Macrophage-targeting Antisenescence nanomedicine enables in-Situ NO induction for Gaseous and antioxidative atherosclerosis intervention

Abstract

Senescent-endothelial cells significantly accelerate atherosclerosis progression, making the mitigation of cellular aging a promising strategy for treating the disease. Nitric oxide (NO), a low molecular weight and lipophilic gas, has been shown to penetrate cell membranes effectively and delay cell senescence. In this study, we designed and engineered osteopontin (OPN)-modified nanoliposomes (CZALO) that encapsulate L-arginine (L-Arg) and cerium-zirconium oxide nanoparticles (CZ NPs), which exhibit enzyme-like activities for targeted atherosclerosis treatment. Following inflammatory chemotaxis and OPN-mediated internalization by macrophages, CZ NPs released from CZALO nanoliposomes significantly scavenge reactive oxygen species, thereby inhibiting cholesterol uptake and promoting macrophage phenotypic transformation, resulting in both antioxidant and anti-inflammatory effects. Additionally, nitric oxide synthase (NOS) overexpressed in macrophages catalyzes L-Arg to produce NO, which is then selectively released in situ and diffuses into endothelial cells, exerting anti-aging effects by regulating senescence-associated secretory phenotype factor secretion, enhancing lysosomal function, alleviating cell cycle arrest, and reducing DNA damage. The antioxidant and anti-aging effects of CZALO nanoliposomes collectively alleviate atherosclerotic burden with minimal toxicity both in vitro and in vivo. This “two-birds-one-stone” nanotherapeutic offers a novel approach for regulating vascular microenvironment homeostasis and improving therapeutic efficiency in atherosclerosis treatment.

Graphical abstract

Highlights

• •The engineered CZALO nanomedicines scavenged ROS, inhibited cholesterol uptake, and promoted macrophage polarization from M1 to M2, inducing strong antioxidant and anti-inflammatory effects.
• •The L-arginine was selectively catalyzed by nitric oxide synthase in macrophages to release nitric oxide into endothelial cells, exerting favorable anti-senescent function.
• •The engineered CZALO nanomedicines demonstrated significant efficacy in reducing plaque formation in vivo through synergistic anti-inflammatory and anti-aging effects.

1. Introduction

Atherosclerosis is the primary cause of peripheral vascular disease, coronary heart disease, and stroke, presenting a major threat to human health [1,2]. Its pathogenesis involves a variety of risk factors and complex mechanisms, including dyslipidemia, inflammation, oxidative stress, and endothelial dysfunction [3,4]. The vascular microenvironment of atherosclerotic plaques consists mainly of endothelial cells, macrophages, smooth muscle cells, and extracellular matrix. Macrophages absorb lipids and become foam cells, driving inflammation and plaque instability [5,6]. Regrettably, numerous exploiting single anti-inflammatory agents have demonstrated only modest therapeutic effects in preventing and treating atherosclerosis (Table S1). Consequently, combination therapy has emerged as a compelling strategy, effectively addressing the complex interplay among pathogenic mechanisms and yielding more favorable therapeutic outcomes.

Endothelial dysfunction is regarded as the initial step in atherosclerosis, with most cases associated with the aging and death of endothelial cells [7,8]. Generally, aging endothelial cells contribute to the progression of atherosclerosis by disrupting barrier function, resulting in the infiltration of low-density lipoprotein cholesterol and immune cells [9,10]. Furthermore, aging endothelial cells enhance their capacity to attract white blood cells by upregulating the expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), thereby perpetuating a chronic inflammatory environment within the atherosclerotic wall [11,12]. Cellular aging refers to a permanent state of arrested development, characterized by changes in the composition of secretory proteins, reshaping of neighboring cells, and alterations in the extracellular matrix microenvironment [13,14].

To date, the commonly used anti-senescence drugs in clinical practice include metformin, rapamycin, and NAD⁺ precursors [15]. However, these drugs still face numerous challenges in clinical applications, particularly regarding their safety, long-term efficacy, and potential side effects. Therefore, there is an urgent need to develop new strategies for intervening in the aging process of endothelial cells. It was reported that the bioavailability of NO within endothelial cells diminishes as individuals age, disrupting cellular homeostasis and promoting the release of endothelin-1 (ET-1) and other vasoactive stimuli, thereby contributing to the development of age-related vascular diseases [16,17]. During the progression of atherosclerosis, age-related disruptions in intercellular adhesion junction complexes, including those within the extracellular matrix of endothelial cells, lead to decreased NO production [18,19]. NO plays a critical role in gas therapy due to its significant biological functions, making it a key therapeutic agent. As an endogenous signaling molecule, NO facilitates vasodilation, reduces blood pressure, and enhances blood flow [20], leukocyte adhesion, and platelet aggregation. These actions help maintain endothelial integrity and decrease the risk of atherosclerosis [3,21]. Additionally, NO has a low molecular weight and is lipophilic, enabling it to easily penetrate cell membranes [22,23]. Consequently, the gas therapy based on NO is a promising strategy that can quickly reverse endothelial cell dysfunction and effectively slow the aging of blood vessels in atherosclerosis. To the best of our knowledge, research on gas therapy for the anti-aging treatment of atherosclerosis remains relatively limited.

Apart from endothelial cells, macrophages within atherosclerotic plaques play a crucial role in plaque formation and progression by engulfing lipids and transforming into foam cells [24,25]. Furthermore, they contribute to plaque instability through the secretion of inflammatory cytokines, which significantly heightens the risk of cardiovascular diseases [26]. Macrophages are critical immune cells that can be classified into two main types: pro-inflammatory M1 and anti-inflammatory M2 [27,28]. During inflammatory responses, M1 macrophages are activated and release significant amounts of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which intensify local inflammation [29,30]. In contrast, M2 macrophages secrete anti-inflammatory cytokines like interleukin-10 (IL-10), helping to suppress inflammation and promote tissue repair and homeostasis [31]. Therefore, regulating macrophage polarization to reduce the release of inflammatory cytokines is crucial for the effective treatment of atherosclerosis. Reactive oxygen species (ROS) play a vital role in various biological processes, but excessive ROS production can cause cellular damage and is closely linked to several pathological conditions [32]. Moreover, ROS activate multiple signaling pathways, such as Toll-like receptor and TNF signaling pathways, which enhance the expression of inflammatory genes, leading to an increase in both the number and activity of M1 macrophages [33]. Extensive research has shown that reducing excess ROS can decrease the number of M1 macrophage and promote their transition to the M2 phenotype, thereby lowering pro-inflammatory release and restraining atherosclerotic plaque formation, highlighting the potential therapeutic benefits of specifically eliminating ROS in atherosclerosis treatment [34]. Therefore, early intervention targeting the ROS within macrophages-aimed at reducing oxidative stress damage, modulating the polarization of microenvironmental macrophages, and clearing intracellular inflammatory factors-represents an effective therapeutic strategy for atherosclerosis.

Herein, we developed an osteopontin (OPN)-modified nanoliposomes, termed CZALO, which incorporates L-arginine (L-Arg) and cerium-zirconium oxide nanoparticles (CZ NPs). Following inflammation-driven targeting and OPN-mediated endocytosis, the CZ NPs are released from the CZALO nanoliposomes and significantly scavenge ROS by mimicking multiple natural enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione peroxidase (GPx). As a result, it inhibits cholesterol uptake, promotes macrophage phenotype transformation and suppresses inflammatory cytokine secretion, thereby achieving antioxidant and anti-inflammatory effects. More interestingly, the overexpressed NOS enzyme within macrophages selectively catalyzes the in situ conversion of L-arginine to nitric oxide (NO), enabling the precise release of NO at sites of inflammation. Once diffusing into endothelial cells, NO effectively regulates the secretion of senescence-associated secretory phenotype (SASP) factors, improves lysosomal function, disrupts cell cycle arrest, and reduces DNA damage in senescent endothelial cells, thereby exerting favorable anti-senescence effects. The CZALO nanoliposome-mediated “two-birds-with-one-stone” strategy for antioxidant and anti-senescence therapy has demonstrated favorable anti-atherosclerosis efficacy both in vitro and in vivo, effectively reducing the burden of atherosclerosis with minimal toxicity (Fig. 1).

Fig. 1.

Schematic illustration indicating the strategy enabled by CZALO nanoliposomes for efficient and safe atherosclerotic treatment. (a) The synthetic route of CZALO nanoliposomes. (b) After OPN-mediated active targeting and cellular internalization by macrophages, CZ NPs released from CZALO nanoliposomes exhibit SOD/CAT/POD/GPx-like activities to efficiently scavenge ROS, thus inhibiting cholesterol uptake and reprograming macrophage phenotype. (c) L-Arg is selectively catalyzed by overexpressed NOS within macrophages to produce NO gas, which further diffuses into endothelial cells to exert the anti-aging function through breaking cell cycle arrest, relieving DNA damage, decreasing lipofuscin and SASP content, and scavenging ROS.

