates and incubated for 12 hours at 37°C in a cell culture incubator with 5% CO2. Lipopolysaccharide (LPS) (final concentration 1 μg / mL) was then used to treat HUVECs. Four hours later, PID, PID NPs, and MPID NPs were added to each well. Twenty-four hours later, cells and nuclei were stained using a reactive oxygen species detection kit and Hoechst 33342 staining solution, respectively, and then photographed using an inverted fluorescence microscope.

[0114] HUVECs were incubated with PID, PID NPs, and MPID NPs for 24 hours before flow cytometry analysis. After 24 hours of incubation with PID, PID NPs, and MPID NPs, the cells were digested and collected in a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0115] 2.10.3 Experiment on the ability of PID NPs to scavenge ROS in RAW 264.7 cells

[0116] RAW264.7 was added at 2 × 10 per well. 4Cells were seeded in 12-well plates and incubated for 24 hours at 37°C in a cell culture incubator with 5% CO2. RAW264.7 cells were treated with lipopolysaccharide (LPS) (final concentration 10 μg / mL) and PIDNPs were added at different concentrations 24 hours later. Cells and nuclei were stained using a reactive oxygen species detection kit and Hoechst 33342 staining solution, respectively, and then observed and photographed under an inverted fluorescence microscope.

[0117] RAW 264.7 cells were incubated with PID NPs for 24 hours before flow cytometry analysis. After 24 hours of incubation, the cells were digested and collected in a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0118] 2.10.4 Experimental study on the ability of PID, PID NPs and MPID NPs to scavenge ROS in RAW 264.7 cells

[0119] RAW264.7 cells were plated at 2 × 10 4 RAW264.7 cells were seeded in a 12-well plate and incubated for 24 hours at 37°C in a cell culture incubator with 5% CO2. Lipopolysaccharide (LPS) (final concentration 10 μg / mL) was then added to each well 24 hours later. PID, PID NPs, and MPID NPs were then added to each well. Cells and nuclei were stained using a reactive oxygen species detection kit and Hoechst 33342 staining solution, respectively, and then observed and photographed under an inverted fluorescence microscope.

[0120] RAW264.7 cells were incubated with PID, PID NPs, and MPID NPs for 24 hours before flow cytometry analysis. After incubation of PID, PID NPs, and MPID NPs with RAW 264.7 cells for 24 hours, the cells were collected by pipetting into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0121] 2.11 Uptake of PID NPs and MPID NPs by HUVECs

[0122] HUVECs were plated at 2 × 10 per well. 4HUVECs were seeded in 12-well plates and incubated for 12 hours at 37°C in a cell culture incubator with 5% CO2. Tumor necrosis factor (TNF-α) (final concentration 50 nM) was used to treat HUVECs. DiD-labeled DPIDNPs and DMPID NPs (concentration 200 μg / mL) were added 12 hours later. After 24 hours, the cells were fixed with paraformaldehyde and stained with phalloidin-FITC and DAPI for the cytoskeleton and nuclei, respectively. Images were then taken using an inverted fluorescence microscope.

[0123] HUVECs were incubated with DPID and DMPID NPs for 24 hours before digestion and collection of cells in a centrifuge tube. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were then resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0124] 2.12 Detection of IL-1β and TNF-α in the supernatant of RAW 264.7 cells by enzyme-linked immunosorbent assay

[0125] RAW 264.7 was added at 3 × 10 per well. 4 The number of cells was inoculated into a 6-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours. The cells were treated with ATP and LPS (ATP concentration was 5 mmol / L, LPS concentration was 0.5 μg / mL). After 12 hours, PID, PID NPs and MPID NPs (concentration was 100 μg / mL) were added to each well. After 24 hours, the culture medium in the well plate was collected into the corresponding 15 mL centrifuge tube and centrifuged at 5000 rpm for 10 minutes. After centrifugation, the supernatant in the corresponding centrifuge tube was collected and the IL-1β and TNF-α content in the culture supernatant was detected using an ELISA kit.

