Introduction

Atherosclerosis (AS) as a potential risk of myocardial infarction and stroke is mainly characterized by thickening of the arterial wall due to fibrous plaque. The progression of AS is a complex process that includes factors such as inflammation and lipid accumulation. When the lipid load in the monocyte-infiltrated subendothelial muscle layer is inadequately processed under the atherosclerotic lesion, overwhelming cholesterol deposition induces macrophages and smooth muscle cells (SMCs) to become pathological foam cells while forming fatty streaks [1, 2]. Subsequently, fibrous plaques are gradually formed, reducing the arterial lumen area. Cholesterol crystal (CCs) deposition played an important role during the course of atherosclerosis [3]. Some studies have shown that CCs initially arise and accumulate in the chocolate-cell lysosomes and then are gradually deposited in atherosclerotic lesions with apoptosis of the chocolate-cells [4, 5]. CCs induces the secretion of inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α), which in turn enhances monocyte infiltration and induces apoptosis in foam cells, leading to chronic inflammation [6,7,8].

It is well-known that only the free cholesterol can be further metabolized and intracellularly efflux by cholesterol transport proteins in lysosome, such as Niemann-Pick C-type associated protein (NPC) 1 or 2. As soon as free cholesterol has been converted to CCs, the CCs cannot be trafficked via NPC-1 or NPC-2, which results in over-loading of CCs in lysosome and consequent apoptosis [9]. Thus, preventing CCs formation or cleavage CCs from lysosomes is a widely used strategy in the clinical treatment of AS. Because of the unique molecular structure of β-Cyclodextrin (β-CD) can efficient loading or enhance cholesterol solubility through “host–guest” interactions, i.e., the β-CD lumen closely matches the cholesterol molecule [10]. Therefore, β-CD is considered the potentially effective therapeutic agent to enhance cholesterol efflux for the promising AS treatment.

Methotrexate (MTX) is an anti-inflammatory drug for the treatment of diseases such as rheumatoid arthritis and psoriatic arthritis. Recently, researchers have suggested that MTX could be effective in treating the chronic inflammatory disease AS [11, 12]. MTX has been proven to inhibit foam cell formation by MTX-induced upregulation of scavenger receptor B1 (SR-B1), ATP binding cassette transporter A1 (ABCA1) and cholesterol 27-hydroxylase (CYP27A1), which efficiently induces the cholesterol efflux [11, 13]. However, further usage of the hydrophobic MTX is greatly hindered because of the low solubility induced by the low bioavailability and the serious adverse effects in the blood [12]. Nanotechnology offers a pathway for the systematic delivery of multiple drugs. Compared to free drugs, nanomedicines can significantly increase their efficacy and reduce side effects [14,15,16,In vivo atherosclerotic plaques targeting and long-term circulation test

ApoE−/− mice were fed a high-fat diet (HFD) for 2 months. DiD NPs and MM@DiD NPs were administered via the tail vein at DiD dose of 2 mg/kg. After 24 h, the mice were euthanized and perfused with precooled PBS containing 4% PFA. Each aorta and the main organs were isolated for imaging using a Xenogen IVIS 200 system (Xenogen, IVIS 200, USA).

C57BL/6 mice weighing 25 ± 2 g were subjected to in vivo long-term cycling tests. Briefly, DiD NPs and MM@DiD NPs (200 μL, 2 mg/mL) were injected intravenously and 30 μL of blood was collected rapidly from the tail after 1 min, 1, 6, 12, 24 and 48 h. Blood samples were diluted with 30 μL of PBS containing EDTA-K2 in a 96-well plate and fluorescence intensity was measured using a microplate apparatus (Tecan, M1000 PRO, USA).

Treatment of atherosclerosis in ApoE−/− mice

ApoE−/− mice after 8 weeks of HFD feeding were randomized into 4 groups (5 mice per group) and dosed for 30 days by tail vein injection every three days. In the treatment groups, mice were administered PBS, free MTX, MTX NPs, or MM@MTX NPs at a dose of 5 mg/kg of MTX.

At the end stage of the treatment, the ApoE−/− mice were euthanized. The pathological evolution was assessed by measuring the lesion area of atherosclerotic plaques in the aorta from the heart to the iliac bifurcation. Briefly, each aorta was fixed with PFA (4% PBS) for 1 h. After washing the peripheral tissue, the aorta was opened longitudinally and the whole aorta was then stained with ORO to quantify the plaque area. The extent of atherosclerotic plaque in the aortic root was determined in the same way. Atherosclerotic plaque areas were quantified using Adobe Photoshop 2021 software.

Aortic root histology and immunohistochemical staining: Aortic roots were fixed in 4% PFA for 1 h. Frozen sections were prepared and stained with toluidine blue to quantify the necrotic core. For analysis of cholesterol and cholesteryl esters, sections were placed in a solution of ferric ammonium sulphate and reacted at room temperature for 3 days. An acetic acid-sulphate mixture was added and subsequently observed under the microscope. For immunohistochemistry, sections were immersed in 3% hydrogen peroxide and 100% methanol for 20 min to inhibit endogenous peroxidase activity, followed by blocking with 1% bovine serum albumin in PBS containing 0.3% Triton X-100 for 60 min. CD68, F4/80, ABCA1, SR-B1, CYP27A1, IL-1β, TNF-α and IFN-β antibodies were co-incubated for macrophage quantification. Histological and immunohistochemical quantification was performed using Adobe Photoshop 2020 software. Hematoxylin–eosin (H&E) staining was used to analyse sections of major organs.

Complete blood routine analysis and serum biochemistry analysis: Blood was collected and analyzed using an automated hematology analyzer (Sysmex Co., Sysmex KX-21, Japan) after treatment.

Statistical analysis

The data obtained are reported as the mean ± SD in this study. GraphPad Prism Version 7.0 software (GraphPad Software, GraphPad Prism 7.0, USA) was used for the statistical analysis. One-way or two-way analysis of variance (ANOVA) by Tukey’s test was used to reveal differences between the groups. The difference significance levels were set at *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. n.s., no significance.