2. Materials and methods

2.1. Materials

The zirconium (IV) acetylacetonate (97 %) and cerium (III) acetylacetonate hydrate were sourced from Sigma Aldrich (St. Louis, MO). Oleylamine was procured from Aladdin (Shanghai, China). Acetone (99.5 % extra pure), H₂O₂ (30 %) (99.5 % extra pure), and chloroform were supplied by Shang Hai University. Various reagents including Lipopolysaccharide (LPS), CCK-8, DCFH-DA probe, DAPI, Annexin V-FITC and PI dual staining kit, Calcein-AM/PI double stain kit, JC-1 double stain kit, ORO detection kit, senescence-associated β-galactosidase, DNA Damage Assay Kit by γ-H2AX, and 3-Amino, 4-aminomethyl-2′,7′-difluorescein diacetate were purchased from Beyotime Biotechnology. The Mouse ELISA Kit was obtained from YOBIBIO (Shanghai, China).

2.2. Characterization

The dispersed CZ NPs and CZALO dispersion were deposited onto a copper grid covered with a carbon film, dried at room temperature, and then analyzed using TEM with a JOEL-2100f instrument (Japan). FTIR spectral analysis was performed using an FTIR 8300 series spectrometer (Shimadzu, Japan). X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA) was employed to determine the elemental composition and valence states of the nanoparticles. The crystal structure of the nanospheres was examined using X-ray diffraction (XRD, Smartlab, Tokyo, Japan).

2.3. Preparation of CZ and CZALO

A mixture of hydrated cerium acetylacetonate and zirconium acetylacetonate (0.5 g, with a molar ratio of 7:3) was added to oleylamine (15 mL). The resulting solution was sonicated at 20 °C for 15 min and subsequently heated to 80 °C at a controlled rate of 2 °C per minute. The reaction mixture was maintained at 80 °C for 24 h, followed by cooling to room temperature. The resulting CZ nanoparticles (CZ NPs) precipitate was washed several times with acetone (100 mL) at 5000 rpm and finally dispersed in chloroform to reach a concentration of 10 mg mL⁻¹.

To prepare phospholipid polyethylene glycol-encapsulated CZ NPs, chloroform-dispersed CZ NPs (5 mL, 10 mg mL⁻¹) were mixed with DSPE-PEG (2000) in chloroform (10 mL, 10 mg mL⁻¹). The resulting mixture was subjected to spin evaporation using a rotary evaporator and then incubated in a vacuum oven at 70 °C to ensure complete removal of the chloroform. Afterward, deionized water (5 mL) was added to the sample, which was subsequently filtered through a 0.4 μm filter and ultracentrifuged to remove excess DSPE-PEG (2000).

The lipid components DOPE, DSPE-PEG2000-Maleimide (Mal), and the positively charged lipid DOTAP were combined with CZ NPs in chloroform at a molar ratio of 1:0.12:0.12. The mixture was transferred to a round-bottom flask, and the organic solvent was removed by rotary evaporation at 55 °C under vacuum (120 rpm), forming a uniform lipid membrane. To hydrate the lipid membrane, L-Arg phosphate-buffered saline (PBS) solution (5 mL, 50 mg) was added, followed by emulsification using an acoustic vibration instrument in an ice bath. The acoustic vibration was set to pulse mode with a power of 400 W for 4 min. Subsequently, the liposome solution was centrifuged at 10,000 rpm for 15 min to remove free lipid components and unencapsulated drugs. Finally, OPN-SH (50 mg) was added to the solution (5 mL) and mixed in an ice bath for 4 h before purification by ultrafiltration to obtain CZALO, which was then stored at 4 °C.

2.4. Drug release assay of CZALO

The in vitro release of L-Arg was measured using the Sakaguchi reaction method. Briefly, CZALO solution (2 mL) was placed into a dialysis bag and immersed in PBS (pH 6.5 and 7.5), then rotated at 200 rpm at 37 °C. At specific time intervals, samples (1 mL) containing L-Arg were withdrawn and transferred to a centrifuge tube. Subsequently, NaOH solution (1.0 M, 1.0 mL), diacetyl/propanol solution (0.5 mL L-1, 1.0 mL), naphthol/propanol solution (0.6 M, 1.0 mL), and L-Arg solution (100 μL) were added successively to the centrifuge tube for color development. This mixture was incubated in a 30 °C water bath for 15 min, and the absorbance was measured at 540 nm using a microplate reader.

2.5. Multienzyme activity assay

2.5.1. SOD mimetic activity assay

The SOD-like activity of CZ NPs was evaluated using a total SOD activity assay kit with the WST-8 method (Beyotime, China), following the manufacturer's instructions. Absorbance at 450 nm was measured using a microplate reader.

2.5.2. POD mimetic activity assay

The POD-like activity of the prepared CZ NPs was determined using TMB as the POD substrate in the presence of H₂O₂. In a typical test, TMB (0.8 mM), H₂O₂ (10 mM), and CZ NPs at varying concentrations were mixed by pipetting at room temperature in an acetate buffer solution. As the reaction progressed, the development of a blue color and changes in absorbance at 652 nm were immediately measured in time-scanning mode to evaluate POD-like activity.

2.5.3. CAT mimetic activity assay

The catalase-like (CAT-like) activity of CZ NPs was assessed by measuring O₂ production using a JPB-607A portable dissolved oxygen meter (INESA Scientific Instruments, Shanghai, China). In brief, CZ NPs solution (2 mL) was mixed with PBS (8 mL) containing varying concentrations of H₂O₂. The reaction was performed at room temperature for 10 min, with O₂ generation monitored at 1-min intervals.

2.5.4. GPx mimetic activity assay

The GPx-like activity of CZ NPs was assessed using a total GPx activity assay kit with the DTNB method (Beyotime, China) according to the manufacturer's instructions. Absorbance at 340 nm was measured using a microplate reader.

2.5.5. DPPH radical scavenging activity assay

The scavenging activity of CZ NPs against DPPH radicals was evaluated using a DPPH radical cation decolorization assay. A DPPH solution (3 mM) in anhydrous ethanol was prepared, and the absorbance at 517 nm was measured with a microplate reader after a 30-min incubation with varying concentrations of CZ NPs. The inhibition rate of DPPH radicals was calculated based on the proportion of neutralized DPPH radicals to the total free radical content.

2.5.6. PTIO radical scavenging assay

A solution of PTIO (3 mg) in distilled water (20 mL) was prepared, and varying concentrations of CZ NPs were added to assess their PTIO· scavenging ability. Distilled water was used to adjust the total reaction volume. The mixtures were then incubated at 37 °C for 2 h, after which the absorbance was measured at 557 nm.

2.5.7. •OH radical scavenging assay

The •OH-scavenging capacity of CZ NPs was evaluated using the salicylic acid (SA) method with a microplate reader. Hydroxyl radicals (•OH) were generated via the Fenton reaction by mixing FeSO₄ (2 mM) and H₂O₂ (5 mM) for 5 min, followed by the addition of CZ NPs. The remaining •OH was quantified by measuring the characteristic absorption peak of 2,3-dihydroxybenzoic acid, formed through the oxidation of SA by •OH, at 510 nm.

2.6. Cell culture of HUVECs and Raw264.7

Human umbilical vein endothelial cells (HUVECs) and macrophages (Raw264.7) were obtained from the Cell Bank/Stem Cell Bank of the Chinese Academy of Sciences. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS, Gibco), 1 % streptomycin, and penicillin. The cells were maintained at 37 °C in a humidified incubator with 5 % CO₂.

2.7. In vitro cytotoxicity of CZALO

The cytotoxicity of CZALO was assessed using Raw264.7 and HUVECs. Briefly, 1 × 10⁴ cells per well were evenly seeded in a 96-well plate. After a 24-h incubation period, various concentrations of CZALO (0, 20, 40, 60, 80, 100, and 120 μg mL⁻¹) were added to the wells. Following an additional 24 h of incubation, the CCK-8 reagent was applied according to the protocol, and the absorbance at 450 nm was measured after a further 30-min incubation at 37 °C.

2.8. Cell viability assay

Raw264.7 cells were seeded onto confocal dishes and allowed to mature for 12 h. The cells were then treated with L, H₂O₂, PBS, CZL, CZAL, and CZALO. Both viable and non-viable cells were stained with calcein-AM and PI, respectively, and co-incubated for 30 min. Following incubation, the cells were washed twice with PBS and examined under a fluorescence microscope.