[0126] 2.13 Ability of PID, PID NPs, and MPID NPs to inhibit pyroptosis in RAW 264.7 cells

[0127] RAW264.7 cells were plated at 1×10 4 Cells were seeded in 12-well plates and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. Cells were treated with ATP and LPS (ATP concentration was 5 mmol / L, LPS concentration was 0.5 μg / mL). 12 hours later, PID, PID NPs, and MPID NPs (concentration was 100 μg / mL) were added to each well. After 24 hours, cells were stained with Calcein AM and PI staining reagents and then observed and photographed under an inverted fluorescence microscope.

[0128] RAW264.7 cells were incubated with PID, PID NPs, and MPID NPs for 24 hours before flow cytometry analysis. After incubation of PID, PID NPs, and MPID NPs with RAW 264.7 cells for 24 hours, the cells were collected by pipetting into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed. The cells were resuspended in 600 μL of 1× PBS and analyzed by flow cytometry.

[0129] Lysosomal escape of PID NPs and MPID NPs in RAW 264.7 cells

[0130] RAW264.7 cells were plated at 1×10 4 Cells were seeded in confocal dishes and incubated in a cell culture incubator at 37°C and 5% CO2. After 24 hours, DiD-labeled DPID NPs and DMPID NPs (at a concentration of 200 μg / mL) were added to each well. After incubation for 2 hours and 6 hours, respectively, lysosomes and cell nuclei were stained using a lysosomal probe and Hoechst 33342 staining solution, and then observed and photographed under a confocal microscope.

[0131] 2.15 CCK-8 experiment

[0132] 2.15.1 CCK-8 assay to detect the ability of PID NPs to inhibit RAW 264.7 cell death

[0133] RAW264.7 cells were plated at 1×10 4 Each well was seeded in a 96-well plate and incubated for 24 hours in a cell culture incubator at 37°C and 5% CO2. RAW264.7 cells were treated with cholesterol (final concentration 20 μg / mL). After 24 hours, various concentrations of PID NPs were added to the culture medium. Three hours later, 100 μL of CCK-8 solution was added to each well. After 1-2 hours, the absorbance at 450 nm was measured using a microplate reader.

[0134] 2.15.2 Detection of the Anti-cell Death Ability of MPID NPs in RAW 264.7 Cells Using CCK-8

[0135] RAW264.7 cells were plated at 1×10 4 Each well was seeded in a 96-well plate and incubated for 24 hours at 37°C in a cell culture incubator with 5% CO2. RAW264.7 cells were treated with cholesterol (final concentration 20 μg / mL). After 24 hours, various concentrations of MPID NPs were added to the culture medium. Three hours later, 100 μL of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured using a microplate reader 1-2 hours later.

[0136] 3 Results

[0137] 3.1 Preparation, characterization, and optimization of Pid NPs

[0138] This study aims to construct a biomimetic nanodrug MPID NPs, which can achieve the purpose of treating AS by targeting and inhibiting the occurrence of macrophage pyroptosis in AS lesions. Oxidative stress in the AS microenvironment is the main cause of its pathogenicity. At the same time, reactive oxygen species (ROS) in oxidative stress have a positive regulatory effect on the formation of GSDMD-N pores. In previous studies, our team successfully connected probucol (Pu) and idibenzoquinone (ID) to form a small molecule prodrug system (Pu-ID) through an oxalate bond with ROS-responsive bond breaking. We used carbon nuclear magnetic resonance spectroscopy, hydrogen nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry to verify the small molecule prodrug Pu-ID, and the results all proved that the small molecule prodrug Pu-ID was successfully prepared. In addition, Pu, ID and Pu-ID were subjected to in vitro RAW264.7 cytotoxicity tests. The results showed that neither Pu, ID nor the two connected with an oxalate bond would cause cytotoxicity to RAW264.7, as shown in the following figure. Figure 1 shown.