2.9. Intracellular ROS levels observation

Intracellular ROS levels were analyzed using fluorescence microscopy, with DCFH-DA as the fluorescence probe. Raw264.7 cells (1.2 × 10⁵) were seeded onto a confocal dish and incubated at 37 °C with 5 % CO₂ for 24 h. After washing with PBS, the cells were treated with various groups (LPS, L, CZL, CZAL, and CZALO) and incubated for another 24 h. DCFH-DA was then introduced and incubated in the dark for 20 min, followed by dilution to the appropriate concentration in serum-free medium. After three washes with culture medium, the cells were observed using confocal microscopy.

2.10. Apoptosis assay

Raw264.7 were cultured in medium for 12 h before being subjected to various treatments, including PBS, H₂O₂, L, CZL, CZAL, and CZALO. After treatment, the cells were collected and stained using the Annexin V-FITC/PI apoptosis detection kit. The degree of cell apoptosis was then measured using flow cytometry, and the results were analyzed with FlowJo software. The entire process was conducted in a dark environment.

2.11. Treatment and staining of Raw264.7

Raw264.7 cells were incubated in confocal dishes for 12 h, followed by simultaneous treatment with LPS (100 ng mL⁻¹), ox LDL (100 μg mL⁻¹), and various treatment groups (L, CZL, CZAL, and CZALO) for an additional 12 h. The cells were then fixed with 4 % paraformaldehyde for 10 min and washed with PBS. Afterward, the cells were stained with Nile red and incubated in the dark for 20 min. Confocal images were captured using a confocal laser scanning microscope (CLSM), with two additional PBS washes performed during the process. The same methods were used for foam cell generation and treatment, followed by incubation for ORO staining. The cells were fixed in 4 % paraformaldehyde for 10 min, washed with PBS, stained using an ORO detection kit, and then imaged under an optical microscope.

To further investigate cholesterol efflux, the supernatants of phagocytes from each group were collected prior to staining using a cholesterol assay kit. The absorbance was measured at a wavelength of 500 nm using a microplate reader.

2.12. Inflammatory cytokine assay

Raw264.7 (1 × 10⁶ cells per well) were induced with LPS (100 ng mL⁻¹). After induction, the cells were plated at a density of 1 × 10⁵ cells per well in 24-well plates. The model group was treated with LPS solution (100 ng mL⁻¹) for 8 h, while the control group received only fresh medium. The other experimental groups were incubated with different treatments (L, CZL, CZAL, CZALO) for 12 h each, followed by coincubation with fresh DMEM containing LPS solution (100 ng mL⁻¹) for 8 h. Inflammatory cytokines, including IL-10, IL-4, TNF-α), and IFN-γ, were measured using ELISA kits.

2.13. Immunofluorescence imaging to assess anti-inflammatory efficacy of CZALO

Immunofluorescence imaging was used to assess the efficacy of CZALO in preventing inflammation. Cells were initially cultured overnight in a confocal dish. The control group received only fresh medium, while the model group was treated with LPS solution (100 ng mL⁻¹) for 8 h. After 2 h of co-incubation with various treatments (L, CZL, CZAL, and CZALO), the experimental groups were further incubated for 8 h with fresh DMEM containing LPS solution (100 ng mL⁻¹).

Following treatment, the cells were washed with PBS and fixed for 30 min with 4 % paraformaldehyde. The cells were then permeabilized with a membrane-breaking solution containing 1 % Triton for 30 min, rinsed with PBS, and incubated with a diluted primary antibody for 12 h. After multiple PBS washes, the cells were incubated in the dark for 1 h with diluted fluorescent dye-conjugated secondary antibodies. Following a final PBS wash, the cells were sealed with DAPI-containing anti-fade mounting medium and examined under a fluorescence microscope.

2.14. Cell treatment and co-culture assay

HUVEC cells were stimulated with LPS (100 ng mL^-1) and exposed to various treatment groups (Control, LPS, L, CZL, CZAL, and CZALO) for 24 h. Raw 264.7 cells (1 × 10⁴ cells per sheet) were cultured overnight on a polyester membrane (Transwell, 6-well, 0.4 μm pore size). To simulate the aging process associated with atherosclerosis, the HUVECs were treated with 400 μM H₂O₂ for 12 h. Following this, a co-culture of HUVECs (1 × 10⁶) and Raw264.7 cells was established for 24 h in each well. After washing the monolayer three times with PBS, it was transferred to a confocal plate for imaging.

2.15. Bio-TEM imaging

The experimental procedures involved multiple iterations of cell preparation, treatment, and incubation. Raw264.7 and HUVECs were initially cultured in medium for 12 h. Subsequently, PBS, CZAL, or CZALO treatments were administered to Raw264.7 for 24 h. After treatment, HUVECs were washed twice with PBS and preserved overnight in a 2.5 % glutaraldehyde solution at 4 °C. The samples were then fixed with 1 % osmium tetroxide for 2 h, washed with PBS, dehydrated through a graded ethanol series, and embedded. Ultrathin sections were prepared using a Leica UC7 ultramicrotome. These sections were doble-stained with 3 % uranyl acetate and lead citrate, then analyzed for subcellular structures using a HITACHI HT770 TEM.

2.16. Senescence-associated secretory phenotype assay

The medium from the upper compartment in the previously mentioned experiment was collected. ELISA kits were then used to assess the levels of IL-1β, IL-6, IL-8, and TNF-α.

2.17. Western blotting analysis

The previously described cell processing procedures were repeated. Following the collection of treated cells, a lysis buffer containing a protease inhibitor cocktail was added. After removing unwanted lysates, proteins were separated by electrophoresis. The samples were then transferred onto a polyvinylidene difluoride (PVDF) membrane, blocked for 1 h with 5 % skim milk, and incubated overnight with primary antibodies against IL-6, IL-8, p16, p53, γ-H2AX, and β-actin. The membranes were then washed three times with TBST and incubated for 45 min with HRP-labeled secondary antibodies. Finally, the membranes were treated with ECL reagent and analyzed using an infrared scanner.

2.18. Atherosclerotic mice

Male C57BL/6 ApoE^−/− mice (6–8 weeks old) were fed a Western diet for eight weeks and housed in a humidity-controlled room with a 12-h light/dark cycle to induce atherosclerotic plaque formation.

2.19. In vivo biosafety evaluation

Nine healthy male C57BL/6 mice (6–8 weeks old) were randomly divided into three groups (n = 3). The control group received an intravenous injection of saline on day 0. The second group received an intravenous injection of CZALO at a dose of 1 mg kg⁻¹. The third group received the injection on day 21 before euthanasia. Subsequently, blood and major organs (heart, liver, spleen, lung, and kidney) were collected from each group for histological examination, routine blood analysis, and blood biochemical index analysis.

2.20. In vivo imaging system (IVIS)

In further studies, ApoE^−/− mice were fed a high-fat diet for 10 weeks to induce the development of atherosclerotic plaques. The biodistribution of L and LO in the heart, aorta, liver, spleen, lung, kidney, and other vital organs was assessed. ApoE^−/− mice (n = 3) received intravenous injections of Cy5.5-labeled L and LO (Ex/Em = 673/707 nm). Six hours post-injection, major organs from the ApoE^−/− mice were excised and imaged using the PerkinElmer IVIS system.

2.21. In vivo therapeutic effects

At the beginning of the study, 35 ApoE^−/− mice were fed a high-fat diet for 8 weeks to induce the formation of atherosclerotic plaques. These atherosclerotic mice were then randomly divided into five groups (n = 7 each): the saline group (0.1 mL), L group, CZL group (1 mg kg⁻¹, 0.1 mL in saline), CZAL group (1 mg kg⁻¹, 0.1 mL in saline), and CZALO group (1 mg kg⁻¹, 0.1 mL in saline). Over the course of eight weeks, the different treatments were administered once a week, for a total of eight sessions.

For the ultrasonic hemodynamic study, a random selection of atherosclerotic mice was made from each group. Using a Canon Aplio i900, spectral Doppler ultrasound was employed to measure the resistance index (RI) of blood flow at the initial site of the mouse aorta. This index, which reflects the degree of stenosis in the downstream blood vessels, is calculated as follows: Resistance Index (RI) = (Peak Systolic Velocity (PSV) - End Diastolic Velocity (EDV))/PSV.

After the various treatments and imaging investigations, the atherosclerotic mice were euthanized. Six atherosclerotic mice were randomly selected from each group, and their entire aortas, from the abdominal aorta to the common carotid arteries, were stained with ORO to assess lesion areas.

To observe the plaque-filled areas in the cross-sections, serial sections of paraffin-embedded aortic sinus were created and stained with ORO. Additionally, H&E and Masson's trichrome staining were performed for histological analysis. Immunohistochemical detection using antibodies against α-SMA and immunofluorescence staining with antibodies against p16, p53, p21, γ-H2AX, CD86, CD206, and F4/80 were also conducted on the paraffin-embedded aortic sinus sections. Furthermore, semi-quantitative analysis was performed using ImageJ software.