[0139] Building on the successful preparation of the small molecule prodrug Pu-ID, we prepared Pid NPs using different Pu-ID to DSF ratios. We prepared the nanoparticles using Pu-ID to DSF mass ratios of 5:4, 5:2, and 5:1. Laser irradiation was then performed on aqueous solutions of the prepared nanoparticles to observe the Tyndall effect. The three nanoparticle solutions were also tested for their hydrodynamic diameters, zeta potential, and stability. Results showed that the nanoparticle solutions prepared at 5:1 and 5:2 ratios exhibited a strong Tyndall effect upon laser irradiation. However, the nanoparticle solution prepared at a 5:4 ratio exhibited no Tyndall effect upon laser irradiation. Measurements of the hydrodynamic diameters, zeta potential, and stability of the three nanoparticle solutions revealed that the particle sizes of the three solutions were similar, at 141.74 nm, 135.063 nm, and 156.51 nm, respectively. The zeta potential and stability of the three nanoparticle solutions were also comparable. The zeta potentials were -25.3467 mV, -25.2833 mV, and -23.85 mV, respectively, and the stabilities were 0.0913, 0.119, and 0.065, respectively. Based on these results, the three nanoparticle aqueous solutions were stored at room temperature and their particle size and stability were measured continuously over 9 days. We found that the particle size and stability of all three nanoparticle aqueous solutions increased upon storage at room temperature, but the particle size and stability of the nanoparticle aqueous solutions prepared at the 5:2 and 5:4 ratios showed an upward and then downward trend. We speculate that this phenomenon is due to the loss of DSF from the Pid NPs upon storage at room temperature. Furthermore, small particles clearly precipitated from the walls of the centrifuge tube containing the nanoparticle aqueous solution prepared at the 5:4 ratio. In comparison, the nanoparticle aqueous solution prepared at the 5:1 ratio showed relatively stable kinetic diameter and stability, and the nanoparticle solution in the centrifuge tube remained clear even after 9 days at room temperature. Therefore, we decided to use a Pu-ID to DSF mass ratio of 5:1 to prepare nanoparticles for subsequent experiments. Figure 2 (ah) shown.

[0140] Preparation and characterization of PID NPs and MPID NPs. We prepared PID NPs and MPID NPs by adding DSPE-PEG and DSPE-PEG-Mannose to a Pu-ID to DSF mass ratio of 5:1. DSPE-PEG can help improve stability while enhancing the long-term circulation ability of nanoparticles. The addition of mannose can further enhance the ability of nanoparticles to target plaques in atherosclerotic lesions. We used transmission electron microscopy (TEM) to analyze the two nanoparticles, PID NPs and MPID NPs, and found that both nanoparticles had spherical morphology. We also used DLS to characterize the water and kinetic diameters, surface potential, and stability of PID NPs and MPID NPs. The results were consistent with the TEM results. The particle sizes of PID NPs and MPID NPs were 141.74 nm and 91.09 nm, respectively. The Zeta potential and PDI of PID NPs are -25.3467mV and 0.0913, respectively, and the Zeta potential and PDI of MPID NPs are -24.6233mV and 0.153, respectively. In order to verify the stability of the two nanoparticles, we added the prepared nanoparticles into culture medium containing 10% fetal bovine serum, 1×PBS and ultrapure water, respectively. After being placed at room temperature for 9 days, their water and kinetic diameters and stability were measured to observe their changes. The results showed that the particle size of MPID NPs and PID NPs in 1×PBS aqueous solution were significantly larger than the particle size of nanoparticles in other aqueous solutions on the first day. This may be due to the presence of sodium chloride and other factors in the 1×PBS solution, which affected the particle size of the nanoparticles. However, based on the overall observation within 9 days, the changes in water and kinetic diameters and stability of MPID NPs and PID NPs were relatively stable, as shown in the following results. Figure 2 (iq) shown.