2.22. Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 and reported as mean ± standard deviation (SD). The significance of the data was assessed using one-way or two-way ANOVA. Significance levels were indicated as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant (P > 0.05).

3. Results

3.1. Synthesis and characterization of CZALO nanoliposome

First of all, Zr⁴⁺-doped ceria nanoparticles, namely CZ NPs, were synthesized using a non-hydrolytic sol-gel method (Fig. 2a). Studies have shown that doping CZ NPs with Zr⁴⁺ significantly increases the Ce³⁺/Ce⁴⁺ ratio, enhancing the rapid regeneration capability of Ce³⁺ and improving ROS scavenging efficiency [35]. The resulting CZ NPs display uniform morphology and discrete distribution as confirmed by transmission electron microscopy (TEM) images (Figs. 2b and S1). The elemental distribution mappings and full X-ray photoelectron spectroscopy (XPS) analysis indicate that the successively synthesized CZ NPs contain both cerium and zirconium components (Figs. 2c and S2). Additionally, the XPS spectrum of Ce verifies the coexistence of Ce³⁺ and Ce⁴⁺ oxidation states within the element, imparting it with enzyme-like activity and ROS scavenging capability (Fig. 2d). Meanwhile, two characteristic peaks, Zr 3d₅/₂ and Zr 3d₃/₂ with binding energies at approximately 182.0 eV and 184.2 eV, respectively, occurred in the Zr 3d spectrum, which were consistent with the characteristic signals of Zr⁴⁺, confirming that the Zr in CZ NPs is in the +4 oxidation state (Fig. S3). These results further validate the material properties of CZ NPs and underscore their remarkable potential in ROS scavenging. X-ray diffraction (XRD) analysis confirms the crystalline phase of the CZ NPs, which validates their successful synthesis (Fig. 2e).

Fig. 2.

Synthesis and characterization of CZALO. (a) Schematic diagram indicating the synthetic process of CZ NPs. (b) TEM images of CZ NPs. (c) Elemental mapping of CZ NPs. (d) XPS spectrum of Ce in CZ NPs. (e) XRD pattern of CZ NPs. (f) SOD-like activity of CZ NPs with various concentrations (n = 5) (left) and ESR assays for evaluating O₂^•- (right). (g) Schematic representation (left) and assays (right) implying the POD-like activity of CZ NPs. (h) O₂ production (left) (n = 3) and H₂O₂ clearance (right) (n = 6) for CAT-like activity evaluation. (i) Schematic representation (left) and assays (right) implying the GPx-like activity of CZ NPs (n = 5). (j) Schematic illustration of enzyme-mimicking activities of CZ NPs, encompassing SOD, POD, CAT, and GPx. (k–n) The radical scavenging performance of CZ NPs, including (k) •OH (n = 3), and (l) ESR results for measuring •OH, (m) DPPH (n = 5), (n) PTIO (n = 3). (o) Scheme showing the preparation protocol of CZALO. (p) TEM images of CZALO. (q) In vitro release profiles of L-Arg at pH 6.5 and pH 7.5. Data in (f, h, i, k, m, n) are expressed as mean ± S.D. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant (P > 0.05).

Subsequently, we explore the multienzyme-like performance of CZ NPs. Firstly, by using the traditional WST-8 method, the ability of CZ NPs to convert superoxide anions (O₂^•-) into oxygen (O₂) and hydrogen peroxide (H₂O₂) was assessed to evaluate their SOD mimetic activity [36]. As shown in Fig. 2f, CZ NPs exhibit favorable SOD-like activity and significantly decrease the O₂^•- content in a concentration-dependent manner, which is consist with the electron spin resonance (ESR) results. Considering that POD, as an important antioxidant enzyme, is capable of detoxifying H₂O₂ into H₂O, we then measured the POD-like activity of CZ NPs by adopting 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate [37,38]. Specifically, CZ NPs catalyze the decomposition of H₂O₂ and simultaneously oxidize colorless TMB into blue-hued oxidized TMB (ox TMB). The results demonstrates that CZ NPs feature apparent POD mimetic activity in a concentration- and time-dependent manner (Figs. 2g and S4). Apart from POD, CAT is able to catalyze the conversion of H₂O₂ into H₂O and O₂. Thus, we investigated the performance of CZ NPs to mimic CAT by monitoring the O₂ production. According to the data in Fig. 2h, CZ NPs can effectively decompose H₂O₂ into O₂ and H₂O. Furthermore, the O₂ production and H₂O₂ decomposition mediated by CZ NPs significantly enhanced with the increase of the concentration of CZ NPs. Glutathione peroxidase (GPx) is essential for maintaining cellular redox balance and safeguarding cells against oxidative damage. In the presence of glutathione (GSH), GPx catalyzes the reduction of H₂O₂ to H₂O [39]. Concurrently, glutathione reductase (GR) uses NADPH as an electron donor to reduce glutathione disulfide (GSSG) back to its reduced form GSH [40], thereby restoring its antioxidant capacity and maintaining the intracellular GSH/GSSG redox balance (Fig. 2i). Our experiment indicated that higher concentrations of CZ NPs resulted in greater GSH clearance, thereby verifying the GPx-like activity of CZ NPs. Taken together, the aforementioned findings provide strong evidence for the multifunctional enzyme-mimicking properties of the as-constructed CZ NPs, including SOD, POD, CAT, and GPx activities, which lays the foundation for subsequent antioxidant function (Fig. 2j).

Next, the radical scavenging capacity of CZ NPs, such as 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS^•+), 2-2 diphenyl-1-picrylhydrazyl radical (DPPH^•), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO^•), was evaluated. As demonstrated in Fig. 2k, l and S5, CZ NPs can efficiently remove ABTS^•+, DPPH^•, and PTIO^•, and their scavenging capability is positively correlated with the concentration of CZ NPs. Especially, •OH exhibits the strongest oxidative potential among all kinds of ROS and can interact with nearly all biological molecules [41]. The results demonstrated a significant elevation in •OH scavenging ability with increasing concentrations of CZ NPs (Fig. 2m). Furthermore, ESR analysis revealed that CZ NPs effectively scavenge •OH (Fig. 2n).

Inspired by the above results, we then employed the thin-film hydration method to prepare a nanoliposome composed of DOPE-PEG-Mal, DOTAP, and DOPE, and loaded it with CZ NPs and L-Arg. To elevate the delivery efficiency of nanoliposomes into arterial plaques, we further covalently modified the surface of liposomes with OPN using a Michael addition reaction (Fig. 2o) [42]. The Fourier Transform Infrared Spectroscopy (FTIR) results suggest that the characteristic peaks of OPN and CZAL at 2329 cm⁻¹ and 2931 cm⁻¹ are observed in the CZALO samples, respectively, indicating the successful modification of OPN onto the surface of nanoliposomes (Fig. S6). TEM image reveals that CZALO are quasi-spherical, with relatively uniform size and distribution. There is no noticeable adhesion between particles, indicating the successful synthesis of CZALO nanoliposome (Figs. 2p and S7). Then, we monitored the particle size of CZALO under different temperatures (4 °C, 25 °C, and 37 °C), and the results showed no significant changes in particle size across the tested storage conditions, demonstrating excellent temperature stability. Furthermore, the stability of CZALO under different pH conditions was investigated (4.5, 5.6, and 7.4). The findings revealed no significant differences in particle size across the tested pH environments, highlighting its stability in varying pH conditions. Collectively, these results demonstrate the excellent stability of CZALO within the physiological environment (Fig. S8).

Additionally, the enzyme-mimicking activity of CZALO nanoliposomes was thoroughly measured, including SOD, CAT, POD, and GPx. The results demonstrated that CZALO nanoliposomes effectively preserved the intrinsic enzyme-mimicking activities of CZ NPs without significant difference, confirming that the enzyme-mimicking activity of CZ NPs is well maintained after being incorporated into the CZALO nanoliposomes (Fig. S9). To evaluate the controlled drug release capability of CZALO, we assessed the drug release kinetics of L-Arg at pH 7.5 and 6.5. As shown in Fig. 2q, L-Arg is rapidly released within the first 12 h, followed by a slow release phase, implying that CZALO can achieve sustained and controlled release of L-Arg in physiological environments with different pH levels.

3.2. CZALO-enabled ROS scavenging and inflammation attenuation in macrophages

We used Raw264.7 mouse macrophages (Raw264.7) and human umbilical vein endothelial cells (HUVECs) as the in vitro models to investigate the antioxidant/anti-inflammatory effect and anti-aging effect of CZALO, respectively. First, the biocompatibility of CZALO was evaluated using the standard cell counting kit-8 (CCK-8) assay on Raw264.7 and HUVECs. CZALO exhibites ignorable cytotoxicity across a dose range of 0–120 μg mL⁻¹ (Fig. S10). Subsequently, we investigated the targeting capability of CZALO towards Raw264.7 by comparing its cellular internalization efficiency between Raw264.7 (high expression of OPN) and HUVECs (low expression of OPN). As shown in Fig. S11, compared to HUVEC, significant green fluorescence was observed in foam cells co-incubated with fluorescein isothiocyanate (FITC)-labeled CZALO, demonstrating that CZALO has a strong targeting ability towards macrophages, with the fluorescence intensity increasing in a time-dependent manner over a period of 10 h.

Reactive oxygen species (ROS) play a crucial role in various biological processes, but excessive ROS production can cause cellular damage and is closely associated with the development of numerous pathological conditions [43]. Elevated levels of ROS exacerbate oxidative stress and amplify inflammatory responses, contributing to plaque instability and accelerating the progression of various diseases [44,45]. Excessive ROS heightens oxidative stress within plaques, amplifying inflammation within the plaques, ultimately leading to increased plaque instability [46,47]. The ROS scavenging ability of CZALO was initially evaluated using the fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA). The findings revealed that CZAL exhibited a higher ROS scavenging capacity compared to CZL, suggesting the synergistic ROS scavenging effect between CZ NPs and L-Arg. Moreover, the LPS + CZALO group displayed a superior ROS scavenging effect compared to the LPS + CZAL group, which was attributed to the active targeting mediated by OPN (Figs. 3a and S12). The protective effect of CZALO on macrophages was subsequently assessed using calcein AM/propidium iodide (PI) staining. In comparison to other treatments, an apparent reduction of PI-labeled dead macrophages was observed in the CZALO group (Figs. 3b and S13). Similarly, the macrophages treated with CZALO exhibits robust anti-apoptotic activity against H₂O₂-induced apoptosis in Raw264.7, compared to treatments with CZAL and CZL (Fig. 3c and S14). The above results collectively confirmed that CZALO can effectively scavenge intracellular ROS and protect macrophages from oxidative stress-induced damage.

Fig. 3.

CZALO-enabled ROS scavenging and inflammation attenuation. (a) Intracellular ROS level in LPS-pretreated Raw264.7 after treatment with different formulations. (b) Representative live/dead staining fluorescence images of Raw264.7 under various treatments. (c) Cellular apoptosis evaluation of H₂O₂-pretreated Raw264.7 after various treatments, stained with an Annexin V-FITC/PI double dye kit and analyzed by flow cytometry. (d) Typical CLSM images and (e) corresponding quantitative analysis of Nile Red staining in Raw264.7 (n = 3). (f) Intracellular cholesterol content in the Raw264.7 after various treatments (n = 3). (g, h) Immunofluorescence of (g) CD86 and (h) CD206 in LPS-pretreated Raw264.7 after various treatments. (i–l) Inflammatory factors levels, including (i) TNF-α, (j) IFN-γ, (k) IL-4, and (l) IL-10 (n = 3). (m) Schematic diagram of CZALO-mediated ROS elimination and macrophage phenotype regulation. Note: L: plain liposomes, CZL: liposomes loaded with CZ NPs, CZAL: liposomes co-loaded with CZ NPs and L-arg, CZALO: CZAL liposomes further modified with OPN, LDL: low-density lipoprotein. Data in (e, f, i-l) were expressed as mean ± S.D. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant (P > 0.05).

The formation of foam cells is a key characteristic in the early stages of atherosclerotic lesions, where excessive cellular uptake of ox LDL plays a critical role in this process. Therefore, we further investigated whether CZALO could affect the uptake of ox LDL by macrophages. Oil Red O (ORO) staining revealed that Raw264.7 treated with LPS (100 ng mL⁻¹) and ox LDL (100 μg mL⁻¹) for 12 h exhibited substantial intracellular lipid droplets and formed numerous foam cells. However, following CZALO treatment, the intracellular lipid droplets and foam cell was significantly reduced (Fig. S15). Similarly, Nile Red, a red fluorescent dye with a specific affinity for lipids, was chosen to assess the cholesterol content in phagocytes using confocal laser scanning microscopy (CLSM). As shown in Fig. 3d and e, the red fluorescence intensity notably decreased in the CZALO-treated groups, indicating effective reduction of lipid retention in macrophages and significant inhibition of foam cell formation. Subsequently, cholesterol levels within Raw264.7 were measured, revealing a significant reduction in intracellular cholesterol levels in the CZALO-treated group (Fig. 3f). Which is consistent with the aforementioned results. All the aforementioned experiments have confirmed that CZALO can inhibit the uptake of ox LDL, thereby preventing the formation of foam cells [48,49]. Therefore, we investigated the impact of CZALO on the regulation of macrophage phenotype using immunofluorescence markers, including CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker). As shown in Fig. 3g and h, the CZALO-treated macrophages exhibited significantly lower CD86 level and higher CD206 level compared to the model group. Furthermore, the potential of CZALO to reduce inflammatory responses was evaluated using the enzyme-linked immunosorbent assay (ELISA). As demonstrated in Fig. 3i and j, the pro-inflammatory cytokines including TNF-α and interferon-γ (IFN-γ) obviously elevated after LPS treatment. Conversely, in the macrophages incubated with CZALO, the excretion level of these pro-inflammatory cytokines significantly decreased. Additionally, the anti-inflammatory cytokines, such as interleukin-4 (IL-4) and IL-10, were increased in the CZALO group compared to other groups (Fig. 3k and l). These findings indicate that CZALO facilitates a transition from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, consequently upregulating anti-inflammatory cytokines and down-regulating pro-inflammatory cytokines (Fig. 3m).

To further explore the potential biological mechanisms underlying the antioxidant/anti-inflammatory effects of CZALO, we conducted a high-throughput transcriptome sequencing analysis to compare the differentially expressed genes (DEGs) between LPS-stimulated macrophages (Model group) and CZALO-treated macrophages (Experimental group) (Fig. 4a). The heatmap results suggested that a total of 5421 genes were differentially expressed after CZALO treatment, indicating significant transcriptomic differences between the CZALO-treated group and the model group (Fig. S16). As shown in the volcano plot, 2519 genes were significantly upregulated and 2932 genes were significantly downregulated following CZALO treatment (Fig. 4b). Gene Ontology (GO) analysis was conducted to elucidate the changes of these genes in biological processes, cellular components, and molecular functions (Fig. 4c). To further elucidate the biological functions of DEGs induced by CZALO, a bubble chart based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis reveals that the enriched signaling pathways was associated with the regulation of inflammatory responses, including the TNF signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, and NOD-like receptor signaling pathway, which might play a significant role in the antioxidant and anti-inflammatory effects of CZALO (Fig. 4d). Gene set enrichment analysis (GSEA) analysis also demonstrated that compared to the model group, DEGs in the CZALO-treated group were significantly enriched in several pathways, including TNF signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, and NOD-like receptor signaling pathway (Figs. 4e and S17). Circular plot further confirms that the above inflammatory signaling pathways are closely linked to inflammation-related genes, indicating a strong antioxidant/anti-inflammatory effect induced by CZALO (Fig. 4f). It is well-known that the TNF signaling pathway and Toll-like receptor signaling pathway significantly amplify local inflammatory responses, promote the secretion of pro-inflammatory cytokines, and drive foam cell formation, thereby accelerating the progression of atherosclerosis. Subsequently, we analyzed the expression level of DEGs in these two pathways and found that DEGs such as interleukin-1beta (IL-1β), CD86, leukemia inhibitory factor (Lif), TNF, IL-6, interleukin-12beta (IL-12β), and nuclear factor kappa b subunit 1 (Nfkb1) were significantly downregulated in Raw264.7 treated with CZALO compared to the model group (Fig. 4g). To further validate these findings, we performed reverse-transcriptase qPCR (RT-qPCR) assays to assess the RNA expression levels of the aforementioned genes. The results indicated that, compared to the H₂O₂-treated group, the relative expression levels of IL-1β, CD86, Lif, TNF, IL-6, IL-12β, and NF-kappa B were significantly downregulated in the CZALO treatment group. These results are consistent with the findings from the transcriptomic sequencing analysis (Fig. S18).

Fig. 4.

High-throughput transcriptome sequencing of Raw264.7 cells following various treatments. (a) Principal component analysis (PCA) of DEGs between the model group and the CZALO group. (b) Volcano plot illustrating upregulated (green) and downregulated (pink) genes in Raw264.7 treated with CZALO compared to the model group, analyzed via RNA-Seq (threshold: log 10 (fold change) ≥ 2.0). (c) GO annotation analysis results of DEGs in Raw264.7 after different treatments. (d) KEGG pathway analysis of DEGs in CZALO-treated Raw264.7 cells compared to the model group. (e) GSEA plots illustrating the enrichment of genes in the TNF signaling pathway (top) and Toll-like receptor signaling pathway (bottom) between the model group and the CZALO-treated group. (f) Circular plot suggesting the interaction between DEGs and key signaling pathways. (g) Circular heatmap representing the expression levels of DEGs associated with the TNF signaling pathway and Toll-like receptor signaling pathway.