[0141] Functional evaluation of MPID NPs on HUVECs

[0142] After systematically characterizing MPID NPs, we evaluated the intracellular delivery of MPID NPs. First, RAW264.7 cells and human umbilical vein endothelial cells (HUVECs) were selected, and the biosafety of the nanoparticles was evaluated using the Cell Counting Kit-8 reagent (CCK-8 method). After different concentrations of PID NPs and MPID NPs were incubated with RAW 264.7 cells and HUVECs, respectively, the cell viability remained above 80%, indicating that they have good biosafety. Next, in order to further evaluate the effects of several nanoparticles on cell viability, we selected PID (Pu-ID+DSF), PID NPs and MPID NPs at the same concentration (100 μg / mL) and incubated them with RAW 264.7 cells and HUVECs at the same time. The results showed that the cell viability remained above 80%, and cell proliferation was promoted. In addition, no obvious hemolysis occurred in the in vitro hemolysis experiment, as shown in the following results. Figure 3 shown.

[0143] In order to clarify the uptake of nanoparticles by HUVECs, we used the red fluorescent probe DiD to label the nanoparticles, used FITC-phalloidin to stain and locate the cytoskeleton, and used blue 4', 6-diamino-2-phenylindole (DAPI) to stain and locate the cell nucleus. HUVECs incubated with nanoparticles for 4 hours were photographed using a confocal fluorescence microscope. The results showed that compared with untreated HUVECs, HUVECs treated with TNF-α showed an increase in intracellular red fluorescence after incubation with nanoparticles. At the same time, regardless of whether HUVECs were treated with TNF-α, their uptake of MPIDNPs was better than that of PID NPs. Flow cytometry results also showed that HUVECs' uptake of MPID NPs was significantly better than that of PID NPs, as shown in the following table. Figure 4 As shown in (ac). At the same time, compared with HUVECs in normal conditions, HUVECs treated with TNF-α will further increase the uptake of nanoparticles. After determining the uptake of nanoparticles by HUVECs, we still used the red fluorescent probe DiD to label the nanoparticles and used blue 4', 6-diamino-2-phenylindole (DAPI) to stain and locate the cell nucleus, and used lysosomal dye (Lysotracker) to locate the lysosomes in the cells. Confocal fluorescence microscopy results showed that when HUVECs were incubated with nanoparticles for 6 hours, the number of nanoparticles reaching the cytoplasm was significantly higher than when they were incubated for 2 hours. This microscopic result shows that nanoparticles can successfully escape from lysosomes in HUVECs and then reach the cytoplasm to exert their effects, thereby preventing the nanoparticles from being degraded by various digestive enzymes in the lysosomes. Figure 5shown.

[0144] We constructed an in vitro Transwell model to simulate the situation after nanoparticles enter the blood vessels. The fluorescence intensity in the culture medium of macrophages in the lower chamber of Transwell was detected using a fluorescence microplate reader and found that the fluorescence intensity in the culture medium of the MPID NPs group was higher than that of the PID NPs group. Next, we used the red fluorescent probe DiD to label the nanoparticles, used FITC-phalloidin to stain and locate the cytoskeleton, and used blue 4', 6-diamino-2-phenylindole (DAPI) to stain and locate the cell nucleus. The fluorescence intensity in RAW 264.7 cells in the lower chamber of Tramswell was detected using a confocal fluorescence microscope. The results showed that the red fluorescence in the cells of the MPID NPs group in the lower chamber was significantly higher than that in the cells of the PID NPs group. Flow cytometry also further verified this result. The results are as follows Figure 4 (dh) shown.