3.3. CZALO-mediated senescence alleviation within endothelial cell

NO synthase, overexpressed in pro-inflammatory macrophages, can selectively catalyze L-Arg to produce NO [50], then the latter gradually diffuses into endothelial cells, where it exerts anti-senescence function by regulating the secretion of SASP factors, improving lysosomal function, disrupting cell cycle arrest, and reducing DNA damage in senescent endothelial cells (Fig. 5a). The CZALO-mediated anti-senescence efficacy within endothelial cells was explored by utilizing H₂O₂-stimulated HUVEC as an in vitro senescence model. Six groups were established for the experiments: the control group, the H₂O₂+PBS group, the H₂O₂+L group, the H₂O₂+CZL group, the H₂O₂+CZAL group, and the H₂O₂+CZALO group. The CCK-8 results showed a significant improvement in cell viability of HUVECs, reaching 64.7 % and 83.7 % after co-incubation with the CZAL and CZALO, respectively (Fig. S19), which is ascribed to the anti-senescence function enabled by CZALO. Subsequently, a co-culturing transwell system with HUVECs and Raw264.7 was employed to mimic the atherosclerotic microenvironment. HUVECs were seeded in the upper compartment and treated with H₂O₂ for 12 h. Meanwhile, Raw264.7 in the lower compartment were pre-stimulated with LPS and then co-incubated with L, CZL, CZAL, and CZALO, respectively. The NO levels within HUVECs were assessed using the fluorescent dye 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FMDA). As anticipated, the CZAL and CZALO groups exhibited higher fluorescence intensities in HUVECs compared to the L and CZL groups (Fig. 5b). The flow cytometry results were consistent with the fluorescent images and further confirmed that CZALO triggered the strongest fluorescence signal, confirming that the NO level in HUVECs significantly increased after treatment with CZALO (Fig. S20).

Fig. 5.

CZALO-mediated senescence alleviation within endothelial cell. (a) Schematic illustration showing the selective release of NO from CZALO to relief the senescence of endothelial cells. (b) Intracellular NO content in HUVECs after treatment with different formulations. (c–f) Excretion levels of (c) IL-1β, (d) IL-6, (e) IL-8, and (f) TNF-α in H₂O₂-induced senescent HUVECs after different treatments (n = 3). (g) Representative images of SA-β-gal staining in senescent and NO gas-treated HUVECs. (h) Bio-TEM images of HUVECs after different treatments. (i) Representative immunofluorescence images of the DNA damage marker γ-H2AX. (j, k) Representative immunofluorescence images of cell cycle arrest markers, including (j) p16 and (k) p53. (l) Western blotting analysis of the expression levels of IL-8, IL-6, p53, p16 and γ-H2AX, in HUVECs with different treatments. Note: L: plain liposomes, CZL: liposomes loaded with CZ NPs, CZAL: liposomes co-loaded with CZ NPs and L-arg, CZALO: CZAL liposomes further modified with OPN. Data in (c–f) are expressed as mean ± S.D. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant (P > 0.05).

Senescent endothelial cells will release a variety of biologically active molecules when exposed to external stress. These molecules, collectively known as the SASP, include inflammatory cytokine, chemokines, growth factors, and ROS produced by senescent cells, leading to permanent growth arrest [51,52]. To evaluate the inflammatory cytokines associated with the SASP, we employed ELISA to measure IL-6, interleukin-1beta (IL-1β), interleukin-8 (IL-8), and TNF-α. Following H₂O₂ treatment, there was a significant increase in the excretion level of inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-8 in HUVECs. The expression of inflammatory cytokines was markedly inhibited when HUVECs were treated with L, CZL, CZAL, and CZALO, respectively (Fig. 5c–f). Meanwhile, to validate these findings, we conducted Western blotting experiments to assess the expression levels of IL-6 and IL-8 in HUVECs subjected to various treatments. Consistent with our previous observations, NO treatment resulted in a significant reduction in the protein expression of IL-6 and IL-8 (Fig. 5l). Additionally, we further assessed the ability of CZALO to eliminate ROS by using the fluorescent dye DCFH-DA. As expected, the fluorescence intensity in the CZAL and CZALO groups significantly decreased (Fig. S21), indicating that the ROS levels in HUVECs were significantly reduced after NO treatment. The above experiments demonstrate that NO produced by CZALO significantly decreases the SASP levels including inflammatory cytokine and ROS.

The lysosome functions as the cell's digestive system, primarily responsible for degrading various biological macromolecules [53]. As cells age, lysosomal function gradually weakens, and the activity of β-galactosidase significantly increases [54], which leads to the accumulation of incompletely degraded substances such as lipofuscin within the cells, resulting in the formation of lipofuscin [55]. Thus, we used senescence-associated β-galactosidase (SA-β-Gal) as an aging-related marker to evaluate the anti-aging performance of CZALO (Fig. 5g). The NO treatment group (CZAL group and CZALO group) significantly reduced the levels of SA-β-Gal in HUVECs. Additionally, biological transmission electron microscopy (bio-TEM) observation revealed that CZALO treatment could reduce the accumulation of lipofuscin in HUVECs (Fig. 5h). As expected, the chromatin accumulation (red circles) and lipofuscin content (white boxes) were significantly reduced in the CZALO-treated HUVECs, suggesting that NO released form CZALO can enhance lysosomal function and reduce SA-β-Gal activity, thereby decreasing the accumulation of harmful substances such as lipofuscin.

During cellular senescence, the accumulation of DNA damage increases, resulting in elevated levels of phosphorylated H2A histone family member X (γ-H2AX) [56]. This persistent DNA damage signal can activate cell cycle checkpoints and induce permanent cell cycle arrest through signaling pathways such as tumor protein p53 (p53), cyclin-dependent kinase inhibitor 1A (p21), and cyclin-dependent kinase inhibitor 2A (p16), ultimately leading to cellular senescence [57,58]. Herein, γ-H2AX is used as an early biomarker for the detection of DNA damage. After inducing senescence in HUVECs with H₂O₂, the expression levels of γ-H2AX in HUVECs were observed using CLSM following NO treatment (CZAL and CZALO groups). CLSM images showed a significant increase in γ-H2AX fluorescence intensity in senescent cells. However, the fluorescence intensity of γ-H2AX was significantly reduced in the CZALO group (Fig. 5i), confirming that CZALO treatment can effectively protect HUVEC from DNA damage. Subsequently, to further confirm the anti-aging performance of CZALO, the expression levels of the cell cycle arrest markers p16 and p53, which are positively correlated with senescence, were evaluated using immunofluorescence staining. The immunofluorescence images demonstrated a significant reduction in the fluorescence intensity of p16 and p53 following CZALO treatment, demonstrating that CZALO-based NO treatment can substantially reduce the expression of p16 and p53 (Fig. 5j and k). Western blotting results were consistent with our previous observations, presenting a substantial decrease in the protein expression of γ-H2AX, p16, and p53 in the H₂O₂ group following CZALO treatment (Figs. 5l and S22). Collectively, NO released from CZALO significantly reduces the expression levels of γ-H2AX, p16, and p53, thereby facilitating DNA repair and alleviating cell cycle arrest.

To further investigate the anti-senescence mechanisms of CZALO-based nanotherapy in H₂O₂-treated HUVECs, we conducted a high-throughput transcriptomic analysis, which revealed significant DEGs between H₂O₂-stimulated HUVECs (Model group) and CZALO-treated HUVECs (Experimental group) (Fig. 6a). Heatmap analysis identified 593 gene with significant differential expression following CZALO treatment, indicating a notable transcriptomic difference between the CZALO-treated group and the control group (Fig. S23). As shown in Fig. 6b, the volcano plot further demonstrated that 202 genes were significantly upregulated, while 391 genes were significantly downregulated after CZALO treatment. To gain deeper insights into the biological functions of CZALO-mediated DEGs, we performed GO analysis to explore changes in biological processes, cellular components, and molecular functions (Fig. 6c). Additionally, a KEGG pathway bubble plot revealed significant enrichment in pathways such as NF-kappa B signaling, AGE-RAGE signaling, MAPK signaling, and TGF-β signaling during CZALO treatment (Fig. 6d). GSEA analysis further confirmed that, compared to the control group, the DEGs associated with these signaling pathways were significantly enriched in the CZALO-treated group (Figs. 6e and S24). A circular plot further corroborated the close association of these signaling pathways with aging-related genes, suggesting that CZALO-induced NO potentially owns benign anti-senescence effects (Fig. 6f). It is well established that the AGE-RAGE, NF-kappa B, and TGF-β signaling pathways collectively drive cellular dysfunction and chronic inflammation during the aging process. The AGE-RAGE pathway exacerbates “inflammaging” by inducing oxidative stress and inflammatory responses, thereby activating the NF-kappa B signaling pathway. Meanwhile, the TGF-β signaling pathway, by regulating cell cycle arrest and tissue fibrosis, cooperates with NF-kappa B, further promoting cellular aging and the progression of age-related diseases. We further analyzed the expression levels of DEGs within these three pathways and found that in CZALO-treated HUVECs, the expression levels of DEGs such as interleukin-1α (IL-1α), transforming growth factor β2 (TGFβ2), forkhead box O1 (FOXO1), and superoxide dismutase 2 (SOD2) were significantly downregulated, which was consist with the previous findings (Fig. 6g). Collectively, the NO generated by CZALO effectively modulates the secretion of SASP factors, enhances lysosomal function, alleviates cell cycle arrest, and reduces DNA damage in senescent endothelial cells, thereby exerting a potent anti-aging effect. Transcriptome sequencing of HUVEC cells revealed a significant downregulation of genes, including IL-1α, TGFβ2, FOXO1, and SOD2, in the CZALO-treated group compared to the model group. To further corroborate these findings, we conducted RT-qPCR experiments, using H₂O₂ to induce cellular senescence in HUVECs. The results demonstrated that, relative to the model group, the expression levels of IL-1α, TGFβ2, FOXO1, and SOD2 were significantly downregulated in the CZALO-treated group, which was consistent with the transcriptomic sequencing results (Fig. S25).

Fig. 6.

High-throughput transcriptome sequencing of HUVECs subjected to different treatments. (a) Principal component analysis (PCA) showing the DEGs between the model group and the CZALO group. (b) Volcano plot depicting the upregulated (blue) and downregulated (green) genes in HUVECs treated with CZALO versus the model group, analyzed using RNA-Seq (threshold: log 10 (fold change) ≥ 2.0). (c) GO annotation analysis of DEGs in HUVECs under various treatments. (d) KEGG pathway analysis of DEGs in CZALO-treated HUVECs compared to the model group. (e) GSEA plots showing gene enrichment in the AGE-RAGE pathway (top) and TGF-β signaling pathway (bottom) between the model group and the CZALO-treated group. (f) Circular plot illustrating the interaction between DEGs and key signaling pathways. (g) Circular heatmap displaying the expression levels of DEGs linked to the AGE-RAGE, NF-kappa B, and TGF-β signaling pathways.

3.4. Anti-atherosclerosis therapeutic efficacy and mechanism of CZALO in ApoE^−/− mice

Firstly, the in vivo biosafety and biocompatibility of CZALO were evaluated in healthy C57BL/6 mice after intravenous administration of CZALO (1 mg kg⁻¹) for days 7 and 21, while control mice received saline injection. Routine blood assessments indicated that all hematological parameters remained within the normal range (Fig. S26). Even at a concentration of 800 μg mL⁻¹, there was neglectable hemolytic phenomena (Fig. S27). Additionally, hematoxylin and eosin (H&E) staining of primary tissues, including heart, liver, spleen, lung, and kidney, revealed no significant pathological changes (Fig. S28). These findings collectively suggest the superior biocompatibility of CZALO and suggest its availability for in vivo application. To confirm the OPN-mediated active targeting and intraplaque enrichment, atherosclerotic mice were intravenously injection of CZALO labeled with Cyanine5.5 (Cy5.5). Ex vivo fluorescence images demonstrated that the fluorescence intensity of thoracic aorta and aortic root in the CZALO group obviously stronger than that of the CZAL group, indicating the effective intraplaque accumulation of CZALO due to the OPN-enabled active targeting (Fig. 7a and b). Meanwhile, there was no significant difference in the biodistribution of other vital organs, such as kidneys, liver, lungs, and spleen (Fig. S29).

Fig. 7.

Anti-atherosclerosis therapeutic efficacy of CZALO in ApoE^−/− mice. (a, b) Ex vivo fluorescence images (a) and corresponding quantitative analysis (b) of hearts and aortas in atherosclerotic mice (n = 3). (c) Schematic illustration of the therapeutic protocols. (d, e) Representative (d) images and (e) corresponding quantitative analysis of ORO-stained aortas from atherosclerotic mouse after varied treatments (n = 6). (f, g) Representative (f) images of aortic arch sections stained with ORO and (g) corresponding quantitative analysis of the aortic sinus (n = 7). (h–k) Representative immunohistochemical images and quantitative analysis of aortic root sections stained with (h, i) H&E, (j, k) Masson's trichrome (n = 7). Note: L: plain liposomes, CZL: liposomes loaded with CZ NPs, CZAL: liposomes co-loaded with CZ NPs and L-arg, CZALO: CZAL liposomes further modified with OPN. Data in (b, e, g, i, k) are expressed as mean ± S.D. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant (P > 0.05).

To further investigate the in vivo anti-atherosclerosis efficacy of CZALO, ApoE^−/− mice received an eight-week high-fat diet to induce atherosclerotic plaques, as shown in Fig. 7c. Then the established atherosclerotic mice were randomly divided into five groups: Saline, L, CZL, CZAL, and CZALO. After 16 weeks of treatment, the in vivo therapeutic performance was firstly measured through ultrasound imaging. The resistance index in the left ventricular outflow tract, a marker indicting the degree of aortic arch stenosis, was reduced during CZALO treatment, suggesting relief of stenosis (Fig. S30). Aortas were then removed from atherosclerotic mice and longitudinally dissected to expose the artery intima. According to the ORO staining visualizing plaques (Fig. 7d and e), the CZALO group exhibited the lowest average plaque area percentage (13.72 %) compared to the saline (32.59 %), L (26.44 %), CZL (23.17 %), and CZAL (16.55 %). Similarly, to further investigate the anti-atherosclerosis efficacy and mechanisms, we collected frozen and paraffin sections at the aortic arch. The frozen sections were stained with ORO, and the results showed a decrease in the ORO-stained area from 14.5 % to 5.8 % (Fig. 7f and g), indicating that CZALO significantly reduces plaque area. These findings confirm that CZALO significantly reduces plaque area both longitudinally and horizontally. H&E staining indicated that atherosclerotic mice receiving CZALO treatment exhibited a reduction in necrotic cores from 42.31 % to 30.25 % compared to the saline group (Fig. 7h and i). Furthermore, the plaque formation and vascular stenosis can be possibly exacerbated by the migration and proliferation of smooth muscle cell and the production of collagen, thus Masson's trichrome staining and alpha-smooth muscle acti (α-SMA) immunohistochemical staining were employed to visualize smooth muscle cell and collagen content, respectively. Both Masson staining (Fig. 7j and k) and immumohistochemical staining of α-SMA antibody (Fig. S31) demonstrated that CZALO effectively reduced vascular smooth muscle cell infiltration and decreased collagen accumulation in the plaques, which is beneficial for stabilizing atherosclerotic plaques.

Encouraged by the above results, we conducted an in-depth exploration of the mechanisms by which CZALO combats atherosclerotic plaques in vivo. We firstly investigated the potential of CZALO to induce macrophage polarization and reprogram immune microenvironment for exerting anti-inflammatory effects. As depicted in Fig. 8a, Multiplex immunofluorescence staining was performed using F4/80, CD86, and CD206 for labeling macrophages, M1 macrophages, and M2 macrophages, respectively. Arteriosclerotic mice in the saline group exhibited high percentages of CD86⁺ macrophages (M1 type) and low percentages of CD206⁺ macrophages (M2 type) in their major arteries. CZALO treatment apparently reduced CD86⁺ macrophages and increased CD206⁺ macrophages, which verified that CZALO promoted polarization from M1 (inflammatory) to M2 (anti-inflammatory) macrophages. Additionally, CZALO demonstrated effective anti-atherosclerosis function by decreasing serum levels of inflammatory factors including TNF-α and IFN-γ, as shown in Fig. 8b–e. The above results indicate that CZALO combats atherosclerotic plaques in vivo by promoting the polarization of macrophages from the inflammatory M1 type to the anti-inflammatory M2 type and reducing serum levels of inflammatory factors.

Fig. 8.

Anti-atherosclerosis mechanism of CZALO in ApoE^−/− mice. (a) Representative immunofluorescence images of aortic root sections from ApoE^−/− mice subjected to different treatments, stained with antibodies for CD86, CD206, and F4/80. (b–e) Inflammatory factor levels of (b) TNF-α, (c) IFN-γ, (d) IL-4, and (e) IL-10 in serum (n = 3). (f–m) Representative immunofluorescence images and corresponding quantitative analysis of aortic root sections stained with antibody against (f, j) γ-H2AX, (g, k) p16, (h, l) p53 and (i, m) p21 (n = 3). Note: L: plain liposomes, CZL: liposomes loaded with CZ NPs, CZAL: liposomes co-loaded with CZ NPs and L-arg, CZALO: CZAL liposomes further modified with OPN. Data in (b–e) and (j–m) are expressed as mean ± S.D. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant (P > 0.05).