[0145] After confirming that the nanoparticles can enter HUVECs and can escape lysosomes, we want to test the ability of nanoparticles to scavenge ROS in HUVECs. We used DCFH-DA to stain and locate ROS in cells and used blue Hoechst33342 dye to stain and locate the cell nucleus. The results of confocal fluorescence microscopy showed that PID NPs at a concentration of 200μg / mL had the best effect on clearing ROS in HUVECs. Flow cytometry also indirectly verified this result. Therefore, we selected different dosing groups with a concentration of 200μg / mL to treat HUVECs. The confocal fluorescence microscopy results showed that compared with the Model group, the ROS green fluorescence in the drug-treated group cells was reduced, and the MPID NPs-dosing group was better than the PID and PID NPs-dosing groups. The flow cytometry results also showed that the MPID NPs group had the best inhibitory effect on ROS in HUVECs. The results are as follows. Figure 6 shown.

[0146] Functional evaluation of 3.3MPID NPs on RAW 264.7 cells

[0147] After evaluating the functional effects of MPID NPs on HUVECs, we further investigated their effects on the phagocytic capacity of RAW264.7 cells. We labeled the nanoparticles with the red fluorescent probe DiD, stained the cytoskeleton with FITC-phalloidin, and stained the cell nuclei with blue 4',6-diamino-2-phenylindole (DAPI). Confocal fluorescence microscopy of RAW264.7 cells incubated with the nanoparticles for 6 hours revealed that LPS-treated RAW264.7 cells phagocytosed MPID NPs more than those treated with PID NPs. Confocal microscopy revealed no significant difference in the phagocytic capacity of untreated cells for PID and MPID NPs. Furthermore, LPS-treated RAW264.7 cells exhibited a further enhanced phagocytic capacity for nanoparticles. Flow cytometry revealed that both LPS-treated and untreated RAW264.7 cells exhibited a superior phagocytic capacity for MPID NPs compared to PID NPs. Moreover, RAW264.7 cells treated with LPS showed more obvious phagocytic behavior towards MPID NPs compared with untreated cells. Figure 7 (ac) shown.

[0148] In order to clarify whether the nanoparticles can escape from the lysosomes and reach the cytoplasm after being engulfed by RAW264.7 cells to exert their effects, we used the red fluorescent probe DiD to label the nanoparticles and used blue 4', 6-diamino-2-phenylindole (DAPI) to stain and locate the cell nuclei. At the same time, we used lysosomal dye (Lysotracker) to locate the lysosomes in the cells. The results of confocal fluorescence microscopy showed that when the nanoparticles were co-incubated with the cells for 2 hours, the red fluorescence content in the cells in the MPID NPs co-incubation group was significantly higher than that in the PID NPs administration group. When the co-incubation time reached 6 hours, the red fluorescence in the cells in the MPID NPs co-incubation group was significantly higher than that in the cells when the MPID NPs were co-incubated for 2 hours. The results are as follows. Figure 7 (d) shown.

[0149] After confirming that MPID NPs can achieve lysosomal escape, we then tested their ability to scavenge ROS in RAW 264.7 cells. We used DCFH-DA to stain and localize ROS in cells, while also using blue Hoechst 33342 dye to stain and localize cell nuclei. Confocal fluorescence microscopy revealed no significant difference in green fluorescence levels between RAW 264.7 cells treated with PID NPs at concentrations of 100 μg / mL and 200 μg / mL for 24 hours. Flow cytometry also confirmed this finding, showing no significant difference in ROS elimination between PID NPs at concentrations of 100 μg / mL and 200 μg / mL. Therefore, we selected RAW 264.7 cells treated with different concentrations of 100 μg / mL to assess their ROS clearance. Confocal fluorescence microscopy revealed that the MPID NPs group exhibited the greatest ROS clearance. Flow cytometry results also verified that the ROS content in the cells of the MPID NPs-treated group was significantly lower than that in the PID and PID NPs-treated groups. Figure 7 (ej) shown.

[0150] 3.4MPID NPs inhibit apoptosis of RAW 264.7 cells

[0151] To further explore the function of MPID NPs on RAW264.7 cells, we treated RAW264.7 cells with cholesterol to induce apoptosis in RAW264.7 cells, and then added different concentrations of PID NPs and MPID NPs to the cells. The CCK-8 method was used to detect the effect of the two nanoparticles at different concentrations on inhibiting apoptosis of RAW264.7 cells. The results showed that the PID NPs group had the best inhibition of RAW 264.7 cell apoptosis at 75 μg / mL. The inhibition of RAW264.7 cell apoptosis in the MPID NPs group was detected, and it was found that 100 μg / mL MPID NPs inhibited RAW274.7 cell apoptosis significantly better than the 75 μg / mL concentration. Based on the above results, we treated RAW264.7 cells after cholesterol treatment with 75, 100 and 200 μg / mL PID NPs, respectively. The treated cells were stained with Annexin V-FITC / PI double staining, and the results of fluorescence microscopy showed that the red fluorescence content after PI staining in RAW 264.7 cells was reduced after treatment with three concentrations of MPID NPs. The results of flow cytometry further showed that 75μg / mL and 100μg / mL were significantly better than 200μg / mL in inhibiting apoptosis of RAW264.7 cells after cholesterol treatment, and there was no significant difference. Therefore, we selected PID, PID NPs and MPID NPs at a concentration of 75μg / mL to treat RAW264.7 cells treated with cholesterol. The results of fluorescence microscopy showed that the red fluorescence content after PI staining in RAW264.7 cells after treatment with each drug group was reduced after staining with Annexin V-FITC / PI double staining. The results of flow cytometry further confirmed that MPIDNPs had the best effect on inhibiting apoptosis of RAW264.7. The results are as follows Figure 8 (af) shown.

[0152] 3.5MPID NPs reduce GSDMD-N content in RAW 264.7 cells

[0153] After investigating the inhibitory effect of MPID NPs on RAW264.7 cell apoptosis, we further investigated the effects of nanoparticles on pyroptosis in RAW264.7 cells. We treated RAW264.7 cells with ATP+LPS to induce pyroptosis. We first used the CCK-8 assay to assess the effects of different concentrations of PID NPs on the viability of ATP+LPS-treated RAW264.7 cells. We found that 100 μg / mL appeared to be a cutoff point; this concentration did not significantly affect the viability of treated RAW264.7 cells compared to higher concentrations. Therefore, we selected 100 μg / mL as the treatment concentration and added PID, PID NPs, and MPID NPs to ATP+LPS-treated RAW264.7 cells. Immunofluorescence staining of GSDMD-N and CD68 was performed in RAW264.7 cells treated with different drug groups, and DAPI was used to stain the nuclei of RAW264.7 cells. Confocal fluorescence microscopy of stained RAW 264.7 cells revealed a significant decrease in GSDMD-N green fluorescence in RAW 264.7 cells treated with MPID NPs, indicating that MPID NPs can reduce GSDMD-N production. Next, immunofluorescence staining of GSDMD-FL and CD68 was performed in RAW 264.7 cells treated with different dosing groups. DAPI was also used to stain the nuclei of RAW 264.7 cells. Confocal fluorescence microscopy revealed an increase in GSDMD-FL green fluorescence in RAW 264.7 cells following MPID NP treatment. We hypothesize that DSF inhibits pyroptosis in RAW 264.7 cells only by inhibiting the formation of GSDMD-N plasma membrane pores after GSDMD-FL cleavage, resulting in reduced GSDMD-FL cleavage and, consequently, decreased GSDMD-N formation and increased GSDMD-FL. We also used enzyme-linked immunosorbent assay to detect the supernatant of RAW264.7 cells after treatment with different drug groups and found that the levels of TNF-α and IL-1β in the supernatant of the drug groups were reduced. Figure 8 (gk) shown.

[0154] 3.6 In vivo evaluation of the efficacy of MPID NPs in the treatment of AS

[0155] Next, we used ApoE - / -The efficacy of MPIDNPs in treating AS was evaluated in vivo in male mice. Mice were randomly divided into saline, PID, PID NPs, and MPID NPs groups. Each group received injections of drug every Tuesday and Friday for seven weeks, totaling fourteen injections. After treatment, the entire aorta was harvested and stained with Oil Red O to assess atherosclerotic plaque growth. Compared with the model group, MPID NPs significantly reduced plaque content. Next, the mouse hearts were harvested, and the tricuspid valve was sectioned and stained with ORO, TB, and Masson staining, and the stained sections were quantified. Quantitative results showed that lipid accumulation, collagen remodeling, and fibrosis were reduced in the MPID NPs-treated group, with the greatest effect observed in the drug-treated group. MPID NPs treatment increased plaque stability and slowed plaque progression in mice with atherosclerosis. To further investigate the effects of MPID NPs on inflammatory cytokines in this mouse model, sections of the tricuspid valve were stained with CD68, IL-1β, and TNF-α, and the staining results were quantified. The results showed that after drug treatment, the expression of CD 68, IL-1β and TNF-α in the tricuspid valve of mice decreased, and the expression of CD68 and IL-1β in the sections of mice in the MPID NPs treatment group was significantly lower than that in the PID and PID NPs treatment groups. The expression of TNF-α in the MPID NPs treatment group was significantly higher than that in the PID treatment group, but there was no significant difference between PID NPs and MPID NPs. Figure 9 To further explore the mechanism of MPID NPs, we used transcriptome RNA sequencing to assess gene expression. Gene volcano plots and heat maps confirmed that MPID NPs altered the expression of many genes.

[0156] 3.7 In vivo safety evaluation of MPID NPs

[0157] In order to evaluate the safety of MPID NPs, we conducted a comprehensive evaluation. The complete blood count test showed that there were no significant changes in the levels of hemoglobin (Hb), platelets (Plt), white blood cells (WBC), and red blood cells (RBC). Histological examination of major organs (heart, liver, spleen, lungs, and kidneys) by HE staining revealed no significant changes or damages with different treatments. Blood biochemical analysis showed that representative indicators including urea (UREA), aspartate aminotransferase (AST), creatinine (CREA), alanine aminotransferase (ALT), triglycerides (TG), cholesterol (CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) did not change significantly, indicating that there was no obvious liver or kidney toxicity. These results collectively demonstrate that MPID NPs have good biosafety, as shown in the following examples. Figure 10 shown.

[0158] 4 Analysis

[0159] This study aimed to construct a novel small-molecule prodrug-coassembled nanodrug that specifically inhibits pyroptosis in macrophages at AS lesions, thereby achieving efficient, targeted, and safe treatment of AS. Specifically, Pu and ID were linked via an oxalate bond that responds to ROS cleavage to synthesize the ROS-responsive heterodimeric small-molecule nanoprodrug, Pu-ox-ID. Furthermore, Pu-ox-ID was coassembled with DSF to form small-molecule nanoprodrugs (PIDNPs). To achieve macrophage-targeted function at AS lesions, the nanodrug was functionalized with DSPE-PEG-mannose. The resulting nanodrugs (MPIDNPs) efficiently targeted and accumulated at AS lesions and were taken up by inflammatory macrophages in the area. Upon entry into inflammatory macrophages, the MPIDNPs, under the influence of high intracellular ROS levels, released Pu, ID, and DSF. Pu and ID effectively inhibited ROS production, thereby synergistically inhibiting GSDMD-NT pore formation and suppressing macrophage pyroptosis. In summary, this nanomedicine can effectively inhibit the occurrence of macrophage pyroptosis in AS lesions, thereby effectively inhibiting the occurrence and development of AS. This study provides a new idea and treatment strategy for the efficient targeted treatment of AS.

Claims

1. A method for preparing a small molecule prodrug co-assembled nanomedicine for targeted inhibition of macrophage pyroptosis, characterized in that: The steps include: S1. Preparation of Pu-ID prodrug: S1.

1. Add idebenone to organic solvent A to obtain an idebenone solution, then add oxalyl chloride dropwise to the idebenone solution and allow to react at 0-4°C. After the reaction is complete, drain the remaining reaction liquid to obtain an intermediate product, which is then dissolved in organic solvent A to obtain an intermediate product solution. S1.

2. Dissolve 4-dimethylaminopyridine (DMAP), triethylamine, and probucol in organic solvent A to obtain a mixed solution. Add the mixed solution dropwise to the intermediate product solution at 0-4°C. After addition, move the reaction flask to 20-25°C for full reaction. After the reaction is complete, add a quencher to quench the reaction. Extract the mixture with organic solvent A and elute the extract with an eluent to obtain the Pu-ID prodrug. S2. Preparation of MPID NPs: Pu-ID prodrug, disulfiram, DSPE-PEG-Mannose and DSPE-PEG-COOH were weighed and fully dissolved in organic solvent B, and the obtained mixed solution was dropped into pure water under stirring to form an MPID NPs solution; the obtained MPID NPs solution was then transferred to a dialysis bag for dialysis; after the dialysis was completed, the MPID NPs solution in the dialysis bag was collected to obtain the small molecule prodrug co-assembled nanodrug.

2. The preparation method according to claim 1, wherein: In step S1.1, the reaction is carried out at 0-4°C for 3-6 hours, the molar ratio of idebenone to oxalyl chloride is 1:4-6, and the idebenone solution is preferably prepared at a ratio of 1 mmol of idebenone to 8-12 ml of organic solvent A; preferably, the intermediate product prepared by 1 mmol of idebenone is dissolved in 8-12 ml of organic solvent A to prepare the intermediate product solution; The organic solvent A is selected from dichloromethane.

3. The preparation method according to claim 1, wherein: In step S1.2, the temperature is moved to 20-25° C. and the reaction is carried out for 10-16 hours. The quencher is pure water; the eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 14-16:1; The molar ratio of DMAP, triethylamine and probucol is 0.15-0.25:1.0-1.2:1, and the usage ratio of probucol to organic solvent A is 1 mmol:4-6 ml.

4. The preparation method according to claim 1, wherein: In step S2, the mass ratio of the Pu-ID prodrug, disulfiram, DSPE-PEG-Mannose, and DSPE-PEG-COOH is (4-6):1:(220-280):(220-280), and the organic solvent B is DMF or DMSO; the molecular weight of PEG of DSPE-PEG-Mannose and DSPE-PEG-COOH is 1000-3000, preferably 1500-2500; The mass volume ratio of the disulfiram to the organic solvent B is (1-2):(0.8-1.2).

5. The preparation method according to claim 1, wherein: The molecular weight cut-off (MWCO) of the dialysis bag is 3000-4000 Da, preferably 3500 Da.

6. The preparation method according to claim 1, wherein: In step S2, the obtained mixed solution is added dropwise into pure water under stirring, and the volume ratio of the mixed solution to pure water is 1:(3-5); The obtained MPID NPs solution was transferred to a dialysis bag and dialyzed in ultrapure water for 4 to 14 hours.

7. A small molecule prodrug co-assembled nanomedicine for targeted inhibition of macrophage pyroptosis, characterized by: The method according to any one of claims 1 to 6 is used for preparation.

8. Use of the small molecule prodrug co-assembled nanomedicine for the targeted inhibition of macrophage pyroptosis according to claim 7 in the preparation of a drug for treating atherosclerosis.

9. The use according to claim 8, characterized in that: The small molecule prodrug co-assembled nanomedicine targets and inhibits macrophage pyroptosis in atherosclerotic lesions.

10. The use according to claim 9, characterized in that: The small molecule prodrug co-assembled nanodrug aggregates to the site of atherosclerotic lesions and is taken up by inflammatory macrophages in the area. Under high intracellular ROS levels, Pu, ID and disulfiram drugs are released. Pu and ID can effectively inhibit the generation of ROS and synergistically inhibit GSDMD-NT pore formation together with DSF, inhibit the occurrence of macrophage pyroptosis, and inhibit the occurrence and development of atherosclerosis.

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