The in vitro cellular experiments have confirmed that NO released from CZALO can achieve significant anti-senescence effects by regulating SASP factor secretion, enhancing lysosomal function, alleviating cell cycle arrest, and reducing DNA damage in senescent endothelial cells. Consequently, we proceeded to explore its anti-aging mechanisms for treating atherosclerosis in vivo. According to the γ-H2AX immunofluorescent staining, a significant bright yellow fluorescent signal was observed in the saline group, while the CZALO group showed a substantial decrease in fluorescent signal within the plaque (Fig. 8f and j). Next, immunofluorescence staining was performed using the biomarkers of cellular aging, including p16, p21, and p53, to evaluate the anti-aging effect of CZALO. According to the experimental results, significant red fluorescence signals of p16 were observed in the saline group, while the fluorescence signals within the plaques were significantly reduced in the CZALO treatment groups (Fig. 8g and k). Similarly, in the p21 and p53 immunofluorescence staining images, the saline group showed strong fluorescence signals, whereas the fluorescence signals in the CZALO group progressively weakened (Fig. 8h–l, i and m). The above results indicate that CZALO exerts anti-aging effects in the treatment of atherosclerosis by downregulating the aging markers γ-H2AX, p21, p16, and p53. To sum up, the in vivo experiments demonstrated that CZALO exhibits synergistically anti-inflammatory and anti-aging effects, thereby effectively treating atherosclerosis.

4. Discussion

Atherosclerosis poses a substantial global health challenge and remains the leading cause of cardiovascular diseases, including coronary artery disease, peripheral arterial disease, and stroke [59]. Its pathogenesis is driven by a complex interplay among dysregulated lipid metabolism, chronic inflammation, oxidative stress, and endothelial dysfunction, culminating in plaque formation and vascular damage [60]. Existing therapeutic strategies predominantly target isolated pathological mechanisms, such as inflammation or oxidative stress. Current therapeutic approaches often struggle to address the complex and interconnected pathways underlying atherosclerosis, emphasizing the critical need for innovative, multi-targeted strategies. By simultaneously modulating key aspects of disease pathology, such integrative approaches have the potential to more effectively slow disease progression, enhance therapeutic efficacy, and overcome the inherent limitations of conventional monotherapies in managing this major contributor to global morbidity and mortality.

In this study, CZALO nanoliposomes were developed as an innovative therapeutic platform to target the multifaceted pathophysiology of atherosclerosis. The experimental results demonstrate that CZALO effectively addresses the limitations of conventional monotherapies by integrating antioxidant, anti-inflammatory, and anti-senescence functionalities. Specifically, CZALO exhibited a robust capacity to scavenge ROS, enabled by the enzyme-like activities of CZ NPs. Fluorescence-based assays confirmed a significant reduction in intracellular ROS levels following CZALO treatment, providing protection against oxidative damage in macrophages (Fig. 3a). Furthermore, CZALO significantly attenuated lipid accumulation and foam cell formation, as indicated by decreased Oil Red O staining and reduced intracellular cholesterol levels (Fig. 3d and f). This antioxidative effect is pivotal for disrupting the early atherogenic processes initiated by oxidative stress, underscoring the therapeutic potential of CZALO in combating atherosclerosis at its molecular roots.

Macrophage polarization is a crucial determinant of the inflammatory microenvironment in atherosclerotic plaques and directly impacts plaque stability [61]. Pro-inflammatory M1 macrophages exacerbate local inflammation and destabilize plaques by producing cytokines such as TNF-α and IFN-γ. In contrast, anti-inflammatory M2 macrophages secrete IL-10 and IL-4, promoting tissue repair and enhancing plaque stability [62]. Shifting the balance from the M1 to the M2 phenotype is therefore a critical therapeutic strategy for mitigating atherosclerosis. CZALO effectively modulated macrophage polarization, inducing a phenotypic transition from M1 to M2 macrophages (Fig. 3g and h). This shift was evidenced by an increased expression of the M2 marker CD206 and a decreased expression of the M1 marker CD86. These changes were accompanied by reduced levels of pro-inflammatory cytokines, such as TNF-α and IFN-γ (Fig. 3i and j), and elevated levels of anti-inflammatory cytokines, including IL-10 and IL-4 (Fig. 3k and l). Transcriptomic analysis further demonstrated that CZALO downregulated key inflammatory pathways, including NF-κB, TNF, and Toll-like receptor signaling, which are central to chronic inflammation and plaque instability (Fig. 4d). By reprogramming the inflammatory microenvironment, CZALO enhanced plaque stability, highlighting its potential as a promising therapeutic strategy for combating atherosclerosis.

Endothelial cell senescence is a key pathological driver of atherosclerosis, contributing to vascular dysfunction and plaque instability. Senescent endothelial cells lose their regenerative capacity and secrete pro-inflammatory factors collectively known as the SASP, including IL-6, IL-8, and TNF-α. These factors exacerbate inflammation, oxidative stress, and endothelial dysfunction, accelerating plaque progression and destabilization. Furthermore, senescence is characterized by lysosomal dysfunction, DNA damage, and elevated expression of cell cycle arrest markers such as p16 and p53, which further compromise vascular homeostasis [63]. Thus, targeting endothelial senescence is critical for restoring vascular function and mitigating atherosclerosis progression. CZALO effectively counteracted endothelial senescence through the selective release of NO (Fig. 5b). This intervention significantly reduced the secretion of SASP factors, including IL-6, IL-8, and TNF-α, thereby alleviating the pro-inflammatory environment (Fig. 5c–f). Moreover, CZALO enhanced lysosomal function by decreasing SA-β-gal activity and lipofuscin accumulation, key markers of cellular aging (Fig. 5g and h). It also mitigated DNA damage, as evidenced by reduced γ-H2AX levels, and suppressed the expression of cell cycle arrest markers such as p16 and p53 (Fig. 5i–k). These findings underscore CZALO's dual ability to protect endothelial cells from senescence-induced dysfunction while restoring their physiological activity, highlighting its potential as an innovative therapeutic strategy for atherosclerosis treatment.

The multifunctionality of CZALO underscores its potential as a comprehensive therapeutic strategy for atherosclerosis. By simultaneously addressing key pathological mechanisms, including oxidative stress, macrophage-driven inflammation, and endothelial dysfunction, CZALO represents a significant advancement over existing therapies. Furthermore, its targeted delivery system minimizes off-target effects, as evidenced by its excellent biocompatibility in vivo. These results suggest that CZALO has the potential to transform the treatment landscape of atherosclerosis, offering a novel, multi-targeted approach to mitigate disease progression and reduce cardiovascular risk. Future studies focusing on the clinical translation of CZALO and its potential integration with existing treatment regimens could further enhance its therapeutic utility and broaden its impact in managing cardiovascular diseases.

5. Conclusion

In summary, we have designed and engineered an innovative CZALO nanoliposome to implement a “two-birds-one-stone” strategy for specifically regulating vascular microenvironment homeostasis in effective anti-atherosclerosis therapy. First, CZALO selectively targets atherosclerotic sites and enters macrophages via OPN-mediated endocytosis, subsequently releasing encapsulated CZ nanoparticles (NPs) and L-Arg. The CZ NPs mimic multiple natural antioxidant enzymes, such as SOD, POD, CAT, and GPx, to effectively scavenge ROS, thereby significantly inhibiting cholesterol uptake and promoting macrophage phenotype transformation, which provides antioxidant and anti-inflammatory effects as the first benefit. Simultaneously, L-Arg is catalyzed by overexpressed NOS in macrophages to produce NO gas, which is selectively released in situ and then diffuses into endothelial cells. This process regulates SASP factor secretion, enhances lysosomal function, disrupts cell cycle arrest, and reduces DNA damage in senescent endothelial cells, thereby achieving anti-aging effects as the second benefit. The combined antioxidant/anti-inflammatory and anti-aging effects synergistically modulate the atherosclerotic microenvironment, reducing atherosclerosis burden with satisfactory biosafety both in vitro and in vivo. As illustrated in Table S1, this work emphasizes CZALO's unique capability to concurrently target and treat atherosclerosis by leveraging its selective antioxidant properties and anti-senescence mechanisms.

CRediT authorship contribution statement

Yuanyuan Peng: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Wei Feng: Validation, Methodology, Formal analysis, Data curation. Hui Huang: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Yu Chen: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Shaoling Yang: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Ethics approval and consent to participate

All experimental procedures were performed under the policies of the National Ministry of Health with the approval of the Ethic Committee of Shanghai University (approved ID: ECSHU-2022-050).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix B. Supplementary data

The following is the Supplementary data to this article: