A hierarchical theranostic nanoagent for multimodal imaging and targeted foam cell intervention in atherosclerosis

Domain: biology
Published: 2026-03-11
Editorial outcome: major_revision

Abstract
Atherosclerosis (AS) is a leading cause of cardiovascular and cerebrovascular disease and is driven by lipid accumulation and chronic inflammation within arterial plaques. Foam cells play a central role in plaque growth and destabilization, yet their precise diagnosis and targeted regulation remain challenging. Here we present a hierarchical nanoagent that integrates multimodal imaging with selective foam cell intervention for improved AS management. The molecular probe enables near-infrared II fluorescence, photoacoustic, and magnetic resonance imaging, providing complementary information on plaque location, depth, and morphology. To achieve targeted therapy, the probe is co-encapsulated with atorvastatin using responsive nanocarriers camouflaged with macrophage membranes and equipped with a foam cell-targeting peptide. This hierarchical strategy enables precise targeting to inflamed plaques and foam cells, where controlled drug release promotes lipid efflux, reduces inflammatory signaling, and enhances plaque stabilization in female AS mouse models. This integrated imaging-guided therapeutic platform holds great promise for precise diagnosis and treatment of cardiovascular disease.

Introduction
Cardiovascular diseases (CVDs) remain the leading global cause of morbidity and mortality, accounting for nearly half of all deaths worldwide annually.[1] Among these, atherosclerosis (AS) stands out as the most prevalent and clinically significant, representing a chronic inflammatory disorder characterized by the progressive, often asymptomatic accumulation of lipid-laden plaques within the arterial walls.[2-4] Over time, these plaques can rupture, precipitating catastrophic cardiovascular events such as myocardial infarction, stroke, and sudden cardiac death.[5,6] Early detection and precise monitoring of AS are paramount for facilitating timely therapeutic interventions, mitigating symptoms, and halting disease progression.[7-9] However, current diagnostic modalities are limited in their capacity to detect early-stage lesions with the requisite sensitivity and specificity, thereby hindering effective clinical management. Traditional plaque imaging techniques, such as intravascular ultrasound and optical coherence tomography suffer from low sensitivity, weak functional signals, restricted imaging depth (typically 1-3 mm), and invasiveness, which significantly restrict their broader clinical application.[10] In contrast, magnetic resonance imaging (MRI), with its non-invasive nature and absence of ionizing radiation, offers a promising alternative for detecting AS plaques.[11,12] However, conventional small-molecule MRI contrast agents, including gadolinium-based compounds, are characterized by low specificity, rapid systemic clearance, and suboptimal image quality, which undermine diagnostic precision.[13-15] These limitations, coupled with the extended acquisition time required for high-resolution imaging further limit their utility for real-time monitoring and detection of smaller plaques.[16,17] To address these challenges, integrating MRI with other complementary imaging modalities emerges as a promising approach to enhance diagnostic accuracy and sensitivity by providing more comprehensive, high-resolution pathological insights.[18,19]

Optical imaging, renowned for its high sensitivity, real-time capabilities, cost-effectiveness, and accessibility, offers a valuable complementary tool to MRI.[20,21] In particular, the second near-infrared (NIR-II) window (1000-1700 nm) has been explored for optical imaging recently, which can minimize tissue autofluorescence and light scattering, thereby enabling superior tissue penetration and enhanced signal-to-noise ratio.[22-24] Furthermore, photoacoustic (PA) imaging, a rapidly advancing non-invasive modality, has garnered increasing attention in the biomedical field.[25,26] PA imaging offers deeper tissue penetration (up to several centimeters) and high spatial resolution, surpassing traditional fluorescence imaging.[27,28] Despite these promising attributes, the sensitivity of PA imaging remains a notable challenge.[29] Given the complex and heterogeneous pathological microenvironment of AS, there is a pressing need for multimodal imaging strategies that leverage the strengths of each imaging modality to provide more comprehensive insights into disease diagnosis, treatment, and therapeutic outcome evaluation.[30,31] In this regard, single-molecule probes capable of integrating multiple imaging functions hold substantial promise, offering distinct advantages over traditional mixed-mode systems. However, optimizing the performance of each imaging modality within a single molecule poses a significant challenge, as different imaging components often interfere with one another.[32,33] Currently, reports of single-molecule probes that simultaneously enable MRI, NIR-II fluorescence, and PA imaging remain rare, highlighting the need for further efforts to overcome these obstacles and fully exploit the potential of such multimodal imaging platforms in AS diagnosis.

Foam cells, along with the inflammatory responses they provoke, play pivotal roles in the pathogenesis of AS.[11] These lipid-laden macrophages accumulate within atherosclerotic plaques, where they contribute significantly to plaque instability and progression.[34] Foam cells are central to necrotic core formation and act as potent producers of pro-inflammatory cytokines, which further recruit immune cells to the plaque site to amplify the inflammatory cascade.[35-38] Moreover, foam cells express surface molecules such as CD47, a "don't eat me" signal, which allows them to evade phagocytosis and clearance by the immune system.[39-41] Given their crucial involvement in the disease process, the precise imaging and regulation of foam cells are vital for both accurate detection and effective treatment of AS. However, the complex microenvironment of atherosclerotic plaques makes targeted imaging and effective modulation of foam cells challenging.[42,43] Inefficient plaque accumulation, limited penetration into the plaque core, and nonspecific targeting of foam cells associated with conventional imaging agents and drugs usually compromise imaging outcomes and lead to undesirable side effects.[44,45] These limitations undermine the effectiveness of both diagnostic and therapeutic strategies in managing AS. Consequently, there is an urgent need to develop advanced, multi-tiered targeting strategies that facilitate more precise imaging of pathological foam cells and enable efficient therapeutic interventions.

In this work, we present a hierarchical nanoplatform for precise multimodal imaging and targeted intervention of AS (Fig. 1). We engineered a high-performance multimodal molecular probe that enabled simultaneous MRI, NIR-II fluorescence, and PA imaging. Through rational molecular design, the signals for each imaging modality were optimized, offering a robust imaging system. In particular, NIR-II fluorescence imaging, with its high sensitivity and real-time visualization capabilities, facilitated rapid localization of the disease and dynamic, in situ tracking of plaque progression. MRI, known for its deep tissue penetration, enabled precise detection of plaque location and volume, aiding in the assessment of plaque stability. PA imaging, with its superior spatial resolution, supplied detailed insights into plaque morphology. By integrating these modalities, this molecular probe offers a versatile platform for comprehensive AS evaluation, which also holds great potential for guiding subsequent therapeutic interventions and offering in situ assessment of therapeutic outcomes. This probe, along with the therapeutic agent atorvastatin (AT), was encapsulated in responsive nanocarriers, which were subsequently coated with biomimetic macrophage membranes and functionalized with a phosphatidylserine-targeting peptide, thereby creating a hierarchical targeting platform. This engineered nanosystem exploits the adhesive bioligands on macrophage membranes for selective targeting of inflamed endothelium, while the targeting peptide further directs the system to foam cells within plaques. The AT released in foam cells promoted lipid efflux and reduced the expression of CD47 demonstrated that the targeted manipulation of foam cells phagocytosis significantly inhibited plaque progression, alleviated the inflammatory burden, and reinforced plaque stability in AS modeled mice. By combining hierarchical targeting, multidimensional visualization, and on-demand manipulation of pathological cells, this platform provides a novel paradigm for advanced management of AS.

Results
Design and property of molecular probes
As shown in Fig. 2a, a low-bandgap molecule was conjugated with Ga-DOTA to create the targeted molecular probe with multimodal imaging capabilities. The probe was designed by synthesizing a donor-acceptor (D-A) type chromophore, in which fluorene and benzobisthiadiazole (BBT) served as the donor and acceptor units, respectively. The electron-rich, highly fluorescent fluorene groups enhance the probe's brightness, while the methoxy-substituted thiophene ring plays a dual role: facilitating electron transfer and extending the conjugation length. Furthermore, the steric hindrance introduced by the methoxy group induces a distorted molecular geometry, which prevents aggregation-caused quenching and further improves the fluorescence brightness.[46] The incorporation of highly conjugated and electron-withdrawing BBT unit results in a long-wavelength-response NIR emitter (FTB), which is ideal for both fluorescence and PA imaging. To endow FTB with MRI properties, Gd-DOTA was conjugated to the carboxyl group of FTB via an amidation reaction. The large Gd-DOTA moiety is expected to avoid strong intermolecular interactions, such as \pi-\pi stacking, leading to brighter fluorescence emission. Moreover, the Gd-DOTA substitution could also provide some room for molecular motion in aggregate state, facilitating PA transformation.[47] Additionally, this conjugation effectively restricts the flipping rate of the gadolinium ion compared to small gadolinium molecules, which in turn enhances the relaxation time and longitudinal relaxivity, resulting in an amplified MRI signal.[48] Detailed syntheses and characterizations of FTBGd are provided in the Supporting Information (Supplementary Fig. 1-16).

The absorption and photoluminescence (PL) spectra of FTBGd in THF revealed maximal absorption and emission at 776 nm and 984 nm, respectively (Fig. 2b), indicating great potential for NIR-II fluorescence and PA imaging. To investigate the emission characteristics in aggregate states, FL spectra of FTB and FTBGd were recorded in THF/water mixtures with varying water fractions (f ). As shown in Fig. 2c and Supplementary Fig. 17, FTB exhibited a 5.2-fold enhancement in PL intensity at high f (95%), while FTBGd demonstrated an even greater 8.6-fold amplification under the same conditions, highlighting the favorable aggregation-induced emission (AIE) feature of FTBGd. Unlike conventional fluorophores that exhibit aggregation-caused quenching effect, AIE-active molecules are non-emissive or weakly emissive in solution but become highly fluorescent upon aggregation. Furthermore, a direct comparison of their PL intensities in the aggregate state (f = 95%) revealed that FTBGd emitted fluorescence over five times stronger than FTB (Fig. 2d). This remarkable enhancement can be attributed to the anti-quenching effect of Gd-DOTA block in FTBGd, which effectively mitigates fluorescence quenching. Additionally, the PA spectrum of FTBGd was further examined, which exhibited a strong correlation with its absorption profile and exhibited a maximum PA signal around 780 nm (Supplementary Fig. 18). To assess the MRI property, we measured the MR signal intensity of both FTBGd, FTB, and Gd-DOTA at an equal concentration (0.05 mM). As shown in Supplementary Fig. 19, FTBGd exhibited a bright MR signal, whereas FTB, lacking Gd-DOTA substitution, produced negligible MR signals, confirming that the MR contrast originates from Gd-DOTA moiety. Furthermore, at the same Gd content, FTBGd displayed significantly enhanced MR signal intensity compared to Gd-DOTA alone. This improvement can be attributed to the larger molecular size of Gd-DOTA conjugate, which not only restricts gadolinium’s molecular tumbling rate but also enhances its ability to amplify MR contrast through increased longitudinal relaxivity.[49] Additionally, the MR signal intensity of FTBGd was found to increase accordingly with probe concentration (Fig. 2e). The stability of FTBGd as a multimodal imaging probe was further evaluated by exposing its solution to common cellular and in vivo interferents, including hydrogen peroxide (H O ), superoxide (O •−), cysteine (Cys), glutathione (GSH), and various metal ions. Notably, FTBGd maintained stable FL and MR signals in the presence of these interferents (Supplementary Fig. 20), demonstrating its resistance to external influences.

Fabrication and characterization of nanoagents
To develop a biocompatible and multifunctional platform, FTBGd and therapeutic drug AT were co-encapsulated within a ROS-responsive polymer matrix, yielding the water-soluble nanoprobe FA-P. AT, a widely used HMG-CoA reductase inhibitor, has been employed for CVD management. Recent studies indicate that AT can also exert local effects within vascular walls and immune cells, including modulation of cholesterol efflux and anti-inflammatory activity.[50,51] Based on these properties, AT was incorporated in this work to locally modulate lipid metabolism and foam cell formation. Foam cells typically exhibit elevated ROS levels due to their uptake of oxLDL and impaired mitochondrial function. Thus, we utilized poly (1,4-phenyleneacetone dimethylene thioketal) (PPADT), a ROS-responsive polymer in which thioketal (TK) bonds undergo cleavage and oxidation to sulfone in the presence of ROS, to serve as both a nanocarrier for ROS neutralization and a controlled drug-release system within ROS-enriched foam cells.[52] The synthesis and characterization of PPADT are detailed in Supplementary Fig. 21. FA-P, co-loaded with FTBGd and AT, were formulated via the nanoprecipitation method. As confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses, FA-P exhibited a spherical morphology with an average hydrodynamic diameter of 138 nm (Supplementary Fig. 22a). To enhance the targeting capability, the nanoprobes were further cloaked with macrophage cell membranes and modified with phosphatidylserine (PS)-targeting peptides (CLIKKPF), resulting in the formation of FA-P@M and FA-P@MC, respectively.[53,54] The biomimetic macrophage membranes, equipped with natural adhesion molecules such as \alpha4\beta1 integrins, facilitate interactions with activated endothelial cells and inflamed atherosclerotic plaques.[55,56] Meanwhile, the PS-targeting peptide promotes the subsequent targeting to the exposed PS on foam cells within the plaque. TEM and DLS measurements revealed that FA-P@M exhibited a spherical morphology with an average diameter of approximately 156 nm (Supplementary Fig. 22b), with a thin membrane layer approximately 10 nm on the surface. Similarly, FA-P@MC was coated with a membrane layer, and the further incorporation of PS-targeting peptides resulted in a slight increase in particle size to 163 nm (Fig. 2f). Furthermore, the zeta potential of FA-P@M decreased to −13.1 mV and then increased to −5.8 mV for FA-P@MC (Fig. 2g), which could be attributed to the introduction of negatively charged cell membranes and positively charged PS-targeting peptides.[57] The peptide coating density on FA-P@MC was estimated, and the NPs synthesized from different batches exhibited good reproducibility (Supplementary Fig. 23). Additionally, Western blotting analysis confirmed the presence of membrane proteins inherited from the macrophage in both the macrophage cell membrane and FA-P@MC (Fig. 2h). The colloidal stability of FA-P@MC was further assessed, and the size remained largely unchanged over 3 days in a physiologically relevant medium containing 10% fetal bovine serum (FBS) (Supplementary Fig. 24).

We next investigated the photophysical properties of FA-P@MC. The maximum absorption of FA-P@MC was observed at 790 nm, with emission spanning from 850 to 1250 nm within the NIR-II region (Supplementary Fig. 25), aligning well with the spectra of FTBGd probe. Excitation-emission mapping also revealed that FA-P@MC could be efficiently excited by NIR light, with strong fluorescence emission in the NIR-II spectral region above 1000 nm (Fig. 2i). Their potential for PA imaging was further examined, and the nanoprobe exhibited maximal PA signal at approximately 790 nm (Fig. 2j), corresponding to its absorption spectrum. Notably, the PA intensity displayed a linear relationship with the concentration of FA-P@MC, highlighting their potential for quantitative analysis (Fig. 2k). The PA signal demonstrated excellent stability, with minimal change in the intensity under continuous 790 nm laser excitation (inset of Fig. 2k). We also compared the photostability of FA-P@MC with the FDA-approved contrast agent, indocyanine green (ICG). The results showed that FA-P@MC exhibited remarkable stability and resistance to photobleaching, while the absorption intensity of ICG significantly decreased with light exposure (Supplementary Fig. 26). Subsequently, MRI analysis demonstrated that FA-P@MC generated strong MR signals, with the intensity nearly 3 times greater than the clinically used Gd-DOTA (Fig. 2l). These findings further validated that the rational design and integration of Gd-DOTA with the probe not only preserved the individual imaging signals but also enhanced their performance, leading to the development of a robust multimodal contrast agent.

Next, we investigated the ROS-triggered release process. As shown in Fig. 2m, in the presence of H O , DLS and TEM measurements revealed that FA-P@MC underwent substantial breakdown, resulting in both small and large aggregates. Furthermore, upon the H O -induced degradation, the MR signal of FA-P@MC increased by approximately 1.5-fold (Supplementary Fig. 27), whereas the non-responsive control nanoplatform, FA@MC (prepared without the ROS-responsive polymer), exhibited minimal changes in signal intensity in the presence of ROS. The observed increase in MR signal intensity following FA-P@MC decomposition was likely attributed to enhanced interaction between water and the paramagnetic Gd-DOTA center, thereby shortening the water molecule retention time and improving T relaxation. Additionally, the release of AT from FA-P@MC was quantified using high-performance liquid chromatography (HPLC). Based on previous reports[58,59], FA-P@MC were incubated with different concentrations of H O to mimic the oxidative microenvironment of atherosclerotic lesions. First, in the presence of H O , AT release increased over time, showing a rapid release compared to the untreated control (Supplementary Fig. 28). Under identical H O conditions, FA-P@MC displayed slightly lower drug release than FA-P lacking membrane coating (Supplementary Fig. 29), indicating that the membrane coating only modestly influenced the H O -responsive release. We also included a physiological H O concentration (2.5 \muM) as a control condition. At this level, cumulative release from both FA-P and FA-P@MC remained below 10% over 36 hours (Supplementary Fig. 30b). This result indicated the drug release was limited under basal physiological conditions but markedly enhanced in high H O environments. Furthermore, pre-treatment with catalase to decompose H O (1.0 mM) effectively inhibited drug release from both FA-P and FA-P@MC, providing evidence that the release process is H O -dependent (Supplementary Fig. 30c). Beyond H O , we further examined the responsiveness of FA-P@MC to other common ROS in atherosclerotic plaques, including hydroxyl radicals (•OH) and superoxide anions (O •−). As shown in Supplementary Fig. 31, both ROS effectively triggered the AT release from FA-P@MC. These findings demonstrate that FA-P@MC are responsive to multiple ROS species, confirming their ROS-sensitive degradation and release properties.

In vitro cell investigation
The safety profile of nanoprobe is paramount for their application in bioimaging. In this context, we first assessed the biocompatibility of FA-P@MC prior to their use in in vivo imaging studies. Cytotoxicity assays were initially conducted using HUVECs and NIH 3T3 cells (Supplementary Fig. 32a). No cytotoxicity was observed in either cell line, even at a concentration of 100 \muM, thereby confirming their low toxicity. Furthermore, to investigate the potential effects of FA-P@MC on cellular membrane integrity, we performed a lactate dehydrogenase (LDH) assay, a widely accepted indicator of plasma membrane damage. The absence of substantial LDH release across all tested concentrations (ranging from 10 to 100 \muM) in any of the cell lines further confirmed the cytocompatibility of FA-P@MC (Supplementary Fig. 32b).

We then focused on assessing the targeting efficacy of FA-P@MC towards foam cells, which is a key component of atherosclerotic plaques. Foam cells form when macrophages uptake lipid material, and during this process, PS externalization becomes a distinctive feature of their pathophysiology, rendering PS an ideal target for selective binding. We then evaluated the targeting capability of the macrophage membrane-coated, PS-binding peptide-modified nanoprobe toward foam cells. Following incubation with various formulations, foam cells were visualized using confocal laser scanning microscopy (CLSM). The results clearly demonstrated that foam cells internalized substantially more FA-P@M compared to FA-P that lacked macrophage membrane coating after 4 h of incubation at 37 °C (Fig. 3a). Furthermore, the incorporation of PS-targeting peptide in FA-P@MC resulted in a 4.1-fold increase in cellular uptake compared to FA-P and a 1.6-fold increase compared to FA-P@M (Fig. 3b). Additionally, the FA-P@MC prepared from different batches showed comparable cellular internalization, indicating consistent foam cell–targeting capability across batches (Supplementary Fig. 33). These results suggested the potential of engineered macrophage membrane-coated delivery systems to enhance targeting.

We subsequently evaluated the transmigration of the nanoparticles (NPs) across inflamed HUVECs and their uptake by foam cells through in vitro transwell migration assays. The transwell model comprised a monolayer of HUVECs in the upper chamber and foam cells in the lower chamber (Fig. 3c). HUVECs in the upper chamber were subjected to the following treatments: (1) LPS + FA-P, (2) LPS + FA-P@M, (3) LPS + FA-P@MC, and (4) FA-P@MC. The internalization of NPs by foam cells in the lower chamber was subsequently assessed using CLSM and flow cytometry. As shown in Fig. 3d, the NP signal in the foam cells of the FA-P-treated group was faint, suggesting limited passive transendothelial migration of the unmodified NPs. In contrast, FA-P@M exhibited a marked increase in NP signal in the basolateral chamber, indicative of enhanced endothelial permeability and foam cell uptake following macrophage membrane coating. The enhancement in NP transmigration is likely attributed to the presence of cell surface proteins, such as integrins, on the macrophage membrane, which could facilitate the efficient passage of encapsulated NPs across the inflamed endothelial barrier. Remarkably, FA-P@MC exhibited markedly superior endothelial crossing and foam cell targeting capabilities, as evidenced by the strongest NP fluorescence observed in foam cells. As depicted in Fig. 3e, quantitative analysis revealed that the quantities of NPs migrating across the endothelial layer into the basolateral foam cells were 5.24- and 1.88- times higher compared to the FA-P and FA-P@M groups, respectively. Moreover, in the absence of LPS pre-treatment to induce inflammatory conditions, there was a significant reduction in the permeability of FA-P@MC, highlighting the enhanced endothelial traversal and foam cell uptake under inflamed conditions. Flow cytometry analysis further corroborated the CLSM findings (Fig. 3f and Supplementary Fig. 34), confirming that the macrophage membrane coating and PS-binding peptide modification significantly facilitated the transport of NPs across the inflamed endothelial barrier and their subsequent internalization by foam cells.

In vitro anti-inflammatory and immunomodulatory effects
AS is a chronic inflammatory disease, with foam cell formation playing a pivotal role in its progression.[11] Foam cells not only contribute to lipid deposition but also act as key contributors to inflammation in atherosclerotic lesions. FA-P@MC were formulated by co-assembling a ROS-responsive polymer with AT, which have the potential to scavenge ROS, reduce lipid accumulation, and mitigate inflammation. To evaluate the therapeutic efficacy of FA-P@MC, an in vitro model of foam cell was established using oxLDL-treated macrophages. After 24 h of incubation with oxLDL (50 \mug/mL), a pronounced oxidative stress response was observed in foam cells, as detected by the ROS fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Fig. 3g, h). In contrast, FA-P@M and FA-P@MC treatment led to a reduction in DCF fluorescence, with the most pronounced effect observed in the FA-P@MC-treated cells. Flow cytometry analysis further demonstrated that FA-P@MC were more effective than FA-P@M in alleviating oxidative stress (Fig. 3i and Supplementary Fig. 35), potentially due to their enhanced internalization by foam cells.

Subsequently, we evaluated the ability of FA-P@MC to reduce the oxLDL uptake and inhibit foam cell formation. RAW264.7 macrophages exposed to 50 \mug/mL oxLDL for 24 h exhibited significant intracellular lipid droplet accumulation and the formation of foam cells, as verified by Oil Red O (ORO) staining (Fig. 3j, k). Importantly, FA-P@MC treatment markedly suppressed foam cell formation in comparison to other groups. Using the DiI-labeled oxLDL (DiI-oxLDL), fluorescence microscopic analysis further confirmed that FA-P@MC markedly inhibited oxLDL uptake by RAW264.7 cells (Supplementary Fig. 36a,b). Mechanistic studies revealed that FA-P@MC treatment downregulated the expression of scavenger receptors CD36 and LOX-1, which were responsible for oxLDL internalization (Supplementary Fig. 36c–f). To assess the cholesterol efflux, we measured cholesterol levels in the culture supernatant of treated foam cells. We found that the cholesterol content in the supernatant of FA-P@MC group was significantly increased (Supplementary Fig. 37a). Immunofluorescence staining further showed the upregulation of cholesterol transporters SR-BI and ABCA1 after FA-P@MC treatment (Supplementary Fig. 37b–e). We also performed functional cholesterol efflux assays in foam cells using BODIPY-labeled cholesterol with ApoA-I (ABCA1-dependent) or HDL (SR-BI/ABCG1-associated) as acceptors. FA-P@MC enhanced cholesterol efflux to both acceptors compared with other nanoformulations, indicating engagement of these transporters in promoting efflux. Pharmacological inhibition of ABCA1 markedly reduced FA-P@MC-enhanced efflux to ApoA-I, whereas SR-BI inhibition reduced efflux to HDL (Supplementary Fig. 38), consistent with their contribution under our experimental conditions. Parallel studies in foam cells induced from bone marrow–derived macrophages (BMDMs) showed similar FA-P@MC–enhanced efflux, which was attenuated by ABCA1 or SR-BI inhibition (Supplementary Fig. 39), supporting involvement of these pathways without suggesting exclusivity.

Collectively, these results indicated that FA-P@MC not only limited lipid uptake but also enhanced cholesterol efflux via upregulation of efflux transporters, thereby suppressing foam cell formation. We then proceeded to evaluate whether FA-P@MC could also mitigate cellular inflammation in foam cells. The PBS-treated foam cells displayed elevated nitric oxide (NO) levels, indicative of an inflammatory response (Supplementary Fig. 40). Compared to free AT alone, FA-P showed improved efficacy in reducing cellular NO production, likely due to the enhanced intracellular delivery of AT via the nanocarriers. Furthermore, treatment with FA-P@M or FA-P@MC significantly reduced NO production in foam cells, with FA-P@MC exhibiting the most potent effect in suppressing NO generation. These results highlighted that FA-P@MC not only inhibited foam cell formation but also alleviated the inflammatory response within foam cells.

Efferocytosis is the process by which macrophages engulf and eliminate dead or dying cells, including foam cells, which is essential for preventing plaque necrosis and instability.[60,61] However, in atherosclerotic lesions, the efficiency of efferocytosis diminishes over time due to the chronic inflammatory environment and cellular dysfunction. Notably, the upregulation of CD47, a "don't eat me" signal, on the surface of foam cells inhibits their clearance by macrophages. As a result, foam cells can persist in the plaque, contributing to the formation of a necrotic core and heightening the plaque's vulnerability to rupture. The oxidative and inflammatory milieu in atherosclerotic plaques promote the upregulation of CD47 on foam cells, thus we next sought to investigate whether the ROS-scavenging, AT-loaded, and foam cell-targeting nanoformulations could enhance efferocytosis by modulating CD47 expression. First, CD47 expression in foam cells across different treatment groups was evaluated by Western blotting and confocal microscopy. As shown in Fig. 4a-c, foam cells exhibited a high level of CD47 expression, which was consistent with previous reports.[39-41] However, following treatment with nanoformulations, particularly FA-P@MC, the expression of CD47 on foam cells was significantly reduced. To study the ROS-dependent regulation of CD47, we employed specific ROS scavengers, including catalase and mannitol, to selectively eliminate H O and •OH, respectively. The effective removal of each ROS was first verified using corresponding fluorescent probes (Supplementary Fig. 41a). We found that the presence of these scavengers partially attenuated FA-P@MC–induced CD47 downregulation, likely due to the reduced AT release. In line with this, the ROS-nonresponsive control NPs (FA@MC) exhibited weak effect on CD47 expression (Supplementary Fig. 41b,c). Collectively, these findings suggested the ROS-responsive CD47 downregulation effect of FA-P@MC. Then we explored whether the reduction in CD47 expression on foam cells could potentially enhance their phagocytosis by macrophages. As illustrated in Fig. 4d, bone marrow-derived macrophages (BMDMs) were harvested and applied as phagocytic cells, while oxLDL-induced RAW264.7 cells served as foam cells. CLSM images revealed that significantly more foam cells treated with FA-P@MC were engulfed by BMDMs compared to those treated with PBS (Fig. 4e). Flow cytometry analysis further confirmed that the efferocytosis rate of foam cells in the FA-P@MC group (~68%) was substantially higher than in the PBS, Free AT, A-P, FA-P, and FA-P@M groups by 8.92, 2.97, 2.02, 2.16, and 1.26 times, respectively (Fig. 4f, g and Supplementary Fig. 42). Additionally, for the non-ROS-responsive control FA@MC, weak CD47 downregulation and limited phagocytosis were observed (Supplementary Fig. 43). These results suggest that ROS-enhanced drug release contribute to the subsequent CD47 downregulation and phagocytosis increase. These findings indicate that FA-P@MC promoted the phagocytosis of foam cells by macrophages, suggesting their potential to promote plaque regression and stabilization.

In vivo biosafety and pharmacokinetics of FA-P@MC
Prior to conducting in vivo bioimaging studies, the biosafety profile and pharmacokinetics of FA-P@MC were evaluated. Healthy C57BL/6 mice were administered PBS or FA-P@MC (10 mg kg-1) by intravenous injection twice weekly for 2 weeks. Histological analysis of major organs, including the heart, liver, spleen, lung, and kidneys (Supplementary Fig. 44), revealed no pathological abnormalities or inflammatory lesions in the FA-P@MC-treated mice. Furthermore, comprehensive blood panel tests indicated that the hematological parameters of the mice treated with FA-P@MC remained within normal physiological ranges (Supplementary Fig. 45). Additionally, liver biomarkers, such as alanine transaminase (ALT) and aspartate aminotransferase (AST), as well as kidney function markers, including blood urea nitrogen (BUN) and creatinine, showed no significant differences between the PBS group and FA-P@MC-treated group (Supplementary Fig. 46). Moreover, after seven weeks of continuous administration, no obvious abnormalities were detected in the major organs, routine blood parameters, or hepatorenal function indicators (Supplementary Fig. 47), indicating their favorable long-term biocompatibility. Additionally, no significant hemolysis was observed, even at a concentration of 100 \muM (Supplementary Fig. 48). Taken together, these results strongly highlighted the favorable biocompatibility of FA-P@MC, positioning it as a promising candidate for in vivo AS imaging and therapeutic applications.

Subsequently, the in vivo circulation profiles of FA-P@MC were investigated. The NPs were intravenously injected into mice, and blood samples were collected at designated time points post-injection to measure the fluorescence signal of the NPs in circulation. As shown in Supplementary Fig. 49, free AT was rapidly cleared from the bloodstream, exhibiting a half-life of ~15 min. In contrast, FA-P@MC demonstrated a significantly extended circulation time, comparable to that of FA-P@M. These findings indicated that the incorporation of foam cell-targeting peptides on the macrophage membrane surface did not substantially alter the circulation time of the NPs. FA-P@MC exhibited prolonged circulation profiles compared to free drug, highlighting their enhanced propensity to accumulate at atherosclerotic sites and sustain therapeutic efficacy. The biodistribution of FA-P@MC was systematically assessed in major organs (liver, spleen, kidneys, heart, and lung) at multiple time points after administration. The results showed that the nanoparticles were cleared from these organs by day 7 (Supplementary Fig. 50, 51). Analysis of fecal and urinary signals also suggested that FA-P@MC was primarily cleared via the biliary pathway within one week (Supplementary Fig. 52).

Correlation between multimodal imaging signals and plaque burden severity
Prior to employing the multimodal nanoprobe for in vivo AS diagnosis, we first assessed the ability of FA-P@MC to detect plaques and differentiate plaque severities (Fig. 5a). This preliminary evaluation aimed to determine whether the multimodal nanoprobe could serve as a reliable indicator for the assessment of plaque progression. The atherosclerotic model used here was induced by feeding apolipoprotein E-deficient (ApoE−/−) mice a high-fat diet for 12 weeks, followed by intravenous injection of FA-P@MC nanoprobe. At 12 h post-injection, the aorta was harvested and first subjected to NIR-II fluorescence imaging using a custom-built NIR-II imaging system. As shown in Fig. 5b, distinct signals were observed across different arterial regions, and the aorta was segmented into regions A through D based on NIR-II fluorescence intensity. Following this, PA and MRI imaging were performed on each segment using dedicated PA and MRI systems. Consistent with the NIR-II fluorescence findings, both PA and MRI signal intensities showed a progressive increase from segment A to segment D (Fig. 5c, d). These results confirmed the consistency of signal intensity across the three imaging modalities.

To validate the correlation between imaging signal intensities and plaque severity, ORO and hematoxylin and eosin (H&E) staining were performed on these aortic segments. ORO staining, widely used to assess lipid deposition in plaques, revealed that segment A showed no signs of plaque formation, while the extent of plaque areas gradually increased in segments B, C, and D (Fig. 5e), reflecting a progression in plaque severity. Key pathological parameters of the plaques in segments A through D, including the necrotic area, the proportion of the necrotic core relative to the total plaque area, and fibrous cap thickness, were quantified according to the H&E-stained images. As anticipated, the necrotic area increased progressively from segment B to segment D (Fig. 5f), while fibrous cap thickness diminished (Fig. 5g), indicating an escalation in plaque instability from segments B to D. Macrophage infiltration and ROS levels, both key indicators of plaque vulnerability, were evaluated via immunofluorescence staining with an anti-F4/80 antibody and DCFH-DA, respectively. Fluorescence signals for both anti-F4/80 and ROS exhibited a progressive increase from segment B to segment D, indicating enhanced macrophage infiltration and ROS accumulation as plaque severity advanced (Fig. 5h). Notably, a strong correlation was observed between the NIR-II fluorescence, PA, and MRI signal intensities and the severity of the plaques (Fig. 5i). Stronger signal intensities were detected in the regions with more advanced and vulnerable plaques, highlighting the potential of the multimodal nanoprobe to accurately delineate plaque lesions and act as a non-invasive method for assessing plaque severity. The ability of the multimodal nanoprobe to detect plaque progression is likely attributed to its targeting affinity for foam cells, a hallmark of plaque lesions, coupled with its high sensitivity and robust signal strength under each imaging modalities.

In vivo, in situ multimodal imaging of AS
We proceeded to investigate the in vivo real-time diagnostic capabilities of FA-P@MC. Atherosclerotic models were induced in ApoE−/− mice by administering a high-fat diet, after which the mice were intravenously administrated with equal doses of FA-P@C, FA-P@M, or FA-P@MC to compare the imaging performance across different nanoformulations. NIR-II fluorescence imaging was performed at various time points following the administration of the nanoprobes. As shown in Fig. 6a, b and Supplementary Fig. 53, in mice treated with FA-P@C (FA-P modified with PS-targeted peptides), weak fluorescence was initially detected in the carotid artery at 3 h post-injection, which peaked at 6 h and gradually decreased as the probes were metabolized. The macrophage membrane-camouflaged biomimetic FA-P@M demonstrated enhanced plaque accumulation, with peak enrichment occurring at 12 h. During the progression of AS, macrophages actively migrate toward atheromatous plaques via inflammatory chemotaxis. The macrophage membrane-coated NPs, inheriting membrane proteins from macrophages, thereby exhibited a natural binding affinity for inflamed plaques. FA-P@MC, which further incorporated the CLIKKPF peptide to bind PS expressed on activated and senescent foam cells, resulted in significantly enhanced plaque accumulation—nearly double that observed with the FA-P@M group. We also conducted a competitive blocking experiment in which free CLIKKPF peptide was injected prior to FA-P@MC administration. Pre-injection of the peptide reduced plaque accumulation of FA-P@MC to the levels comparable to non-targeted FA-P@M (Supplementary Fig. 54). This result suggests that PS-binding peptide interactions promote plaque targeting. Moreover, the fluorescence images acquired at 12 h in FA-P@MC group displayed the highest signal-to-noise ratio (SNR) (more than 100) in comparison with FA-P@C (~23.3) and FA-P@M (~56.8) group (Fig. 6c). Ex vivo fluorescence imaging further demonstrated that FA-P@MC accumulated substantially more in the aortic root and thoracic aorta compared to FA-P@C and FA-P@M (Fig. 6d, e), validating the in vivo results. Major organs, including the heart, liver, lungs, spleen, and kidneys, from mice injected with either FA-P@M or FA-P@MC were harvested to assess the biodistribution of the nanoprobes. No significant differences in organ distribution were observed between the two groups (Fig. 6f, g).

To visualize the biodistribution of AT, we labelled AT with Cy5, and prepared FA-P@M and FA-P@MC loaded with Cy5-AT. Twelve hours after intravenous injection, the aorta was collected for ex vivo fluorescence imaging. In the AT alone group, only weak fluorescence was detected in the aorta, consistent with the rapid clearance of small-molecule AT. Importantly, FA-P@MC exhibited substantially higher accumulation in aortic plaques compared with the non-targeted FA-P@M and free AT groups (Supplementary Fig. 55). To further support these observations, AT content in different tissues was quantified via homogenization and extraction. Consistently, plaque tissues from the FA-P@MC group contained approximately 12.7-fold higher drug levels than free AT and about 2-fold higher than FA-P@M (Supplementary Fig. 56). No significant differences in AT signals were detected in other major organs, including the liver, spleen, or kidneys, between the FA-P@MC and FA-P@M groups. Additionally, confocal microscopy of atherosclerotic aortic root sections revealed enhanced foam cell targeting by FA-P@MC in vivo. As shown in Supplementary Fig. 57, 58, the proportions of Cy5+F4/80+BODIPY+ foam cells in plaques were increased in mice treated with FA-P@MC compared with other groups. Additionally, the Cy5-labeled FA-P@MC signals exhibited obvious co-localization with PS, CD68, and PLIN2, supporting their preferential association with PS-exposing foam cells (Supplementary Fig. 59). These observations highlighted the enhanced targeting efficiency of FA-P@MC for atherosclerotic plaques and foam cells, which is crucial for advancing both the diagnostic accuracy and therapeutic efficacy of the nanoprobe.

Given the remarkable in vitro PA imaging capabilities and plaque-targeting properties of FA-P@MC, the in vivo non-invasive PA diagnostic performance was further assessed. At 12 h after administering equivalent doses of FA-P@C, FA-P@ML, or FA-P@MC, anatomical localization was assessed through two-dimensional grayscale ultrasound imaging of the carotid artery, captured in both transverse and sagittal sections views. Compared to the PBS group, the carotid artery in FA-P@C group exhibited a faint PA signal upon excitation at 790 nm, suggesting that foam cell-targeting alone was insufficient for effective plaque lesion targeting. The FA-P@M-treated mice exhibited stronger PA signals in the carotid plaques, attributed to the macrophage membrane biomimetics that enhanced plaque accumulation. Interestingly, plaques in the carotid arteries of mice treated with FA-P@MC were distinctly detected with intense PA signals (Fig. 7a,b), suggesting that the combination of macrophage membrane coating and foam cell-targeting peptide modification significantly boosted NPs enrichment in the plaques. Quantification of PA intensity revealed that plaques from FA-P@MC-injected mice exhibited PA signals approximately 4.9 times and 1.8 times stronger in the transverse section than those seen in the FA-P@C and FA-P@M groups, respectively, and 6.1 times and 2.1 times stronger in the sagittal section compared to the FA-P@C and FA-P@M groups (Fig. 7c, d). FA-P@MC enabled enhanced PA imaging of carotid plaques with strong contrast relative to normal tissue, showing the highest SNR compared to other groups (Fig. 7e).

In addition to PA imaging, the nanoprobe with concurrent incorporation of Gd complex can be further employed for in vivo MRI, enabling comprehensive detection of AS. As a proof of concept, in vivo MRI of AS was performed before and at 12 h after the injection of Gd-DOTA, FA-P@M or FA-P@MC into atherosclerotic ApoE−/− mice, with a focus on the carotid region. As shown in Fig. 7f, a pronounced heterogeneous T -MRI signal enhancement was observed in both the left and right common carotid artery walls at 12 h post-injection of FA-P@MC, compared to pre-injection images, indicating the presence of plaque formation in these regions. Furthermore, mice injected with FA-P@MC exhibited a significant increase in MRI signals, which was 2.1- and 3.2-fold higher than those treated with FA-P@M and Gd-DOTA (Fig. 7g). These results highlighted the potential of FA-P@MC for both PA and MRI-based plaque detection. The nanoprobe's favorable multi-modal imaging performance was attributed to its superior AS-targeting capability, as well as the amplified signal intensity in each imaging modality. Considering the complementary strengths of each technique—such as high sensitivity, spatial resolution, and anatomical detail—the combination of NIR-II fluorescence, PA, and MRI imaging facilitates precise, non-invasive assessment of plaque progression, providing a comprehensive overview of plaque characteristics.

In vivo therapeutic efficacy of FA-P@MC in AS
Building on the encouraging results outlined above, we further conducted a thorough investigation into the in vivo therapeutic efficacy of FA-P@LMC in treating AS. As detailed in the treatment protocol (Fig. 8a), ApoE−/− mice were initially fed a high-fat diet for 8 weeks to induce the development of atherosclerotic plaques at arterial bifurcations. Following this, the atherosclerotic mice were randomly assigned to six treatment groups, including PBS, free AT, A-P, FA-P, FA-P@M, and FA-P@MC. Each group received intravenous injections of the designated treatment twice a week while maintaining the high-fat diet for an additional 8 weeks. At the end of the treatment period, the entire aortas were excised, and the effectiveness of each treatment in reducing atherosclerotic plaque size was assessed using ORO staining of the aortic arch, which highlighted lipid accumulation (Fig. 8b). As shown in Fig. 8c, the PBS group exhibited the largest ORO-positive area (~38.5%), indicating significant plaque formation. Treatment with free AT resulted in a moderate reduction in plaque size, with the ORO-positive area decreasing to approximately 26.0%. Both A-P and FA-P groups showed slight therapeutic improvements over free AT, likely due to enhanced delivery of AT via the nanocarriers. Mice treated with FA-P@M, which utilized macrophage membrane biomimicry for targeted delivery to inflammatory plaques, displayed a significantly smaller plaque area (~12.4%) compared to those treated with A-P and FA-P. Notably, the FA-P@MC treatment led to a dramatic reduction in the ORO-positive plaque area to approximately 7.5%, exhibiting the smallest ORO-stained lesions in the aortas. The average plaque-to-total aortic area ratio in the FA-P@MC group was 5.1-, 3.5, 2.5-, 2.4-, and 1.6- times lower than those in the PBS, AT, A-P, FA-P, and FA-P@M groups, respectively. These results highlighted the potent anti-atherosclerotic efficacy of FA-P@MC. A consistent trend was observed in the ORO-stained frozen sections of aortic root, with FA-P@MC demonstrating the most pronounced anti-atherosclerotic effect (Supplementary Fig. 60). Additionally, FA-P@MC could effectively promote cholesterol efflux, lowering cholesterol levels in the plaques (Supplementary Fig. 61). Subsequently, we examined the composition of atherosclerotic plaques in different experimental groups through histochemical and immunohistochemical analyses. As shown in Fig. 8d, H&E staining of the aortic root plaques revealed that plaques in both the PBS and AT-treated groups predominantly consisted of acellular, lipid-rich necrotic cores. While treatment with A-P, FA-P, or FA-P@M reduced the size of the necrotic core, the overall inhibition was limited. In contrast, the FA-P@MC-treated group exhibited a significant reduction in both plaque size and necrotic core area, outperforming all other groups.

FA-P@MC inhibit CD47 expression and inflammatory burden, and modulate immune cell infiltration in plaques
We further explored the underlying mechanism by which FA-P@MC exerted their anti-atherosclerotic effects. The excessive production of ROS within plaques contributes to local tissue damage and perpetuates inflammation. To assess the ROS levels within the plaques, aortic root sections were stained with the ROS-sensitive fluorescent probe dihydroethidium (DHE) following treatment with various formulations (Fig. 8f). Compared to the intense DHE-positive staining observed in the aortic sections of PBS-treated mice, treatment with different nanoformulations resulted in notable reductions in ROS-positive areas. These findings aligned with the ROS-scavenging properties of the polymeric carriers, as demonstrated in our in vitro studies. Remarkably, the DHE staining in the aortic root sections of the FA-P@MC-treated group was significantly lower, showing a 4.6- to 1.9-fold reduction compared to other groups (Fig. 8g), emphasizing their potent ROS scavenging ability within plaques.

Next, we assessed the anti-inflammatory and immunomodulatory efficacy of FA-P@MC in atherosclerotic mice. Foam cells are lipid-laden macrophages that play a pivotal role in the formation and progression of atherosclerotic plaques. Foam cells contribute to plaque inflammation by secreting pro-inflammatory cytokines (such as TNF-α, IL-6 and IL-1β) and chemokines that recruit additional immune cells, thereby perpetuating a cycle of inflammation. As a statin drug, AT facilitates the removal of excess cholesterol from foam cells, thereby alleviating the lipid burden within these cells. Furthermore, our in vitro studies indicated that the combination of polymer carrier’s anti-ROS properties with AT’s anti-inflammatory effects could downregulate CD47 expression on foam cells. This, in turn, enhances the immune clearance of foam cells, reshaping the inflammatory and immune landscape within the plaque microenvironment. The expression of CD47 in plaque tissue across different treatment groups was then assessed. Western blot analysis confirmed that FA-P@MC notably decreased CD47 levels in plaque tissue (Fig. 8h). Additionally, the immunofluorescence images indicated that the plaques from the PBS-treated control group exhibited relatively high levels of the "don't eat me" molecule, CD47, which allowed apoptotic foam cells to evade macrophage-mediated clearance (Fig. 8i,j). In contrast, FA-P@MC treatment resulted in a significant reduction in CD47 expression within the plaques. The enhanced foam cell modulation exhibited by FA-P@MC, in comparison to other nanoformulations, aligns with its improved affinity for atherosclerotic plaques and foam cell-targeting capacity, which together facilitate the targeted delivery of the ROS-scavenging polymer and AT into plaque foam cells for synergistic treatment.

Subsequently, the potential of FA-P@MC intervention to mitigate immune cell infiltration and inflammatory responses was investigated. Since the recruitment of monocytes and macrophages to the plaque is closely linked to plaque progression and disease severity, inhibiting their recruitment can help attenuate plaque development.[62,63] Immunostaining staining for anti-CD68 antibodies demonstrated that FA-P@MC significantly reduced macrophage accumulation within plaques in the aortic arch, showing superior efficacy over other formulations (Supplementary Fig. 62). Similarly, immunohistochemical staining for monocytes using an anti-CD14 antibody revealed a notable decrease in monocyte presence within the plaques following FA-P@MC treatment. In particular, the CD14-positive staining in aortic root sections from the FA-P@MC group was approximately 1.5%, significantly lower than those observed in the PBS (11.7%), free TA (9.1%), A-P (6.4%), FA-P (6.1%), and FA-P@M (3.7%) groups (Supplementary Fig. 63). T lymphocytes, recruited by antigen-presenting cells, have been reported to play an important role in driving lesion inflammation.[64] During plaque progression, CD4+ and CD8+ T cells typically increase and release pro-inflammatory cytokines, whereas regulatory T cells (Tregs) remain relatively low, providing insufficient anti-inflammatory regulation. In the FA-P@MC–treated group, flow cytometry analysis revealed that the proportions of infiltrating CD8+ and CD4+ T cells within plaques were reduced by approximately 4.68- and 2.53-fold, respectively, compared with the model group (Supplementary Fig. 64, 65). Conversely, the proportion of CD25+Foxp3+ Treg cells was increased in the FA-P@MC group relative to other groups. Key pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, are known to be overexpressed by activated immune cells. To assess the impact of various treatments on these cytokines, their serum levels were quantified using ELISA. As shown in Supplementary Fig. 66, inflammatory marker levels were significantly high in the PBS group. The treatment with A-P, FA-P, or FA-P@M resulted in a modest reduction of these cytokine concentrations. Remarkably, FA-P@MC once again exhibited the most pronounced suppression of inflammatory cytokine expression compared to other groups. Collectively, these results demonstrated that FA-P@MC could effectively attenuate AS progression through a combination of targeted drug delivery, oxidative stress reduction, and immune-inflammatory modulation.

Therapeutic impact on enhancing plaque stability
AS is a leading cause of cardiovascular disease, with plaque stability serving as a critical determinant of patient prognosis.[65] The therapeutic efficacy of various formulations was further assessed by evaluating plaque vulnerability. The expression of metalloproteinase-9 (MMP-9) is closely associated with plaque instability.[65] Unlike the pronounced positive MMP-9 staining observed in the aortic root sections from the PBS group, administration with FA-P@MC resulted in a significant reduction in MMP-9 expression (Fig. 9a). Specifically, MMP-9 staining in the FA-P@MC group decreased by 2.0- to 5.9-fold compared to other treatment groups (Fig. 9b). Furthermore, Masson's trichrome staining revealed a considerable increase in collagen content around the plaques and thicker fibrous caps in the FA-P@MC-treated group, thereby strengthening the structural integrity of plaques (Fig. 9c,d). Previous studies have demonstrated the role of vascular smooth muscle cell (VSMC) proliferation in enhancing plaque stability and reducing the risk of rupture. To assess VSMC accumulation, \alpha-smooth muscle actin (\alpha-SMA) staining was performed. As indicated in Fig. 9e,f, an increase in VSMC presence was observed in the plaques treated with FA-P@MC, suggesting the formation of a more robust fibrous cap and improved plaque stability. Additionally, angiogenesis is often a sign of plaque vulnerability, linked to heightened inflammation, plaque rupture, and an increased risk of thrombosis. CD31 immunohistochemistry staining revealed that FA-P@MC treatment significantly reduced the number of perivascular CD31+ neovessels (Supplementary Fig. 67). The observed increases in collagen and \alpha-SMA, alongside reductions in MMP-9 expression and angiogenesis, collectively suggested the therapeutic efficacy of FA-P@MC in enhancing plaque stability. Taken together, these results indicated that FA-P@MC not only halted plaque progression but also fostered a more resilient plaque environment, thereby lowering the potential risk of plaque rupture.

Non-invasive monitoring of therapeutic outcomes using the theranostic nanoprobe
The feasibility of the multimodal nanoprobe to monitor plaque progression following treatment was further evaluated. At the end of the treatment regimen, mice were intravenously injected with FA-P@MC nanoprobe, and NIR-II fluorescence imaging was first conducted at 12 h post-injection. As illustrated in the representative NIR-II fluorescence images (Fig. 9g), the carotid artery of AS mice in the PBS group displayed a strong NIR-II fluorescence signal, indicative of severe plaque development. In contrast, mice treated with FA-P@MC exhibited only a faint NIR-II fluorescence signal in the carotid artery, suggesting a reduction in plaque formation following treatment (Fig. 9h). Further PA imaging was performed to gain deeper insights into the progression of plaque development. PA imaging was carried out at 12 h post-injection of the nanoprobe under 790 nm laser excitation. Compared to the PBS group, a marked reduction in PA signal was observed from the aortas of AS mice following FA-P@MC treatment (Fig. 9i,j). The PA imaging results mirrored those obtained from NIR-II fluorescence imaging, with both showing trends consistent with H&E analysis and plaque stability assessment of isolated aortic roots section (Fig. 9k-n), which also validated that plaque progression was substantially attenuated after FA-P@MC treatment. Taken together, these results demonstrated that FA-P@MC not only effectively mitigated AS and increase plaque stability but also served as a powerful multimodal imaging agent for real-time evaluation of treatment response and plaque progression, providing valuable insights for precision therapy.

Methods
Materials
All chemicals and reagents were provided by commercial sources. Dulbecco’s Modified Eagle’s Medium (DMEM) culture medium, fetal bovine serum (FBS), and penicillin streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). Cell culture dishes and confocal dishes were purchased from Corning Inocorporated Co., Ltd (Beijing, China). 3-(4,5-Dimethylthiazil-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Saiguo biotech Co., Ltd (BioFROXX, Germany). Griess reagent, reactive oxygen species assay kit (DCFH-DA), Oil Red O Staining Kit (ORO), nitric oxide assay kit (DAF-FM DA), and Masson's Trichrome Staining Kit were purchased from Beyotime Biotechnology (Shanghai, China). DiI, DiD, carboxyfluorescein diacetate, and succinimidyl ester (CFSE) were obtained from Beyotime Biotechnology (Shanghai, China). Cy5-labelled AT was obtained from Xi'an Qiyue Biotechnology Co., Ltd. Hematoxylin-eosin staining solution (alcohol soluble) and LDH Cytotoxicity Assay Kit were purchased from Leagene Biotechnology Co., Ltd (Beijing, China). Centrifugal filters were obtained from Merck Millipore Co., Ltd (Merck, America). Immunofluorescence analysis: Anti-CD47 (ABclonal, A1838, 1:200), recombinant anti-CD68 antibody (Abcam, ab283654, 1:200), anti-CD36 antibody (Huabio, ET1701-24, 1:100), anti-LOX-1 antibody (Huabio, ET1706-31, 1:100), anti-SR-BI antibody (Abcam, #ab217318, 1:100), anti-ABCA1 antibody (Huabio, R1510-42, 1:100), Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) (1:1000, Abcam, #ab150077), and Goat anti-Rabbit IgG H&L (Alexa Fluor® 594) (Cell Signaling Technology, #8889, 1:100,). Immunohistochemical analysis: Recombinant anti-MMP9 antibody (Abcam, ab283575, 1:200), \alpha-SMA (Abcam, ab5694, 1:200), Anti-CD31 antibody (Abcam, ab222783, 1:200), Anti-CD14 (Abcam, ab182032, 1:200). TNF-α ELISA kit, IL-6 ELISA kit and IL-1β ELISA kit were purchased from Dakewe Co., Ltd (Beijing, China). OxLDL (YB-002) and HDL (YB-003) were purchased from Yiyuan biotechnology Co., Ltd. (Guangzhou, China). BODIPY-cholesterol (HY-125746), Probucol (HY-B0388), ApoA-I (HY-P72833), and BLT-1 (HY-116767) were obtained from MedChemExpress (MCE).

Cell lines and animals
The RAW264.7 murine macrophage cell line and the mouse embryonic fibroblasts line (NIH 3T3) were purchased from the Chinese Academy of Sciences Cells Bank (Shanghai, China). The human umbilical vein endothelial cell line (HUVEC) was purchased from Cyagen Biosciences (Guangzhou, China) Inc. All cells were cultivated in a humidified atmosphere at 37 °C with 5% of CO .

C57BL/6 mice (6 weeks, female) were purchased from Beijing Vital River Laboratory Animal Technology. ApoE−/− mice (6 weeks, female) were purchased from SPF (Beijing) Biotechnology Co., Ltd. High-fat diet (HFD) feeding of AopE−/− mice began at six weeks of age. All mice were housed in the same specific pathogen-free (SPF) facility and maintained under identical environmental parameters, including cage type, temperature (25 °C), humidity, handling procedures, and a 12-hour light/dark cycle. All model groups received the same HFD throughout the study, and food and water were provided ad libitum, with diet replaced twice weekly. The high-fat diet used in this study was sourced from SPF (Beijing) Biotechnology Co., Ltd., catalog number SFD004. The diet has an energy density of 4.7 kcal/g, with 41% of total kcal from fat, 42% from carbohydrate, and 17% from protein. By weight, the diet contained 21.2% lard, 49.1% carbohydrates, 19.8% protein, and 0.2% cholesterol. Carbohydrates in the formulation are primarily composed of corn starch, sucrose, and maltodextrin, and the fiber component contains 31 g/kg crude fiber based on 90% dry matter. The primary sources of fat are lard (42% saturated fatty acids, 48% monounsaturated fatty acids, 10% polyunsaturated fatty acids) and soybean oil (15% saturated fatty acids, 24% monounsaturated fatty acids, 61% polyunsaturated fatty acids). The diet contains 0.2% cholesterol by weight (equivalent to 2 g/kg), and sodium cholate is not included in the formulation. All procedures involving animals were conducted by the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals, and approved by the Animal Ethics Committee of Nankai University (2024-SYDWLL-000693).

Characterizations
1H and 13C nuclear magnetic resonance (NMR) spectra were acquired using a Bruker-DPX 400 spectrometer. High-resolution mass spectra (HRMS) were performed on a Bruker AutoflexIII LRF200-CID mass spectrometer in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mode. The absorption spectra were detected using a Shimadzu UV-1800 spectrometer. Edinburgh FS5 was used to measure the photoluminescence (PL) spectra. Transmission electron microscope (TEM) images were captured using a Talos L120C G2 instrument by FEI, Czech. Dynamic light scattering (DLS) measurements were performed on a Malvern Zeta sizer Nano ZS-90. PA imaging was conducted on a commercial small-animal optoacoustic tomography system (Vevo LAZR, FujiFilm VisualSonics, America). A short-wavelength infrared (SWIR) imaging system (Wuhan Grand-imaging Technology Co., LTD) was utilized for NIR-II fluorescence imaging of animals and organs. A bruker 9.4 T BioSpec 94/30 MRI installations for small animal magnetic resonance imaging test.

Fabrication of nanoformulations
First, RAW264.7 cells were dispersed in the membrane protein extraction reagent (Beyotime, China) containing 1 mM PMSF and cooled down at 4 °C for 30 min, and subsequently broken by ultrasound (80 W) for 10 min. The mixture was repeatedly frozen and thawed at −80 °C and 37 °C for 3 times, and then centrifuged at 3,000 g for 10 min. The resulting supernatant was centrifugated at 12,000 g × 10 min, followed by a final centrifugation at 100,000 g for 1 h to obtain membrane in precipitates. The membrane was stored at −80 °C for further use.

The targeting molecule (DSPE-PEG-CLIKKPF) was synthesized according to previous reports.[28,67] Briefly, CLIKKPF was reacted with DSPE-PEG-maleimide via thiol-Michael addition reaction in water/methanol solution (90:10, v/v) for 8 h at room temperature. Then the residual CLIKKPF peptide was removed by centrifugal filters (Merck Millipore Co., Ltd, Shanghai China) and the final product DSPE-PEG-CLIKKPF was obtained by lyophilizing.

The molecular probe and AT-loaded nanoformulations were fabricated using a modified nanoprecipitation technique. The synthesis was carried out at 20 °C, where FTBGd (1 mg), AT (1 mg), DSPE-PEG (2 mg), and PPADT (4 mg) were dissolved in 1 mL of DMSO under constant stirring. After achieving complete dissolution, the resulting stock solution was gradually introduced into 15 mL of deionized water under vigorous stirring. During nanoprecipitation, DSPE-PEG was incorporated as a stabilizing surfactant. Owing to its amphiphilic structure, DSPE-PEG anchors its hydrophobic lipid tail within the nanoparticle core while presenting a hydrophilic PEG corona on the surface, thereby enhancing colloidal stability. To eliminate the organic solvent, the mixture underwent dialysis against water for 12 h using a dialysis membrane with an MWCO of 3,500 Da, ultimately producing the final solution, termed FA-P. The encapsulation efficiencies of FTBGd and AT were found to be 63.7% and 59.5%, respectively. To synthesize FA-P@M, a solution of FA-P (100 \muL, 1 mg mL‒1 based on FTBGd) was combined with 0.5 mg of membrane and subjected to ultrasound treatment (100 W, 50 Hz) for 5 min. The mixture was then repeatedly extruded through a micro-extruder with a 200 nm pore size to form FA-P@M. Uncoated membranes were eliminated by centrifugation at 3,000 g, followed by washing until no residual protein was detected in the supernatant. The obtained FA-P@M was stored at 4 °C for future use. After obtaining FA-P@M, FA-P@MC was prepared by incorporating DSPE-PEG-CLIKKPF into its lipid membrane.

Drug release from FA-P@MC
The AT release profile from FA-P@MC or FA-P was investigated under varying H O levels (0, 2.5 \muM, 0.1 mM, 1.0 mM, or 1.0 mM + catalase). Prior to the release studies, both nanoformulations were confirmed to exhibit similar particle sizes and identical drug loading, ensuring a valid comparison. 2 mg of FA-P@MC or FA-P was suspended in 5 mL of deionized water and sealed inside a dialysis bag (MWCO = 10,000). This bag was subsequently submerged in 15 mL of buffer solution and placed in a shaker set to 37 °C with a rotation speed of 30 × g. At predetermined time points, 2 mL of the incubation medium was withdrawn for HPLC analysis, and an equivalent volume of fresh medium was added back to the buffer solution. The percentage of cumulative AT release was determined using the equation: Cumulative drug release (%) = (M/M ) × 100%, where M represents the drug quantity released at time t, and M corresponds to the initial drug content in FA-P@MC. All release experiments were performed in triplicate.

The H O levels in plaque tissues were measured using a commercially available Hydrogen Peroxide Content Assay Kit (Solarbio, BC3595), following the manufacturer's protocol. Briefly, freshly isolated plaque tissues were homogenized in the supplied buffer on ice and centrifuged, and the resulting supernatants were used for analysis. Absorbance was measured at 415 nm using a UV–Vis spectrophotometer (SHIMADZU), and H O concentrations were calculated based on a standard curve generated with known H O standards. The results are reported as mean ± SD (n = 6 individual plaque samples), yielding an H O level of 0.34 ± 0.05 mM, corresponding to approximately 0.34 \mu mol per gram of wet tissue.

Cellular uptake behavior
Cellular uptake of different formulations by foam cells was evaluated. To generate foam cells, RAW264.7 cells were exposed to a medium supplemented with 50 \mug/mL oxLDL. Following this, the medium was substituted with fresh medium containing DiI-labeled nanoparticles—FA-P, FA-P@M, or FA-P@MC—at an FTGd concentration of 50 \muM. After incubation, the cells underwent three PBS washes, were fixed using 4% tissue fixative for 15 min, and then washed three additional times with PBS. The nuclei were stained with DAPI for 10 min, followed by another PBS wash to eliminate residual dye. Finally, confocal laser scanning microscopy (CLSM) (LSM 800 with Airyscan, ZEISS, Germany) was utilized for imaging, and analysis was conducted using ZEN 2012 software.

The transmigration of the NPs across inflamed HUVECs and their subsequent uptake by foam cells were evaluated using in vitro transwell migration assays. HUVECs were cultured in the upper chamber of a Transwell insert (0.3 \mu m) and exposed to 1 \mug/mL LPS for 24 h to induce an inflammatory response. At the same time, RAW264.7 cells (1 × 105 per well) were seeded in the lower chamber of a 24-well plate and treated with 50 \mug/mL oxLDL for 24 h to form foam cells. Subsequently, FA-P, FA-P@M, or FA-P@MC (50 \muM based on FTGd) were added to the upper chamber and incubated for 4 h. After incubation, the foam cells were rinsed three times with PBS and stained with DAPI for 15 min. Imaging was carried out using CLSM, and flow cytometry (BD LSR Fortessa, USA) was used for quantitative analysis.

In vitro safety evaluation
The cytotoxicity of FA-P@MC was evaluated in vitro using MTT assays with NIH 3T3 and HUVEC cell lines. In brief, the cells were plated in a 96-well plate at a concentration of 1 × 105 cells/mL. After 24 h of incubation, the medium was replaced with fresh medium containing nanoparticles at varying concentrations (0, 20, 40, 60, 80, and 100 \muM). Following another 24-hour incubation period, 100 \muL of MTT solution was added to each well. After 4 h, the MTT solution was removed, and 150 \muL of DMSO was added to each well, followed by shaking at room temperature. The optical density at 570 nm was then recorded using a microplate reader. For the LDH cytotoxicity assay, cells were exposed to various concentrations of the nanoparticle formulation. LDH release was then quantified using an LDH Cytotoxicity Assay Kit (Leagene Biotechnology Co., Ltd.) following the manufacturer’s instructions.

In vitro anti-inflammatory effect
Macrophages were simultaneously treated with 50 \mug/mL ox-LDL and different drug formulations (free AT, A-P, FA-P, FA-P@M, and FA-P@MC) at a concentration of 50 \muM for 24 h. After the treatment, the cells were washed three times with PBS. To assess intracellular ROS, the cells were incubated with 10 \muM of DCFH-DA in the dark. After removing the excess probe, the cells were further incubated at 37 °C for 30 min. Confocal microscopy was employed with 488 nm excitation and 530 ± 20 nm emission to observe the dichlorofluorescein (DCF) fluorescence resulting from the reaction of DCFH-DA with ROS. The release of nitric oxide (NO) from cells was assessed using the fluorescent NO probe, DAF-FM DA. The cells were treated with 50 \mug/mL ox-LDL and various drug formulations (free AT, A-P, FA-P, FA-P@M, and FA-P@MC) at a concentration of 50 \muM for 24 h, followed by three PBS washes. The NO probe, DAF-FM DA (final concentration: 5 \muM), was added, and the cells were incubated at 37 °C for 30 min, after which they were washed three times with PBS. Confocal microscopy was employed to visualize the cells. Statistical analysis and graphing were conducted using ZEN 2012 (Version 1.1.2.0).

Inhibition of lipid uptake and foam cell formation
RAW264.7 cells were seeded in confocal dishes at 1 × 105 cells per dish and allowed to adhere overnight. Cells were stimulated with 1 \mu g mL−1 of LPS for 6 h, followed by treatment with different formulations for an additional 12 h. Subsequently, cells were incubated with 50 \mu g mL−1 of DiI-oxLDL for 4 h. The cells were then washed with PBS, and fixed with 4% paraformaldehyde. Fluorescence imaging was performed using CLSM to evaluate the cellular uptake of oxLDL. The expressions of CD36 and LOX-1 were evaluated by immunofluorescence using primary antibodies against CD36 and LOX-1, followed by incubation with fluorescently labeled secondary antibodies.

For foam cell formation and lipid accumulation assays, RAW264.7 cells were seeded in 12-well plates at 1 × 105 cells per well. Cells were then treated with 50 \mu g mL−1 of oxLDL in combination with various formulations for 12 h. For Oil Red O (ORO) staining, the foam cells were treated with the freshly prepared ORO working solution for 15 min and then rinsed with 60% isopropanol. Images of the cells were captured using confocal microscopy. After imaging, ORO was extracted using isopropanol, and its absorbance was measured at 492 nm using a microplate reader. Cholesterol efflux was quantified by collecting culture supernatants post-treatment. The expressions of SR-BI and ABCA1 were evaluated by immunofluorescence using primary antibodies against SR-BI and ABCA1, followed by incubation with fluorescently labeled secondary antibodies.

For functional cholesterol efflux assay, foam cells were loaded with 1 \mu g/mL BODIPY-labeled cholesterol (HY-125746, MedChemExpress) for 6 h to allow intracellular labeling. After washing, cells were treated with FA-P@MC or relevant control formulations in the presence of ApoA-I (10 \mu g mL−1, ABCA1-dependent) or HDL (40 \mu g mL−1, SR-BI/ABCG1-associated, YB-003, Yiyuan biotechnology) as cholesterol acceptors in serum-free medium for 24 h. As a control, cells were pre-incubated with pharmacological inhibitors of ABCA1 (probucol, 20 \mu M) or SR-BI (BLT-1, 1 \mu M) for 2 hours prior to efflux to assess the contribution of these transporters. Fluorescence intensity was determined in both supernatant and cell lysate using a microplate reader (excitation/emission 485/510 nm). The percent of cholesterol efflux was calculated as (fluorescence in the supernatant/the sum of the fluorescence in the medium and the fluorescence remaining in the cells) × 100%. Parallel cholesterol efflux studies were also conducted in foam cells induced from bone marrow–derived macrophages (BMDMs). Bone marrow cells were flushed from femurs and tibias of 8-week-old C57BL/6 mice, filtered through a 70 \mu m strainer, and plated in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, and 20 ng/mL M-CSF. Cells were differentiated for 7 days at 37 °C with 5% CO and used for cholesterol efflux assays. Macrophage differentiation was confirmed by F4/80 immunostaining.

CD47 expression analysis and in vitro efferocytosis evaluation
RAW264.7 cells were incubated with 50 \mu g/mL oxLDL for 24 h, followed by treatment with different NPs for an additional 24 h. Cells were first harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay, and equal amounts of protein were mixed with 4 × loading buffer and denatured by heating at 95 °C for 10 min in a metal bath. Samples were then separated by 10% SDS-PAGE and electrotransferred onto a 0.4 \mu m PVDF membrane. The membrane was blocked with BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature to minimize nonspecific binding. The membrane was then incubated overnight at 4 °C with a primary antibody against CD47, while GAPDH was used as a loading control. Following three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system, and band intensities were quantified using ImageJ software to determine relative CD47 expression levels.

For phagocytosis study, bone marrow–derived macrophages (BMDMs) were labeled with CFSE to serve as phagocytic cells, while oxLDL-treated RAW264.7 cells were used as foam cells. BMDMs and foam cells were co-cultured for 6 h. Following incubation, the medium was discarded, and the cells were washed with PBS, then fixed with 4% formaldehyde for 15 min. Fluorescence images were acquired using CLSM. For flow cytometry, treated cells were washed with PBS, detached, and subsequently analyzed on a BD LSR Fortessa (USA). Flow cytometric data were analyzed using FlowJo version 10.8.1.

In vivo biosafety and pharmacokinetics analysis
To assess the potential toxicity of FA-P@MC, 6-week-old healthy C57BL/6 mice were intravenously injected with PBS or FA-P@MC (10 mg kg-1) by intravenous injection twice weekly for 2 weeks (Day 0–14). One week after treatment (Day 22), the mice were euthanized, and blood samples were collected for analysis. For long-term biosafety evaluation, mice received weekly intravenous injections of either PBS or FA-P@MC (10 mg/kg based on FTBGd) for seven weeks (Day 0–49). On Day 50, hematological parameters, including white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, mean red blood cell volume, platelet count, mean corpuscular hemoglobin content, and mean corpuscular hemoglobin concentration, were measured using an automated hematology analyzer. Serum samples were obtained to evaluate various biochemical markers in the blood, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine concentrations. Additionally, key organs, including the heart, liver, spleen, lungs, and kidneys, were collected for histopathological examination. In brief, tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sliced into 5 \mu m sections for H&E staining. The stained tissue sections were subsequently analyzed using a digital microscope (Leica QWin).

For pharmacokinetic evaluation, 6-week-old C57BL/6 mice were given FA-P@MC intravenously at a dose of 10 mg/kg. Blood samples were taken at different time intervals (0, 4, 8, 12, 24, 48, and 72 h) from the inner eye socket following intravenous injection of different nanoparticles. The fluorescence intensity of the blood samples was quantified using a SWIR imaging system (Wuhan Grand-imaging Technology Co., LTD) to assess the plasma signals of the nanoparticles. The clearance of free AT in the bloodstream was determined by HPLC analysis. To study the in vivo metabolic fate and clearance routes, FA-P@MC was labeled with the near-infrared dye DiR. Fecal and urinary samples were collected at days 1, 3, and 7 after intravenous administration, and their fluorescence signals were monitored using IVIS.

Multimodal imaging and analysis of the aorta ex vivo
To perform ex vivo fluorescence and PA imaging of the aorta, ApoE−/− mice were fed a high-fat diet (HFD; SPF Biotechnology Co., Ltd., Beijing) for 12 weeks (Day 0–84) to induce atherosclerosis. On Day 85, atherosclerotic mice were intravenously administered FA-P@MC. Twelve hours after injection, the mice were euthanized, and the aortas were excised for ex vivo fluorescence and PA imaging to evaluate the probe’s ability to detect plaques and differentiate plaque severity. NIR-II fluorescence imaging was performed using a short-wave infrared (SWIR) system with 808 nm excitation and emission collected through a 1000 nm long-pass filter. PA imaging was acquired at 790 nm excitation using a VisualSonics Vevo LAZR multimodal imaging system (FUJIFILM VisualSonics, Canada). For fluorescence imaging, the laser power was 50 mW cm-2; for PA measurements, pulse energy = 20 mJ cm-2, frequency = 30 MHz, PA gain = 35 dB, pulse repetition rate = 20 Hz, and duration of pulsed laser = 7 ns. 790 nm was selected for PA imaging as it coincides with the probe’s absorption peak and yields strong PA responses. Endogenous absorbers such as hemoglobin exhibit reduced absorption in this spectral region compared to their strong visible-band absorption, and water shows low absorption at this wavelength before its absorption increases at longer wavelengths. For fluorescence imaging, 808 nm light was employed as the excitation source because the probe has strong absorption at this wavelength and can be efficiently excited using the widely available commercial 808 nm laser. This wavelength selection ensures sufficient excitation efficiency while maintaining compatibility with commonly available NIR laser. MRI was carried out on a 9.4 T small-animal MRI scanner (BioSpec 94/30, Bruker).

To validate the correlation between imaging signal intensities with plaque severity, the aorta was subsequently embedded in optimal cutting temperature (OCT) compound, frozen at −80 °C for 30 min, and sliced into 10 \mu m sections. For ORO staining, a working solution was prepared by diluting 6 mL of the stock solution with 4 mL of deionized water. Cryosections of the aorta were incubated with 10 \mu L of this solution for 2 to 5 min, then washed with 60% isopropanol and deionized water. Hematoxylin was used as a counterstain for 5 min. For immunofluorescence, sections were rinsed, blocked, and incubated with anti-F4/80 primary antibody (1:500, Bio-Rad, MCA497GA), followed by washing and incubation with fluorescently labeled secondary antibody for 60 min at room temperature. Nuclei were counterstained with DAPI for 10 min. Images were acquired using a Zeiss fluorescence microscope (FITC: excitation at 488 nm; DAPI: excitation at 405 nm). For ROS staining, aorta sections were treated with DCFH-DA (10 \mu M) for 60 min at 37 °C, followed by a 10-min DAPI incubation at 37 °C. Fluorescent images of DCFH-DA were captured using a laser confocal microscope (DCFH-DA: excitation at 488 nm; DAPI: excitation at 405 nm).

In vivo NIR-II fluorescence, PA, and MR imaging
AopE-/- mice were fed a HDF for eight weeks (Day 0–56), and then received intravenous injections of FA-P@C, FA-P@M, or FA-P@MC (150 \mu L, 4 mg/kg based on FTBGd) via the tail vein on day 57. For the competitive blocking control, mice were pretreated with free CLIKKPF peptide (2 mg/kg, i.v.) 4 hours prior to FA-P@MC administration on Day 57. At predefined time points post nanoformulation injection (0, 3, 6, 12, 24, and 36 hours post-injection), mice were anesthetized with 2% isoflurane and then imaged using a SWIR system equipped with an InGaAs scientific-grade camera and a 1000 nm long-pass filter. To validate plaque visualization, a surgical incision was made to expose the carotid arteries. To evaluate drug distribution and plaque-targeting efficiency, atherosclerotic mice were intravenously administered Cy5-labeled AT, Cy5-AT-loaded FA-P@M, or Cy5-AT-loaded FA-P@MC on Day 57. Twelve hours post-injection, major organs and the aorta were harvested for ex vivo fluorescence imaging using an IVIS system. For quantitative analysis, tissues were weighed, homogenized, and centrifuged to collect supernatants. Cy5 fluorescence intensity in the supernatants was measured using a spectrofluorometer, and background fluorescence from untreated control tissues was subtracted. The AT content in each tissue was calculated based on a standard curve generated from known concentrations of Cy5-AT under identical conditions. To study lesion distribution, plaque tissues were cryosectioned, fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Foam cells were identified by immunostaining with anti-F4/80 and counterstaining with BODIPY to visualize intracellular lipids. Additional staining was performed for PS (Sigma-Aldrich, 05-719, 1:200), CD68 (Abcam, ab283654, 1:200), and PLIN2 (Huabio, ET1704-17, 1:200) to evaluate co-localization with FA-P@MC. Images were acquired using a confocal laser scanning microscope under identical settings for all groups, and co-localization was analyzed using ImageJ.

PA imaging was conducted on living mice on day 57 (12 hours post-injection of different nanoprobe) using the VisualSonics Vevo LAZR multimodal system (Canada) under the following parameters: pulse energy = 20 mJ cm-2, wavelength range = 790 nm, frequency = 30 MHz, PA gain = 35 dB, pulse repetition rate = 20 Hz, and duration of pulsed laser = 7 ns. Ultrasound and PA signals were acquired simultaneously, and merged PA/US images were generated by overlaying pseudo-colored PA signals onto grayscale ultrasound images. MRI was performed on a 9.4T Bruker BioSpec 94/30 system before and 12 h post-injection of Gd-DOTA, FA-P@M, or FA-P@MC (150 \mu L, 4 mg/kg based on FTBGd) (day 57). Mice were anesthetized with 2% isoflurane and positioned in the MRI coil for baseline and post-injection scans. T1-weighted axial images were acquired for analysis.

In vivo therapeutic efficacy study
ApoE−/− mice were initially fed a high-fat diet for 8 weeks to induce the development of atherosclerotic plaques at arterial bifurcations (experimental days 0–56). On day 57, the atherosclerotic mice were randomly assigned to six treatment groups (n = 6 mice per group), including PBS, free AT, A-P, FA-P, FA-P@M, and FA-P@MC (at a dose of 3 mg/kg AT). Each group received intravenous injections of the designated treatment twice a week (days 57, 61, 64, 68, 71, 75, 78, 82, 85, 89, 92, 96, 99, 103, 106, and 110) while maintaining the high-fat diet for an additional 8 weeks (Day 57–112). A standard chow-fed control group was included for comparison. For the evaluation of therapeutic effects via in situ imaging at the end of treatment, mice were intravenously administered the FA-P@MC nanoprobe on Day 113. NIR-II fluorescence and PA imaging were performed 12 hours post-injection following the procedure described above. After imaging, mice were euthanized, and the entire aortas were carefully excised for subsequent analyses (Day 113). Atherosclerotic plaque burden was evaluated by ORO staining of the aortic arch, following established protocols as described above, to quantify lipid-rich plaque areas. Histological assessment of aortic root sections was performed after paraffin embedding. Sections were stained with H&E to examine general tissue morphology, while dihydroethidium (DHE) staining was applied to assess ROS levels within plaques. Immunofluorescence staining was carried out to evaluate the expression of CD47 and CD68, markers of phagocytosis and macrophage content, respectively. Plaque stability was further assessed by immunohistochemical staining of aortic root sections with Masson’s trichrome for collagen content, MMP-9 for matrix degradation, CD31 for angiogenesis, and \alpha-SMA for smooth muscle cell content. Quantitative analysis of stained sections was performed using Image-Pro Plus 6.0. The inflammation was evaluated by collecting fresh blood post-treatment, centrifuging to obtain serum, and measuring TNF-α and IL-6 levels using ELISA kits according to the manufacturer’s instructions. Immune cell profiling within plaques was performed by isolating plaque tissue, enzymatically digesting to obtain single-cell suspensions, and analyzing CD4+ (CD3+CD4+), CD8+ (CD3+CD8+), and regulatory T (Treg) cell (CD25+Foxp3+) populations using flow cytometry.

Statistical analysis
All data were presented as mean value ± the standard deviation of independent experiments. One-way ANOVA and two-way was utilized for statistical analysis. Values of *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 were applied to annotate statistical significance.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors report a hierarchical nanoplatform for multimodal imaging and targeted therapy of atherosclerosis (AS). They\nfirst constructed a molecular probe integrating MRI, NIR-II fluorescence, and PA imaging capabilities, enabling\ncomprehensive evaluation of atherosclerotic lesions and facilitating real-time monitoring of therapeutic outcomes. This\nimaging probe was further combined with a therapeutic agent capable of enhancing lipid efflux and downregulating CD47\nexpression, ultimately leading to inhibition of plaque progression, attenuation of inflammation, and improvement of plaque\nstability in AS mouse models. This research is interesting and holds promise for clinical application. Therefore, I suggest its\npublication after some revisions, as detailed below.\nMajor points:\n1. Although the authors have provided short-term biocompatibility data, the prolonged treatment course of AS necessitates\nevaluation of long-term in vivo biosafety. It is also crucial to investigate the metabolic fate and clearance pathways of the\nnanoplatform in vivo to assess the potential for accumulation or chronic toxicity.\n2. The H2O2-responsive drug release is conducted on macrophage membrane-camouflaged nanoparticles. However, it is\nnot clear to me how much the envelope affects its drug-releasing ability, so the effect of H2O2 on nanoparticles without\nmacrophage encapsulation should be investigated.\n3. The nanoparticle response was tested under H2O2 concentrations ranging from 0.01 to 1 mM. However, the physiological\nconcentration of H2O2 in atherosclerotic lesions is not clearly established. The authors should cite relevant literatures or\ninclude justification for the selected concentration range to confirm its pathophysiological relevance.\n4. Please clarify whether all experimental mice were fed the same high-fat diet throughout the study. A detailed description\nof the composition and source of the high-fat diet would help in reproducing the study and understanding the model’s\nrobustness.\n5. There appears to be an inconsistency between the 790 nm excitation used for PA imaging and the 808 nm excitation for\nfluorescence imaging. The rationale for selecting these two close but distinct wavelengths should be addressed.\nMinor points:\n1. After the biomimetic modification, data on the nanoprobe's colloidal stability under physiological conditions should be\npresented.\n2. In Fig. 1c, the fluorescence intensity of the compounds increases in THF/water mixtures with a water fraction up to 95%.\nThe authors should explain this phenomenon.\n3. In Figure 1m, need to give a clear response time.\n4. Please add color bar in Fig. 8i.\n5. In addition to the responsive polymer, the authors added DSPE-PEG to the system. What is the role of this polymer?", "reviewer_2": "The investigators have performed an elegant biomedical engineering feat- with a single molecule can be used for molecular\nimaging to detect early atherosclerosis with 3 separate advanced imaging modalities- MRI, NIR-II, and photoacoustic\nimaging.\nThey also perform elegant experiments in mouse models to demonstrate efficacy. This manuscript presents a scientifically\nrigorous and impactful study, with strong translational potential. Addressing the mechanistic and quantitative gaps would\nsignificantly strengthen the manuscript’s clarity.\nMy major concern is that this tool and approach does not take into consideration the reality of screening for early\natherosclerosis. The biggest cause of cardiovascular mortality is coronary artery disease. If a patient is asymptomatic-\nscreening even with established techniques as CACS, CT coronary angiography (+/- with photon counting capabilities) has\nyet to be accepted. The proposal that a major unmet need is optimizing the performance of a molecular imaging contrast\nagent for use in all three modalities of MRI, NIR-II and photoacoustic imaging is a ”significant challenge” relevant to\nscreening and treating early CAD is not well founded or backed up by reference to the current clinical literature. Whilst\ncarotid or peripheral artery disease may have some chances in the near future with techniques such as these- this fails to\naccurately predict CAD and CAD-related MI/death. Thus, my concern is that this theranostic tool may not be addressing a\nquestion that we need addressed from a diagnostic perspective.\nIn regard to potential therapeutic value of the tool, the investigators state that: probe, coupled with atorvastatin, is\nencapsulated in responsive carriers, further camouflaged with macrophage membranes, and functionalized with a foam cell-\ntargeting peptide. The hierarchical targeting strategy leverages the biomimetic affinity of macrophages for inflamed plaques\nand the selective targeting of foam cells through a phosphatidylserine (PS)-binding peptide, ensuring precise foam cell\nnavigation. The on-demand release of atorvastatin within foam cells promotes lipid efflux and reduces CD47 expression,\nthereby enhancing macrophage-mediated phagocytosis. This intervention effectively inhibits plaque progression, alleviates\ninflammation burden, and stabilizes plaques in AS mouse models”. This sounds fantastic, however, most of the steps\noutlined in this, and what is shown in the graphical abstract, is not backed with specific experimental data and appropriate\ncontrols, and is theoretical, based on the engineering. Eg. “while the targeting peptide directs the system to foam cells within\nplaques. Atorvastatin release within foam cells promotes lipid efflux and reduces CD47 expression, thereby enhancing\nmacrophage-mediated phagocytosis”. Causality is implied here regarding the specific targeting release of atorvastatin within\nfoam cells promotes the lipid efflux and reduces CD47, thereby enhancing macrophage- mediated\nphagocytosis. There are many layers of this that need specific control to declare causality. These are not well described in the abstract, or the\ngraphical abstract.\nWhen the reader dives in, there is some effort to address this- particularly in regard to the\nnanoparticle itself in the in vitro model of OxLDL induced endothelial inflammation and\nfoam cell interaction. While Western blotting confirms the presence of\nmacrophage membrane proteins like F4/80, there's no quantification done on the peptide density or coating efficiency on the\ncells. This lack of quantification limits the validation of levels or modifications that could happen on the surface. Also batch\nvariability on uptake efficiency by macrophages was not addressed.\nGiven this reviewer’s thoughts that the proposed urgent need for the diagnostic tool of nanoparticle with 3 different imaging\nmodality possibilities combined is not consistent with clinical need, the focus really should be on the therapeutic benefits of\nthis approach. The investigators suggest ways this may be the case- and provide some evidence for improved targeting. The\ninvestigators do compare the most functional version of their nano-particle to free atorvastatin. They report 3 animals per\ngroup, and a significant difference. It is not clear how the dose was established for atorvastatin- and how this compared to\nlevels in clinical practice. It is well reported and wonderful to see the half-life in circulation, but tissue-specific distribution is\nnot quantitatively reported. To claim the plaque specific delivery and reduced off target effects reported, including\nquantitation by fluorescence or radiolabelling in plaque vs organs(liver/spleen) is suggested.\nThe investigators state their approach “facilitates the targeted delivery of the ROS-scavenging\npolymer and AT into plaque foam cells for synergistic treatment”. It is not generally\nunderstood that atorvastatin or any statin has direct mechanism of benefit within the foam cell\n( vs targetting HMGCoreductase in the liver). Whilst off-target, non-specific effects have been\nproposed- this needs further introduction and discussion. For translational relevance, it is\nsuggested to consider immunological profiling beyond cytokines focussing on T-cell\nactivation after repeated dosing in animal model. Also, the study heavily emphasize on H₂O₂ as H₂O₂ is only one of the several ROS species elevated. Expanding ROS profiling to\nother superoxides, peroxides and including relevant ROS scavenger controls is needed to confirm\nROS specific CD47 effects.\nOverall strengths and additional comments in addition to above:\n- Beautifully written with clear linkage between each method and its corresponding result. Sophisticated imaging validation.\n- Beautiful graphical abstract and well written logical progression from molecular design to diagnostic and therapeutic\napplication.\n- The finding that macrophage membrane coating and PS-binding peptide modification significantly facilitated the transport\nof NPs across the inflamed endothelial barrier and their subsequent internalization by foam cells- is of interest and potential\nbroader relevance for atherosclerosis.\n- Use of appropriate models- in vitro, ApoE−/− mouse model. In Figure 5- why is the\napoe-/e- mouse only having carotid\nplaque detected? What about the classic finding of atherosclerosis in the arch of the aorta? Also- why is there only 1 visible\ndot of uptake- not bilateral uptake?\n- From a statistical perspective- there appear to only be n=3 of each treatment group- which\nseems to be below what would be appropriate for the tests used.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "The authors construct a multifunctional molecular probe capable of simultaneously generating near-infrared II (NIR-II)\nfluorescence, photoacoustic (PA), and magnetic resonance imaging (MRI) signals to enable comprehensive,\nmultidimensional assessment of AS plaques. They claim this probe can achieve on-demand release of atorvastatin within\nfoam cells promotes lipid efflux and reduces CD47 expression and thereby enhances macrophage-mediated phagocytosis,\nafter coupled with atorvastatin, encapsulated in responsive carriers, camouflaged with macrophage membranes, and\nfunctionalized with a foam cell-targeting peptide.\nMajor concerns:\n1）The authors use a phosphatidylserine-targeting peptide (PS-binding peptide) to mediate the targeted delivery of FA-\nP@MC to foam cells. However, since PS is externalized to the external leaflet in all apototic cells, the PS-targeting peptide\n(CLIKKPF) used in this study binds not only to foam cells but also to other apototic cells in theory. Fig. 5f,g data also indicate\nthat FA-P@M and FA-P@MC were not only detected in carotid plaques but also highly accumulated in liver and less\naccumulated in spleen and other tissues. Thus, the rationale using PS-binding peptide to target foam cells seems to be\nunreasonable in this case.\n2）In vitro anti-inflammatory and immunomodulatory effects section, the authors state that FA-P@MC treatment markedly\nsuppressed foam cell formation in comparison to other groups, likely due to the therapeutic effect of AT in promoting lipid\nefflux. This speculation is not convincing. The authors need to clarify the underlying mechanism by which FA-P@MC can\nfunctionally promote lipid efflux in foam cells, and identify the transporter/receptors that mediate cholesterol efflux in foam\ncells in this case.\nMinor concerns:\n3）Preparation of macrophage membrane and fabrication of FA-P@MC section. The targeting molecule (DSPE-PEG-\nCLIKKPF) was synthesized according to our previous protocol. Please cite the reference.\n4）List of abbreviations needed to be added."}, "author_response": "Responses to reviewers’ comments for the manuscript titled “A hierarchical theranostic\nnanoagent for multimodal imaging and targeted foam cell intervention in atherosclerosis”.\nWe sincerely thank the reviewers for their positive feedback and valuable comments\nconcerning our article. These comments and suggestions are of great importance to improve\nthe quality of our article. The point-by-point responses to those comments are presented as\nbelow and all changes to the manuscript are highlighted by using blue colored text. We hope\nthat the reviewers are satisfied with these changes and the manuscript will be accepted for\npublication in Nature Communications.\nResponses to the Comments and Suggestions of Reviewer #1\nReviewer #1 (Remarks to the Author):\nThe authors report a hierarchical nanoplatform for multimodal imaging and targeted therapy\nof atherosclerosis (AS). They first constructed a molecular probe integrating MRI, NIR-II\nfluorescence, and PA imaging capabilities, enabling comprehensive evaluation of\natherosclerotic lesions and facilitating real-time monitoring of therapeutic outcomes. This\nimaging probe was further combined with a therapeutic agent capable of enhancing lipid\nefflux and downregulating CD47 expression, ultimately leading to inhibition of plaque\nprogression, attenuation of inflammation, and improvement of plaque stability in AS mouse\nmodels. This research is interesting and holds promise for clinical application. Therefore, I\nsuggest its publication after some revisions, as detailed below.\nResponse: We sincerely appreciate your positive feedback and insightful comments on our\nwork. Your constructive and professional suggestions have greatly enhanced the quality of\nour manuscript. Below, we provide detailed responses to each of your comments.\nMajor points:\n1. Although the authors have provided short-term biocompatibility data, the prolonged\ntreatment course of AS necessitates evaluation of long-term in vivo biosafety. It is also\ncrucial to investigate the metabolic fate and clearance pathways of the nanoplatform in vivo\nto assess the potential for accumulation or chronic toxicity.\nResponse: We sincerely thank the reviewer for the insightful comment regarding the\nimportance of long-term biosafety assessment and metabolic fate studies of the nanoplatform.\nTo address the concern regarding long-term biosafety, we performed a seven-week repeated\nadministration study in mice, during which FA-P@MC nanoformulations were administered\nweekly. At the end of treatment, major organs (heart, liver, spleen, lung, and kidneys) and\nblood samples were collected for comprehensive evaluation. H&E staining revealed no\npathological abnormalities or inflammatory lesions in the examined organs (Figure 1a).\nFurthermore, routine hematological parameters as well as hepatic and renal function\nindicators showed no significant differences compared with the control PBS group (Figure\n1b,c). These results demonstrated the favorable long-term biocompatibility of the FA-P@MC\nnanoformulations. The corresponding data have been added and discussed in the revised\nmanuscript (new Supplementary Fig. 40, page 17).\nFigure 1. a) H&E staining images of major organ sections collected from mice after 7 weeks\nof treatment with PBS or FA-P@MC. Scale bars: 100 μm. b) Hematological parameters of\nmice following 7-week treatment with PBS or FA-P@MC, including white blood cell count\n(WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean\ncorpuscular volume (MCV), platelet count (PLT), mean corpuscular hemoglobin (MCH), and\nmean corpuscular hemoglobin concentration (MCHC). c) Serum biochemical analysis of\nhepatic and renal function indicators after 7-week treatment, including alanine\naminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and\ncreatinine (CREA). Data are expressed as mean ± SD (n = 3).\nIn addition, to investigate the in vivo metabolic fate and clearance pathways, FA-P@MC was\nlabeled with a near-infrared dye DiR. Fecal and urinary samples were collected at\npredetermined time intervals post-administration. As shown in Figure 2, intense fluorescence\nsignals were observed in fecal samples on days 1 and 3, whereas no significant signals were\ndetected in urine. By day 7, the fluorescence intensity in feces had markedly decreased,\nindicating that the nanoparticles were predominantly eliminated via the biliary pathway and\nwere nearly completely excreted within one week. These results are consistent with\npreviously reported findings (Proc. Natl. Acad. Sci. USA 2008, 105, 1410; Nat.\nCommun. 2024, 15, 10395; ACS Nano 2017, 11, 7177). The results have been added (new\nSupplementary Fig. 43) and discussed in the revised manuscript (page 18).\nWe greatly appreciate the reviewer’s valuable suggestions, which have helped us to\nstrengthen our study by providing additional data on the long-term safety and metabolic\nclearance of the nanoplatform.\nFigure 2. Fluorescent images of the collected feces and urine at various time points after\nintravenous injection of FA-P@MC (loaded with DiR) into mice.\n2. The H O -responsive drug release is conducted on macrophage membrane-camouflaged\n2 2\nnanoparticles. However, it is not clear to me how much the envelope affects its drug-releasing\nability, so the effect of H O on nanoparticles without macrophage encapsulation should be\n2 2\ninvestigated.\nResponse: Thank you for your valuable suggestion. In the revised manuscript, we have\nexamined the release profiles of AT drug from the nanocarriers without membrane coating.\nAs shown in Figure 3, incubation with 1 mM of H O for 36 h resulted in a cumulative drug\n2 2\nrelease of about 90% from FA-P NPs. Under the same conditions, FA-P@MC exhibited a\nslightly lower release (~84%). This result indicates that the membrane coating only modestly\ninfluences the H O -responsive release behavior, which could be attributed to the rapid\n2 2\npermeation of small molecules such as H O across the lipid layer. The detailed results and\n2 2\nthe corresponding discussion have been included in the revised manuscript (new\nSupplementary Fig. 29, page 9).\n) % 100\n(\ne s 80\na\ne le 60 0 mM H 2 O 2\nr\ne\n1.00 mM H\n2\nv 40\nita\nlu 20\nm\nu\nC 0\n0 6 12 18 24 30 36\nTime (h)\nFigure 3. AT release behavior from FA-P NPs in the presence or absence of H O . Data are\n2 2\npresented as mean ± SD (n = 3).\n3. The nanoparticle response was tested under H O concentrations ranging from 0.1 to 1 mM.\n2 2\nHowever, the physiological concentration of H O in atherosclerotic lesions is not clearly\n2 2\nestablished. The authors should cite relevant literatures or include justification for the\nselected concentration range to confirm its pathophysiological relevance.\nResponse: We sincerely appreciate the reviewer’s insightful comment. In this study, H O\n2 2\nwas chosen as a kind of representative ROS to evaluate the responsive drug release behavior\nof the nanocarriers, as it is one of the predominant and relatively stable ROS in\natherosclerotic lesions. We quantified the H O concentrations in atherosclerotic plaques\n2 2\nusing a commercial H O Assay Kit. The results demonstrated that the H O level in plaque\n2 2 2 2\ntissues was approximately 0.3-0.4 mM. Accordingly, in our in vitro experiments, we selected\na concentration range of 0.1–1 mM, which not only reflects the measured pathological levels\nbut also aligns with the range commonly adopted in previous studies to evaluate\nROS-responsive therapeutic systems for atherosclerosis (J. Am. Coll. Cardiol. 2018, 72, 2591;\nACS Nano 2018, 12, 8943). The corresponding explanation has been included in the revised\nmanuscript (page 9).\n4. Please clarify whether all experimental mice were fed the same high-fat diet throughout the\nstudy. A detailed description of the composition and source of the high-fat diet would help in\nreproducing the study and understanding the model’s robustness.\nResponse: We thank the reviewer for this valuable comment. All experimental mice used in\nthis study were maintained on the same HDF throughout the experimental period to ensure\nconsistency. The HFD was purchased from SPF (Beijing) Biotechnology Co., Ltd and\nconsisted of 21.2% lard, 49.1% carbohydrate, 19.8% protein, and 0.2% cholesterol by weight,\na formulation widely used for establishing atherosclerosis models. The detailed composition\nand supplier information have now been included in the revised Methods section to facilitate\nreproducibility and better support the robustness of our experimental model.\n5. There appears to be an inconsistency between the 790 nm excitation used for PA imaging\nand the 808 nm excitation for fluorescence imaging. The rationale for selecting these two\nclose but distinct wavelengths should be addressed.\nResponse: We sincerely thank the reviewer for this insightful comment. The selection of\n790 nm for PA imaging and 808 nm for fluorescence imaging were based on a careful\noptimization of both the probe’s optical properties and the availability of excitation sources.\nSpecifically, 790 nm was chosen for PA imaging because it aligns with the probe’s absorption\npeak and corresponds to a strong photoacoustic signal, as confirmed by PA spectroscopy,\nmaking 790 nm the optimal choice for high-contrast PA imaging. For NIR-II fluorescence\nimaging, 808 nm excitation was selected because commercially available lasers at this\nwavelength provide efficient excitation, and the probe also showed strong absorption at\n808 nm, enabling effective fluorescence emission.\nThus, the slight difference in excitation wavelengths reflects a balance between optimal\nmolecular signal performance and instrumental availability. This strategy is consistent with\nprevious reports in the literatures; for example, ACS Nano 2017, 11, 12276 employed 700 nm\nexcitation for PA imaging and 808 nm excitation for fluorescence imaging in vivo. We have\nclarified this rationale in the revised Methods section to address the reviewer’s concern.\nMinor points:\n1. After the biomimetic modification, data on the nanoprobe's colloidal stability under\nphysiological conditions should be presented.\nResponse: According to the reviewer’s suggestion, the colloidal stability of FA-P@MC was\nevaluated in a physiologically relevant medium containing 10% fetal bovine serum (FBS).\nAs depicted in Figure 4, the nanoparticle size remained largely unchanged within 3 days,\ndemonstrating the formulation's stability under physiologically relevant conditions. The\nresults have been added (new Supplementary Fig. 24) and discussed in the revised manuscript\n(page 8).\n200\n)150\nm\nn\n(\ne 100\nz\niS\n50\n0\n1 2 3\nTime (day)\nFigure 4. The colloidal stability of FA-P@MC in 10% FBS measured by DLS. Data were\npresented as mean ± SD (n = 3).\n2. In Fig. 1c, the fluorescence intensity of the compounds increases in THF/water mixtures\nwith a water fraction up to 95%. The authors should explain this phenomenon.\nResponse: We sincerely thank the reviewer’s insightful question. The observed fluorescence\nenhancement of the compound in THF/water mixtures with high water fractions is attributed\nto the aggregation-induced emission (AIE) effect, a characteristic photophysical property of\nour designed molecule. Unlike conventional fluorophores that exhibit aggregation-caused\nquenching effect, AIE-active molecules are non-emissive or weakly emissive in solution but\nbecome highly fluorescent upon aggregation (Nat. Commun. 2023, 14, 5216). In good\nsolvents (such as THF), the AIE molecules are weakly emissive due to non-radiative energy\ndissipation through intramolecular motions. As the fraction of a poor solvent, such as water,\nincreases, the compound’s solubility decreases, leading to molecular aggregation.\nAggregation restricts intramolecular motions, suppressing non-radiative decay pathways and\npromoting radiative transitions, which results in a marked increase in fluorescence intensity.\nThis AIE-induced fluorescence enhancement is a photophysical characteristic of our probe\nand suggests its suitability for imaging in aqueous biological environments.\n3. In Figure 1m, need to give a clear response time.\nResponse: Thanks for this kind reminder. The response time of FA-P@MC in Fig. 1m has\nnow been clearly indicated in the figure caption of the revised manuscript.\n4. Please add color bar in Fig. 8i.\nResponse: We sincerely thank the reviewer for the careful reading and helpful suggestion.\nThe color bars have been added to Fig. 8i in the revised manuscript, and we have also\ncarefully checked all other figures to ensure that color bars are properly included where\nnecessary.\n5. In addition to the responsive polymer, the authors added DSPE-PEG to the system. What is\nthe role of this polymer?\nResponse: We sincerely thank the reviewer for this important question. In our system,\nDSPE-PEG was introduced during nanoparticle preparation via the nanoprecipitation method\nto function as a stabilizing surfactant. Owing to its amphiphilic structure, DSPE-PEG anchors\nits hydrophobic lipid tail within the nanoparticle core while presenting a hydrophilic PEG\ncorona on the surface (Nat. Commun. 2022, 13, 758; ACS Nano 2023, 17, 955). This\nprevents excessive aggregation of the hydrophobic probe and the ROS-responsive polymer,\nthereby promoting uniform nanoparticle formation and enhancing colloidal stability. The\ncorresponding explanation has also been added to the revised Methods section.\nReviewer #2 (Remarks to the Author):\nThe investigators have performed an elegant biomedical engineering feat- with a single\nmolecule can be used for molecular imaging to detect early atherosclerosis with 3 separate\nadvanced imaging modalities- MRI, NIR-II, and photoacoustic imaging.\nThey also perform elegant experiments in mouse models to demonstrate efficacy. This\nmanuscript presents a scientifically rigorous and impactful study, with strong translational\npotential. Addressing the mechanistic and quantitative gaps would significantly strengthen\nthe manuscript’s clarity.\nResponse: We sincerely appreciate your positive feedback and insightful comments on our\nwork. Your constructive and professional suggestions have greatly enhanced the quality of\nour manuscript. Below, we provide detailed responses to each of your comments.\nMy major concern is that this tool and approach does not take into consideration the reality of\nscreening for early atherosclerosis. The biggest cause of cardiovascular mortality is coronary\nartery disease. If a patient is asymptomatic- screening even with established techniques as\nCACS, CT coronary angiography (+/- with photon counting capabilities) has yet to be\naccepted. The proposal that a major unmet need is optimizing the performance of a molecular\nimaging contrast agent for use in all three modalities of MRI, NIR-II and photoacoustic\nimaging is a “significant challenge” relevant to screening and treating early CAD is not well\nfounded or backed up by reference to the current clinical literature. Whilst carotid or\nperipheral artery disease may have some chances in the near future with techniques such as\nthese- this fails to accurately predict CAD and CAD-related MI/death. Thus, my concern is\nthat this theranostic tool may not be addressing a question that we need addressed from a\ndiagnostic perspective.\nResponse: We sincerely thank the reviewer for this insightful comment. We fully agree that\nearly screening for coronary artery disease (CAD) in asymptomatic patients remains highly\nchallenging. As the reviewer mentioned, even well-established modalities such as CACS and\nCT coronary angiography are not yet widely accepted in this context, as they are usually less\nsensitive for detecting non-calcified plaques and are constrained by radiation exposure and\ncost, which restrict repeated monitoring in high-risk populations—particularly since\nearly-stage atherosclerosis often lacks calcification (Cardiovasc. Interv. Ther. 2025, 40, 536;\nCirc. Cardiovasc. Interv. 2025, 28, e015529).\nIn this study, our goal was to establish a proof-of-concept multimodal molecular imaging\nstrategy integrating MRI, NIR-II FL, and photoacoustic imaging for multidimensional\nevaluation of atherosclerosis. For feasibility assessment, we primarily focused on carotid\natherosclerosis, which are widely used in proof-of-concept studies and are\npathophysiologically relevant to systemic atherosclerosis and CAD risk. Our results\ndemonstrated that integrating complementary imaging modalities within one probe enhance\ndiagnostic accuracy and sensitivity by providing comprehensive, high-resolution insights into\nplaque. A key advantage of our platform is that it targets foam cells and combines\ncomplementary imaging modalities within a single molecular probe. As such, it is not\ndependent on calcification and does not involve ionizing radiation, potentially overcoming\nsome of the limitations of current approaches for early-stage plaque monitoring.\nTo further address the reviewer’s comments, we conducted preliminary experiments to\nexplore the potential applicability of the platform in CAD. Early results suggested that our\nplatform-based NIR-II fluorescence imaging could be applied to visualize coronary stenosis\nin high-fat diet-fed ApoE⁻/⁻ mice (Figure 1a,b). Histological analyses (H&E and ORO\nstaining) confirmed coronary plaque formation and lipid accumulation (Figure 1c-f), with\nNIR-II signal correlating with plaque burden. These findings demonstrate the potential of\nextending this imaging strategy to coronary models.\nOverall, the current study provides a proof-of-concept demonstrating the feasibility of a foam\ncell-targeted multimodal imaging platform for atherosclerosis diagnosis. Moving forward, we\nplan to further optimize the imaging probe and multimodal acquisition protocols to further\nimprove sensitivity and specificity for CAD detection, thereby bridging the gap between\npreclinical AS models and clinically relevant coronary imaging. In the revised manuscript, we\nhave expanded the discussion to clarify these points (new Supplementary Fig. 56, page 34).\nFigure 1. a) Representative bright-field and NIR-II fluorescence images of coronary arteries\nfrom healthy and atherosclerotic mice. Scale bars: 1 cm. b) Quantitative analysis of NIR-II\nFL intensity in healthy versus atherosclerotic mice. c) Representative H&E-stained coronary\nsections and d) corresponding quantification of plaque area in healthy and atherosclerotic\nmice. Scale bar: 100 μm. e) Representative ORO-stained coronary sections and f)\ncorresponding quantification of lipid-rich plaque ratio. Scale bar: 100 μm. Data were\npresented as mean ± SD (n = 6). Statistical significance was calculated using two-tailed\nStudent’s t-test.\nIn regard to potential therapeutic value of the tool, the investigators state that: probe, coupled\nwith atorvastatin, is encapsulated in responsive carriers, further camouflaged with\nmacrophage membranes, and functionalized with a foam cell-targeting peptide. The\nhierarchical targeting strategy leverages the biomimetic affinity of macrophages for inflamed\nplaques and the selective targeting of foam cells through a phosphatidylserine (PS)-binding\npeptide, ensuring precise foam cell navigation. The on-demand release of atorvastatin within\nfoam cells promotes lipid efflux and reduces CD47 expression, thereby enhancing\nmacrophage-mediated phagocytosis. This intervention effectively inhibits plaque progression,\nalleviates inflammation burden, and stabilizes plaques in AS mouse models”.\nThis sounds fantastic, however, most of the steps outlined in this, and what is shown in the\ngraphical abstract, is not backed with specific experimental data and appropriate controls, and\nis theoretical, based on the engineering. Eg. “while the targeting peptide directs the system to\nfoam cells within plaques. Atorvastatin release within foam cells promotes lipid efflux and\nreduces CD47 expression, thereby enhancing macrophage-mediated phagocytosis”. Causality\nis implied here regarding the specific targeting release of atorvastatin within foam cells\npromotes the lipid efflux and reduces CD47, thereby enhancing macrophage- mediated\nphagocytosis. There are many layers of this that need specific control to declare causality.\nThese are not well described in the abstract, or the graphical abstract.\nResponse: We sincerely thank the reviewer for the thoughtful suggestions and constructive\ncomments highlighting the importance of providing more detailed experiment evidence to\nsupport the causal relationships proposed in our study. In response, we have reorganized the\ndata presentation and performed additional experiments to address these concerns, as outlined\nbelow:\n1. Targeting and uptake by foam cells: We demonstrated that FA-P@MC could efficiently\ntraverse the inflammatory endothelial barrier and be preferentially internalized by foam cells\n(Figure 2a–f in the manuscript). These results provide direct evidence that the nanoplatform\npossesses foam cell–targeting capability, establishing the basis for drug delivery to foam\ncells.\n2. Effects on lipid uptake: We have demonstrated that FA-P@MC treatment significantly\nreduced lipid uptake by foam cells using ORO staining (Figure 2g,h in the manuscript). To\nfurther validate this effect, we performed additional experiments using DiI-labelled oxLDL.\nFluorescence imaging confirmed that oxLDL uptake in the FA-P@MC group was\n1.84–4.65-folds lower than those in the other treatment groups (Figure 2a-b below).\nTo further elucidate the underlying mechanism, we conducted additional experiments\nexamining the expression of key lipid uptake receptors. Our results indicated that FA-P@MC\ntreatment downregulated the scavenger receptors CD36 and LOX-1 relative to control\ntreatments, which are known to mediate oxLDL internalization, thereby limiting lipid\naccumulation in foam cells (Figure 2c-f below).\n3. Effects on cholesterol efflux: To further investigate cholesterol efflux, we analyzed the\nculture supernatant from foam cells following different treatments. Quantitative analysis\nrevealed a significant increase in cholesterol efflux in the supernatant from\nFA-P@MC–treated group compared with PBS controls and other groups (Figure 3a). In\naddition, immunofluorescence staining demonstrated upregulation of SR-BI and ABCA1,\ntwo\ncritical transporters involved in cholesterol efflux (Figure 3b–e). These results indicate that\nFA-P@MC promotes cholesterol efflux in foam cells by enhancing the expression of efflux\ntransporters.\n4. Regulation of CD47 expression and phagocytosis：Both in vitro and in vivo analyses,\nincluding Western blotting and immunofluorescence staining, demonstrated that targeted\natorvastatin delivery via FA-P@MC significantly downregulated CD47 expression on foam\ncells (Figure 3a–c, Figure 7h–j in the manuscript). Functionally, this downregulation was\nassociated with enhanced phagocytic clearance of foam cells by macrophages, as confirmed\nthrough immunofluorescence imaging and quantitative flow cytometry analysis (Figure 3d–g\nin the manuscript).\n5. In vivo enhanced plaque delivery: Both the in vivo multimodel imaging and ex imaging of\nharvested carotid samples indicated the FA-P@MC had improved targeting toward the plaque\nlesion compared to the non-targeted nanoformulations.\nWe further conducted additional experiment indicated that FA-P@MC can penetrate into\nplaques and be preferentially uptake by foam cells (Figure 4 below). Additionally,\nquantitative analysis of drug accumulation in plaque tissues was conducted, which also\nconfirmed that the FA-P@MC group had a higher AT content within plaques compared with\nnon-targeting control formulations (Figure 5 below), indicating the enhanced drug delivery to\nplaques.\n6. In vivo therapeutic outcomes: In ApoE⁻/⁻ mice, ORO staining of the aortic arch and H&E\nstaining demonstrated that FA-P@MC treatment significantly reduced plaque progression\ncompared with other groups. Analyses of inflammatory cytokines and immune cell\ninfiltration indicated that FA-P@MC mitigated plaque-associated inflammation.\nImmunohistochemical staining also suggested improved plaque stability following treatment.\nTo corroborate these findings, we further performed additional ORO staining of the aortic\nroot and biochemical assays, which confirmed that FA-P@MC administration markedly\nreduced plaque lipid content and total cholesterol levels (Figures 6, 7 below). These results\nare consistent with the observed modulation of lipid uptake, enhanced cholesterol efflux, and\nfoam cell clearance, collectively supporting the therapeutic efficacy of FA-P@MC in\nattenuating atherosclerotic plaque development.\nTogether, these results provide experimental support for the sequential pathway we proposed:\ntargeted delivery of atorvastatin into foam cells → reduced lipid uptake and enhanced\ncholesterol efflux → downregulation of CD47 → improved phagocytic clearance →\nattenuation of plaque burden. These new results and corresponding discussions have been\nadded to the revised manuscript (new Supplementary Fig. 33-34, page 14; new\nSupplementary Fig. 46-50, pages 21, 27). We sincerely thank the reviewer for these\nconstructive comments, which have substantially strengthened our study.\nFigure 2. a) Representative confocal laser scanning microscope (CLSM) images of\nDiI-oxLDL uptake in RAW 264.7 cells after different treatments. Scale bar: 50 μm. b) The\nmean fluorescence intensity (MFI) of DiI-oxLDL in different treatment groups. c)\nRepresentative CLSM images of CD36 expression in RAW 264.7 cells treated with oxLDL\nand various formulations for 24 h. Scale bar: 25 μm. d) The MFI of CD36 expression in\ndifferent treatment groups. e) Representative CLSM images of LOX-1 expression in RAW\n264.7 cells treated with oxLDL and various formulations for 24 h. Scale bar: 25 μm. f) The\nMFI of LOX-1 expression in different treatment groups. Data are expressed as mean ± SD (n\n= 3). Statistical significance was analyzed using one-way ANOVA.\nFigure 3. a) Cholesterol content in foam cell supernatant in different treatment groups. b)\nRepresentative CLSM images of SR-BI expression in foam cells treated with various\nformulations for 24 h. Scale bar: 25 μm. c) The MFI of SR-BI in different treatment groups. d)\nRepresentative CLSM images of ABCA1 expression in foam cells treated with various\nformulations for 24 h. Scale bar: 25 μm. e) The MFI of ABCA1 in different treatment groups.\nData are expressed as mean ± SD (n = 3). Statistical significance was analyzed using one-way\nANOVA.\nFigure 4. Confocal microscopy images of aortic root sections from mice injected with\ndifferent Cy5-AT-loaded formulations. The foam cells were stained using F4/80 antibody and\nBODIPY. The plaque lesion is outlined by a white dotted line. Scale bar: 20 μm.\nPBS\nFA-P@M\ng 30 FA-P@MC\n/%\np＜ 0.0001\nD 20\nI\n10\nFigure 5. Quantitative analysis of AT content in aorta and main organs of ApoE⁻/⁻ mice at 12\nh post administration of different formulations. Data are expressed as mean ± SD (n = 3).\nStatistical significance was analyzed using one-way ANOVA.\nFigure 6. a) Representative images and b) quantitative analysis of ORO-stained plaque lesion\nin aortic root sections. Scale bar: 500 μm. Data are expressed as mean ± SD (n = 6).\nStatistical significance was analyzed using one-way ANOVA.\nlo\nre\n)th\np < 0.0001\nts\ne\ng\nie 12\np = 0.0274\nlow\nh\nc\nte\n8\nla\nto\nt\nc\nw\ng\n/lo\nm\n4\nitro\nA\nm\n( 0\nS T P PM C\nP F B r e e AA- F F A A - - P A @ - P @ M\nF\nFigure 7. The levels of total cholesterol in the aorta after different treatments, measured\nusing Cholesterol Assay Kit. Data are expressed as mean ± SD (n = 6). Statistical significance\nwas analyzed using one-way ANOVA.\nWhen the reader dives in, there is some effort to address this- particularly in regard to the\nnanoparticle itself in the in vitro model of OxLDL induced endothelial inflammation and\nfoam cell interaction. While Western blotting confirms the presence of macrophage\nmembrane proteins like F4/80, there's no quantification done on the peptide density or\ncoating efficiency on the cells. This lack of quantification limits the validation of levels or\nmodifications that could happen on the surface. Also batch variability on uptake efficiency by\nmacrophages was not addressed.\nResponse: We sincerely thank the reviewer for this constructive suggestion. To address these\nissues, we estimated the peptide coating density on nanoparticles prepared from different\nindependent batches using HPLC. The results indicated that the nanoparticles synthesized by\nour nanoprecipitation method exhibited good reproducibility and generally consistent peptide\ncoating across batches (Table 1). Additionally, we further evaluated the uptake efficiency of\nnanoparticles from different batches at the cellular level. Fluorescence-based assays showed\nsimilar cell internalization among the batches (Figure 8).\nThese results have been incorporated into the revised manuscript (new Supplementary Fig. 23,\npage 8; Supplementary Fig. 32, page 11) and discussed accordingly. Collectively, these\nadditional data strengthen the validation of nanoparticle surface modifications and their\nreproducible biological interactions.\nTable 1. Hydrodynamic diameter and peptide density of nanoparticles prepared from\ndifferent batches.\nFigure 8. a) Representative CLSM images showing foam cell uptake of nanoparticles from\ndifferent batches. Scale bar: 20 μm. b) Quantitative analysis of cellular uptake efficiency\namong NPs from different batches. Data are expressed as mean ± SD (n = 5). Statistical\nsignificance was analyzed using one-way ANOVA.\nGiven this reviewer’s thoughts that the proposed urgent need for the diagnostic tool of\nnanoparticle with 3 different imaging modality possibilities combined is not consistent with\nclinical need, the focus really should be on the therapeutic benefits of this approach. The\ninvestigators suggest ways this may be the case- and provide some evidence for improved\ntargeting. The investigators do compare the most functional version of their nano-particle to\nfree atorvastatin. They report 3 animals per group, and a significant difference. It is not clear\nhow the dose was established for atorvastatin- and how this compared to levels in clinical\npractice. It is well reported and wonderful to see the half-life in circulation, but tissue-specific\ndistribution is not quantitatively reported. To claim the plaque specific delivery and reduced\noff target effects reported, including quantitation by fluorescence or radiolabelling in plaque\nvs organs(liver/spleen) is suggested.\nResponse: We sincerely thank the reviewer for these constructive comments and suggestions.\nRegarding the dosing strategy, for the clinical prevention and management of cardiovascular\ndisease, statins are mainly administered orally at doses ranging from 10–80 mg/day. To allow\ncomparison with our animal studies, we referred to the FDA-recommended body surface area\nconversion method to estimate the human equivalent dose. The dose of 3 mg/kg used in our\nApoE⁻/⁻ mouse model corresponds to approximately 14 mg for a 60 kg adult, which falls\nwithin the clinically relevant range. Additionally, our regimen (3 mg/kg, twice weekly,\nintravenously) is lower than those used for several previously reported intravenous nano-AT\nformulations in animal studies (e.g., 7 mg/kg, twice weekly in J. Control. Release 2018, 283,\n241; 8.5 mg/kg every other day in Nanoscale 2020, 12, 9541), yet it still achieved efficient\ntherapeutic benefit. Thus, the dosing strategy we adopted provides a reasonable assessment of\nthe therapeutic potential of our system.\nTo assess tissue distribution and plaque targeting, we further performed biodistribution\nstudies using Cy5-labelled AT. Different formulations were intravenously injected into\nApoE⁻/⁻ mice, and major organs as well as the aorta were harvested at 12 h post-injection for\nanalysis. In the AT alone group, only weak fluorescence was detected in the aorta, consistent\nwith the rapid clearance of small-molecule AT (Figure 9a,b). In contrast, FA-P@MC\ndemonstrated markedly enhanced accumulation in aortic plaques compared with the\nnon-targeted FA-P@M and free AT groups. To further confirm these findings, we quantified\nthe AT content in different tissues through homogenization and extraction. Both FA-P@M\nand FA-P@MC exhibited notable liver distribution due to nanoparticle uptake by the\nreticuloendothelial system, consistent with common nanoparticle biodistribution patterns.\nImportantly, the drug concentrations in plaque tissues of FA-P@MC group were\napproximately 12.7-fold higher than free AT and about 2-fold higher than FA-P@M group\n(Figure 10). Together, these results provide evidence of improved plaque delivery of AT via\nFA-P@MC, consistent with its therapeutic advantage. The data have been added to the\nrevised manuscript (new Supplementary Fig. 45 and Fig. 46) and discussed accordingly (page\n21).\nFigure 9. a) Ex vivo FL images of aorta collected from atherosclerotic mice at 12 h\npost-injection of PBS, free AT, FA-P@M or FA-P@MC (AT in all groups were labeled with\nCy5 dye). b) Quantitative analysis of Cy5 FL intensity in aorta. Data are expressed as mean ±\nSD (n = 3 mice per group). Statistical significance was analyzed using one-way ANOVA.\nPBS\nFA-P@M\ng 30 FA-P@MC\n/%\np＜ 0.0001\nD 20\nI\n10\nFigure 10. Quantitative analysis of AT content in aorta and main organs of ApoE⁻/⁻ mice at\n12 h post administration of different formulations. Data are expressed as mean ± SD (n = 3).\nStatistical significance was analyzed using one-way ANOVA.\nThe investigators state their approach “facilitates the targeted delivery of the ROS-scavenging\npolymer and AT into plaque foam cells for synergistic treatment”. It is not generally\nunderstood that atorvastatin or any statin has direct mechanism of benefit within the foam cell\n(vs targetting HMGCoreductase in the liver). Whilst off-target, non-specific effects have been\nproposed- this needs further introduction and discussion. For translational relevance, it is\nsuggested to consider immunological profiling beyond cytokines focussing on T-cell\nactivation after repeated dosing in animal model. Also, the study heavily emphasize on H₂O₂ as H₂O₂ is only one of the several ROS species elevated. Expanding ROS profiling to\nother superoxides, peroxides and including relevant ROS scavenger controls is needed to confirm\nROS specific CD47 effects.\nResponse: We sincerely thank the reviewer for these insightful suggestions. While the\nprimary therapeutic effect of atorvastatin is usually mediated via inhibition of HMG-CoA\nreductase in the liver, increasing evidence indicates that it can also exert local effects within\nthe vascular walls and immune cells, including modulation of cholesterol efflux and\nanti-inflammatory activity (Clin. Rev. Allergy Immunol. 2020, 60, 175; Nat. Rev. Immunol.\n2006, 6, 358). Consistent with these reports, both our in vitro and in vivo experimental data\nindicated that AT can effectively reduce lipid uptake, enhance cholesterol efflux in foam cells,\nand reduce the foam cells, thus alleviating the plaque burdens. According to the reviewer’s\nsuggestion, we have included a related discussion in the revised manuscript (page 7).\nTo further address the reviewer’s comments regarding immunological profiling on T cells,\nwe\nevaluated T cell populations within atherosclerotic plaques from different treatment groups. T\nlymphocytes, recruited by antigen-presenting cells, are closely associated with lesion\ninflammation. During plaque progression, CD4+ and CD8+ T cells generally increase and\nsecret pro-inflammatory cytokines, whereas regulatory T cells (Tregs) are relatively low or\ninsufficient, providing limited anti-inflammatory control (Nat. Med. 2019, 25, 1576;\nImmunobiology 2016, 22, 1014; Life 2023, 13, 1931). In the FA-P@MC–treated group, the\nproportions of CD8+ and CD4+ T cells within plaques were found to reduce by approximately\n4.68- and 2.53-fold, respectively, compared with the model group, as determined by flow\ncytometry (Figure 11a-c). In contrast, the number of CD25+Foxp3+ Treg cells in the\nFA-P@MC group increased by approximately 5.19-fold relative to the model group (Figure\n11d,e).\nThese changes in T cell composition were consistent with the observed reduction in\npro-inflammatory cytokines reported in our manuscript. Collectively, these findings suggest\nthat FA-P@MC treatment can further affect T cell populations within plaques, providing\nadditional insight into its immunomodulatory effects. The data have been added to the revised\nmanuscript (new Supplementary Fig. 53) and discussed accordingly (page 32).\nFigure 11. a) Representative flow cytometry plots showing the proportions of CD8⁺ and\nCD4⁺ T cells in aortas from mice receiving different treatments. Quantitative analysis of b)\nCD8⁺ T cell and c) CD4⁺ T cell proportions across treatment groups. d) Representative flow\ncytometry plots and e) quantitative analysis showing the CD25+Foxp3+ Treg cell proportions\nin aortas after different treatments. Data are expressed as mean ± SD (n = 6). Statistical\nsignificance was determined using one-way ANOVA.\nRegarding ROS species, we agree that H O represents one of several ROS elevated in\n2 2\natherosclerotic plaques, and other species such as superoxide anion (O •−) and hydroxyl\n2\nradicals (•OH) also play important roles. In our initial experiments, H O was used as a\n2 2\nrepresentative ROS for studying ROS-responsive drug release from FA-P@MC due to its\naccessibility and widespread use in studies of oxidative stress.\nTo address the reviewer’s concern, we have expanded our studies to include additional ROS\nspecies and incorporated specific ROS scavenger controls. First, cumulative drug release\nexperiments demonstrated that both O •− and •OH could effectively trigger AT release from\nhigher oxidative reactivity. These results indicate that the nanoparticles are responsive to\nmultiple ROS species, rather than H O alone.\n2 2\nAt the cellular level, fluorescence probes confirmed that both H O and •OH were generated\n2 2\nby inflamed foam cells. The addition of their respective scavengers—catalase (for H O ), and\n2 2\nmannitol (for •OH)—effectively reduced the levels of the corresponding ROS.\nImmunofluorescence analysis further revealed that ROS-triggered AT release from\nFA-P@MC led to the obvious downregulation of CD47 expression on foam cells. Notably,\nthe presence of respective ROS scavengers partially reversed this CD47 downregulation,\nlikely due to the decreased AT release. Consistently, ROS-nonresponsive control\nnanoparticles (FA@MC) exhibited a diminished effect on CD47 modulation.\nWe sincerely thank the reviewer for the constructive suggestions. These new results and the\ncorresponding discussion have been incorporated into the revised manuscript (new\nSupplementary Fig. 30, page 9; Supplementary Fig. 36, page 15), providing a more\ncomprehensive understanding of ROS-mediated drug release and CD47 regulation.\n) % 100\n(\ne s 80\na\ne\nle 60\nr\ne\nv ita\nlu\nm\n2\n4\n0\nO\n•O\no\n•\nn\n-\ntrol\nu\nC 0\n0 6 12 18 24 30 36\nTime (h)\nFigure 12. Cumulative drug release profile from FA-P@MC in PBS and in the presence of\n•OH and O •−. Data are presented as mean ± SD (n = 3).\nprobe in foam cells, with or without treatment using the corresponding ROS scavengers.\nScale bar: 50 μm. b) Immunofluorescence images of CD47 expression in foam cells after\ntreatment with FA@MC (ROS-nonresponsive control), FA-P@MC, or FA-P@MC in\ncombination with different ROS scavengers. Scale bar: 50 μm. c) Quantitative analysis of the\nCD47 expression under different treatment conditions. Data are expressed as mean ± SD (n =\n3). Statistical significance was analyzed using one-way ANOVA.\nOverall strengths and additional comments in addition to above:\n- Beautifully written with clear linkage between each method and its corresponding result.\nSophisticated imaging validation.\n- Beautiful graphical abstract and well written logical progression from molecular design to\ndiagnostic and therapeutic application.\n- The finding that macrophage membrane coating and PS-binding peptide modification\nsignificantly facilitated the transport of NPs across the inflamed endothelial barrier and their\nsubsequent internalization by foam cells- is of interest and potential broader relevance for\natherosclerosis.\nResponse: We sincerely thank the reviewer for the thoughtful and encouraging comments on\nour manuscript, graphical abstract, and imaging validation. We are also very grateful for the\nrecognition of our findings on macrophage membrane coating and PS-binding peptide\nmodification facilitating nanoparticle delivery to foam cells, which we believe may have\nbroader implications for atherosclerosis therapy.\n- Use of appropriate models- in vitro, ApoE−/− mouse model. In Figure 5- why is the\napoe-/e- mouse only having carotid plaque detected? What about the classic finding of\natherosclerosis in the arch of the aorta? Also- why is there only 1 visible dot of uptake- not\nbilateral uptake?\nResponse: We sincerely appreciate the reviewer’s insightful questions regarding the imaging\nresults in our ApoE−/− mouse model.\nIn Figure 5, we employed NIR-II FL imaging to compare the plaque-targeting capabilities of\ndifferent nanoformulations. We focused on carotid artery plaques, which are more accessible\nfor high-resolution NIR-II FL imaging. Moreover, the carotid artery is a well-established and\nwidely used model for atherosclerotic plaque imaging in ApoE⁻/⁻ mice (Adv. Mater. 2022, 34,\n2106994; Nat. Commun. 2025, 16, 3970).\nRegarding the apparent “single dot” signal, we would like to clarify that the fluorescent\nsignals are in fact bilateral in the carotid arteries; however, due to the scale of the original\nimages, both sides are not easily distinguishable. We have provided enlarged images in the\nrevised manuscript (new Supplementary Fig. 44) to allow readers to better visualize the\nbilateral carotid signals. Thank you for your insightful question, which allows us to further\nimprove the presentation of our data.\n- From a statistical perspective- there appear to only be n=3 of each treatment group- which\nseems to be below what would be appropriate for the tests used.\nResponse: Thanks for your comment on the sample size. Additional experiments were\nconducted and the number of mice of each treatment group has been extended to 6 per group\n(n = 6). Accordingly, Fig. 7, 8 and Supplementary Fig. 49-55 have been updated.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate the information regarding the co-review process and fully\nsupport the Nature Communications initiative to involve and recognize Early Career\nResearchers in peer review. We would like to thank both the reviewer and the co-reviewer for\ntheir constructive feedback, which has been highly valuable in improving the quality of our\nmanuscript.\nReviewer #4 (Remarks to the Author):\nThe authors construct a multifunctional molecular probe capable of simultaneously\ngenerating near-infrared II (NIR-II) fluorescence, photoacoustic (PA), and magnetic\nresonance imaging (MRI) signals to enable comprehensive, multidimensional assessment of\nAS plaques. They claim this probe can achieve on-demand release of atorvastatin within\nfoam cells promotes lipid efflux and reduces CD47 expression and thereby enhances\nmacrophage-mediated phagocytosis, after coupled with atorvastatin, encapsulated in\nresponsive carriers, camouflaged with macrophage membranes, and functionalized with a\nfoam cell-targeting peptide.\nResponse: We sincerely thank you for your positive feedback and insightful comments on\nour work. Your constructive suggestions have significantly improved the quality of our\nmanuscript. Below, we provide detailed responses to each of your comments.\nMajor concerns:\n1）The authors use a phosphatidylserine-targeting peptide (PS-binding peptide) to mediate the\ntargeted delivery of FA-P@MC to foam cells. However, since PS is externalized to the\nexternal leaflet in all apototic cells, the PS-targeting peptide (CLIKKPF) used in this study\nbinds not only to foam cells but also to other apototic cells in theory. Fig. 5f, g data also\nindicate that FA-P@M and FA-P@MC were not only detected in carotid plaques but also\nhighly accumulated in liver and less accumulated in spleen and other tissues. Thus, the\nrationale using PS-binding peptide to target foam cells seems to be unreasonable in this case.\nResponse: We sincerely thank the reviewer for this thoughtful and important comment. In\nour design, the macrophage membrane coating endows the nanoplatform with inflammatory\nplaque-homing capability, while the PS-targeting peptide (CLIKKPF) was introduced to\nfurther enhance selective recognition of foam cells. Unlike transient PS exposure in classical\napoptotic cells, foam cells within atherosclerotic plaques exhibit prolonged and amplified PS\nexposure. Mechanistically, lipid peroxidation in foam cells inactivates flippases and activates\nscramblases, leading to largely irreversible PS exposure (Nat. Commun. 2020, 11, 4646),\nwhile inflammatory signaling (e.g., TNF-α/NF-κB) further suppresses PS internalization\n(Cell Death Dis. 2018, 9, 261). Consequently, PS-targeting peptides have been adopted as a\nfeasible strategy to enhance foam cell–specific delivery within plaques (J. Am. Chem. Soc.\n2024, 146, 10093; Adv. Sci. 2024, 11, 2307441).\nRegarding the biodistribution results (Fig. 5f,g), the liver accumulation is primarily due to\nreticuloendothelial system (RES) clearance, a phenomenon commonly observed for most\nnanomedicines (Adv. Drug Deliv. Rev. 2020, 165–166, 1–14; J. Control. Release 2018, 270,\n165). Importantly, in vivo fluorescence imaging demonstrated that FA-P@MC exhibited\nobviously higher plaque accumulation than non-PS-targeted FA-P@M, despite both\nformulations experiencing liver uptake. Quantitative analysis of drug accumulation in plaque\ntissues was further conducted, which also confirmed that the FA-P@MC group had a higher\nAT content within plaques compared with FA-P@M, indicating that PS-targeting\nmodification enhanced drug delivery to plaques.\nWhile these findings support the rationale that the PS-binding peptide improves plaque\nenrichment, we also acknowledge the reviewer’s point that PS exposure is not entirely unique\nto foam cells, which actually reflects a broader challenge shared by many targeting strategies.\nIn future work, we plan to explore more specific ligands to further enhance both the\nselectivity and efficiency of plaque targeting. The results have been added (new\nSupplementary Fig. 46) and discussed in the revised manuscript (page 21).\nPBS\nFA-P@M\ng 30 FA-P@MC\n/%\np＜ 0.0001\nD 20\nI\n10\nFigure 1. Quantitative analysis of AT content in aorta and main organs of ApoE⁻/⁻ mice at 12\nh post administration of different formulations. Data are expressed as mean ± SD (n = 3).\nStatistical significance was analyzed using one-way ANOVA.\n2）In vitro anti-inflammatory and immunomodulatory effects section, the authors state that\nFA-P@MC treatment markedly suppressed foam cell formation in comparison to other\ngroups, likely due to the therapeutic effect of AT in promoting lipid efflux. This speculation is\nnot convincing. The authors need to clarify the underlying mechanism by which FA-P@MC\ncan functionally promote lipid efflux in foam cells, and identify the transporter/receptors that\nmediate cholesterol efflux in foam cells in this case.\nResponse: We sincerely thank the reviewer for this valuable comment. Following the\nreviewer’s suggestion, we further investigated the mechanism by which FA-P@MC inhibits\nfoam cell formation and promotes lipid/cholesterol efflux by examining the expression of\nrelevant transporters and receptors.\nFirst, we found that FA-P@MC significantly inhibited oxLDL internalization by RAW cells.\nAs shown in Figure 2a,b, oxLDL uptake in the FA-P@MC group was 4.65–1.84-fold lower\nthan those in other groups. Mechanistic studies revealed that FA-P@MC treatment markedly\ndownregulated the expression of scavenger receptors CD36 and LOX-1 (Figure 2c–f), which\nare key mediators of lipid internalization. These results indicate that FA-P@MC effectively\nsuppresses oxLDL uptake by modulating scavenger receptor expression.\nIn addition, FA-P@MC treatment enhanced cholesterol efflux from foam cells. The\ncholesterol concentration in the supernatant increased by 8.6-fold compared with the PBS\ngroup (Figure 3a). Immunofluorescence analysis further showed that the expression of the\nefflux transporters SR-BI and ABCA1 was upregulated by 7.6-fold and 9-fold, respectively,\nin FA-P@MC-treated cells (Figure 3b–e). These findings demonstrate that FA-P@MC not\nonly limits lipid uptake but also actively promotes cholesterol efflux via SR-BI and ABCA1,\nthereby functionally suppressing foam cell formation.\nThe corresponding data have been added to the revised manuscript (new Supplementary Fig.\n33, 34) and discussed in detail (page 14). We greatly appreciate the reviewer’s insightful\nsuggestion, which has significantly strengthened our study.\nFigure 2. a) Representative confocal laser scanning microscope (CLSM) images of\nDiI-oxLDL uptake in RAW 264.7 cells after different treatments. Scale bar: 50 μm. b) The\nmean fluorescence intensity (MFI) of DiI-oxLDL in different treatment groups. c) \nRepresentative CLSM images of CD36 expression in RAW 264.7 cells treated with oxLDL\nand various formulations for 24 h. Scale bar: 25 μm. d) MFI of CD36 expression in different\ntreatment groups. e) Representative CLSM images of LOX-1 expression in RAW 264.7 cells\ntreated with oxLDL and various formulations for 24 h. Scale bar: 25 μm. f) The MFI of\nLOX-1 expression in different treatment groups. Data are expressed as mean ± SD (n = 3).\nStatistical significance was analyzed using one-way ANOVA.\nFigure 3. a) Cholesterol content in foam cell supernatant in different treatment groups. b)\nRepresentative CLSM images of SR-BI expression in foam cells treated with various\nformulations for 24 h. Scale bar: 25 μm. c) MFI of SR-BI in different treatment groups. d)\nRepresentative CLSM images of ABCA1 expression in foam cells treated with various\nformulations for 24 h. Scale bar: 25 μm. e) MFI of ABCA1 in different treatment groups.\nData are expressed as mean ± SD (n = 3). Statistical significance was analyzed using one-way\nANOVA.\nMinor concerns:\n3）Preparation of macrophage membrane and fabrication of FA-P@MC section. The targeting\nmolecule (DSPE-PEG-CLIKKPF) was synthesized according to our previous protocol. Please\ncite the reference.\nResponse: We sincerely thank the reviewer’s valuable suggestion. In the revised manuscript,\nwe have cited the corresponding reference (Nat. Commun. 2023, 14, 6881; Sci. Adv. 2021, 7,\neabd6889) to support the synthesis method of the DSPE-PEG-CLIKKPF peptide conjugate.\n4）List of abbreviations needed to be added.\nResponse: We sincerely thank the reviewer for this constructive suggestion. In response, we\nhave added a list of abbreviations in the revised manuscript as a separate table for clarity and\nease of reference.\nAS: Atherosclerosis\nNIR-II: Near-infrared II\nPA: Photoacoustic\nMRI: Magnetic resonance imaging\nPS: Phosphatidylserine\nCVD: Cardiovascular diseases\nCD47: Cluster of differentiation 47\nAT: Atorvastatin\nFTBGd: FTB-Gd-DOTA\nPL: Photoluminescence\nf : Water fraction\nw\nH O : Hydrogen peroxide\n2 2\nO •−: Superoxide\nGSH: Glutathione\nROS: Reactive oxygen species\nOxLDL: Oxidized low-density lipoprotein\nPPADT: Poly(1,4-phenyleneacetone dimethylene thioketal)\nTK: Thioketal\nA-P: AT-PPADT\nFA-P: FTBGd-AT-PPADT\nCLIKKPF: Cys-Leu-IIe-Lys-Lys-Pro-Phe\nFA-P@M: FTBGd-AT-PPADT@macrophage cell membranes\nFA-P@MC: FTBGd-AT-PPADT@macrophage cell membranes-CLIKKPF\nICG: Indocyanine green\nNPs: Nanoparticles\nORO: Oil Red O\nNO: Nitric oxide\nSNR: Signal-to-noise ratio\nDHE: Dihydroethidium\nMMP-9: Metalloproteinase-9\nVSMC: Vascular smooth muscle cell\nα-SMA: α-smooth muscle actin"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The comments have been well addressed.", "reviewer_2": "I remain concerned about the overly optimistic, and non-evidence based claim about clinical translation. I believe this needs\nto be toned down substantially.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "The authors have responded to my comments well. I don't have further comments."}, "author_response": "Responses to the reviewers’ comments for the manuscript titled “A hierarchical theranostic\nnanoagent for multimodal imaging and targeted foam cell intervention in atherosclerosis”.\nWe sincerely appreciate the reviewers for their positive feedback and valuable comments regarding\nour article. The point-by-point responses to those comments are outlined below and all\nmodifications made to the manuscript are highlighted by using blue colored text. We hope that\nthese revisions meet the reviewers' expectations and the manuscript will be accepted for\npublication in Nature Communications.\nResponses to the Comments and Suggestions of Reviewer #1\nReviewer #1 (Remarks to the Author):\nThe comments have been well addressed.\nResponse: We sincerely appreciate the reviewer’s positive feedback and are grateful for the\nsupportive evaluation of our revision. Thank you for recognizing that the comments have been\nwell addressed.\nResponses to the Comments and Suggestions of Reviewer #2\nReviewer #2 (Remarks to the Author):\nI remain concerned about the overly optimistic, and non-evidence based claim about clinical\ntranslation. I believe this needs to be toned down substantially.\nResponse: We sincerely appreciate the reviewer's comments on the gap between preclinical\nfeasibility and clinical application. Following the reviewer’s valuable suggestion, we have\ncarefully moderated our wording related to clinical translation. Particularly, in the Discussion\nsection, we now clearly clarify that while our findings present a promising proof-of-concept in\nanimal models, translating this platform to the clinic will require extensive future studies to\ncomprehensively evaluate its efficacy, safety, and overall feasibility in humans.\nResponses to the Comments and Suggestions of Reviewer #4\nReviewer #4 (Remarks to the Author):\nThe authors have responded to my comments well. I don't have further comments.\nResponse: We sincerely thank the reviewer for your careful reading of our manuscript and for\nyour positive evaluation. We greatly appreciate your support for the publication of our work.\nResponses to the Comments and Suggestions of Reviewer #3\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate the co-reviewer for the constructive feedback, which has been\nhighly valuable in improving the quality of our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "Thank you for your efforts to improve the manuscript, and to downplay the \"closeness\" to clinical translation, and to better\nidentify the limitations.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "The authors have undertaken extensive revisions and added new data in response to reviewer\nfeedback, which has strengthened the manuscript in several areas. Mechanistic studies,\nimmunological profiling, and partial biodistribution data now provide improved support for the\ntherapeutic potential of their nanoparticle platform. The presentation of imaging data has also been\nclarified.\nDespite these improvements, significant concerns remain. The claim that this platform addresses\nan urgent unmet diagnostic need for CAD screening is still not convincingly justified and should\nbe moderated. While additional experiments have been provided, the mechanistic pathway from\ntargeted atorvastatin delivery through lipid efflux, CD47 downregulation, phagocytosis, and\nplaque regression remains only partially supported with ROS claims need strengthening.\nWhile the authors have provided additional discussions on proof-of-concept scope, and added\nsupportive preclinical data, the central concern regarding the true clinical use of this platform for\nearly CAD screening remains insufficiently addressed. The revisions partially improve the\nmanuscript but do not fully resolve the disconnect between preclinical feasibility and the unmet\ndiagnostic need in asymptomatic CAD. Further clarification and stronger clinical justification are\nrequired before this work can be considered adequately responsive to the concerns raised.\nIn conclusion, while the revisions strengthen the manuscript and improve its scientific depth,\nimportant translational, and methodological gaps remain. Additional revisions on methodological\nclarity and transparency are needed to moderate claims.\nResponse: We sincerely appreciate the reviewer’s thorough evaluation of our revised manuscript\nand your recognition of the improvements made. We also thank you for outlining the remaining\nconcerns, which are valuable for strengthening our work. According to the suggestion, we have\nimplemented the following revisions to the manuscript:\nFirst, according to the reviewer’s suggestion, we have moderated the relevant statements\nregarding the potential application for early CAD screening. Specifically, we now mentioned that\nwhile this proof-of-concept study demonstrates promise in preclinical models, its potential utility\nfor CAD diagnosis still requires extensive future validation in rigorous clinical studies (Page 35 in\nthe revised manuscript). These revisions aim to ensure that our statements are properly balanced.\nRegarding the mechanistic pathway, we first thank the reviewer for recognizing our efforts to\nstrengthen the supporting evidence in the revised version. Following the reviewer’s guidance, we\nhave incorporated additional experiments to further support the role of ROS responsiveness in\ndownstream effects. A non-ROS-responsive control nanoparticle (FA@MC), prepared with carrier\nmaterials lacking the ROS-sensitive moiety, was used for comparison. The uptake experiments\nindicated that FA@MC and FA-P@MC exhibited similar levels of cellular internalization (Figure\n1a, b). In foam cells, FA@MC induced only weak CD47 downregulation, whereas the ROS-\nresponsive FA-P@MC significantly reduced CD47 expression (Figure 1c, d). Consistently,\nphagocytosis assays showed that FA-P@MC treatment markedly enhanced efferocytosis by\nBMDMs, while FA@MC had limited effect (Figure 1e, f). These data have been added in the\nrevised manuscript (Supplementary Fig. 40), suggesting that ROS-enhanced drug release\ncontributes to the observed CD47 downregulation and increased phagocytosis.\nAt the same time, we acknowledge that fully establishing a complete and linear causal chain\nfrom targeted drug delivery to plaque regression within a single study is inherently challenging.\nTherefore, in the revised Discussion, we have carefully refined our interpretation to more\naccurately reflect the scope of the available evidence. We now describe the observed biological\noutcomes as interconnected and collectively contributive effects of our nanoplatform, while also\nrecognizing that other concurrent mechanisms may additionally participate in the overall\ntherapeutic benefits (Page 36 in the revised manuscript).\nRegarding the need for better methodological clarity, we have carefully revised the relevant\nMethods section to provide clearer descriptions of the experimental procedures, quantification\napproaches, and animal experimental timeline (as highlighted in blue in the revised Methods\nsection). These revisions aim to improve reproducibility and enhance the transparency of the\nmanuscript.\nFigure 1. a) CLSM images and b) corresponding quantitative analysis of FA@MC or FA-P@MC\nuptake (DiI-labeled) in foam cells after 4 h incubation. Nuclei were stained with DAPI (blue).\nScale bar: 20 μm. c) Immunofluorescence images of CD47 expression in foam cells under different\ntreatments. Scale bar: 50 μm. d) Quantification of CD47 expression across treatment groups. e)\nThe phagocytosis of foam cells by BMDMs determined by flow cytometry. f) Quantification of\nfoam cell phagocytosis by BMDMs according to flow cytometry analysis. Data in (b, d, f) are\npresented as mean ± SD (n = 3 independent experiments). Statistical significance was determined\nusing one-way ANOVA.\nComment 1: Add at least one organ biodistribution timepoint.\nResponse: We sincerely thank the reviewer for this valuable suggestion. In response, we have\nconducted a time-course organ biodistribution study. The distribution of the nanoplatform was\nsystematically evaluated in major organs (including liver, spleen, kidneys, heart, and lung) at\nmultiple time points: 0, 12, 24, 48, 72, and 168 hours (7 days) post-administration. The results\nshowed that the nanoparticles were mostly cleared from major organs by day 7 (Figures 2 and 3).\nThese results, together with our previously submitted long-term biosafety data (organ histology\nand hematological/biochemical parameters) and clearance profiles, collectively support the\nfavorable safety profile of the material. A detailed description of these results has been added to\nthe Results section under the subsection “In vivo biosafety and pharmacokinetics of FA-P@MC”\nin the revised manuscript (Page 18 in the revised manuscript, and new Supplementary Fig. 47 and\n48).\nFigure 2. Representative ex vivo IVIS images of major organs at different time points after FA-\nP@MC administration. Scale bar: 5 mm.\n40\n0 h\n30 12 h\ng 24 h\n/%\n20 48 h\nD\nI 72 h\n10 168 h\nFigure 3. Quantitative analysis of drug content in major organs at different time points post-\nadministration of FA-P@MC. Data are presented as mean ± SD (n = 3 mice).\nComment 2: Plot both materials±H O together and add stats at key time points. Confirm equal\n2 2\nloading and similar size prerelease. It is good to include at least one lower H O level i.e.\n2 2\nphysiological level or discuss. What is the specificity control (e.g. catalase/NAC).\nResponse: We sincerely thank the reviewer for the constructive feedback. In response, we have\nplotted the drug release profiles of both materials under H O conditions together, with statistical\n2 2\nsignificance at key time points clearly indicated in the graphs (Figure 4a). We confirmed that prior\nto release experiment, both nanoparticles had similar particle sizes and identical drug loading,\nensuring a valid comparison. We have also included a physiological H O concentration (2.5 µM)\n2 2\nas a control condition (Clin. Chim. Acta 2003, 334, 205e209). The results showed that at this low\nlevel, cumulative release from both FA-P and FA-P@MC remained below 10% over 36 hours\n(Figure 4b). This result indicated that the drug release was limited under basal physiological\nconditions but was greatly promoted in high H O environments. Furthermore, we incorporated a\n2 2\nspecificity control using catalase. As shown in Figure 4c, pre-treatment with catalase for 30\nminutes to decompose H O (1.0 mM) effectively inhibited drug release from both FA-P and FA-\n2 2\nP@MC, providing evidence that the release process was H O -dependent.\n2 2\nThese new results have been incorporated into the revised manuscript as new Supplementary Fig.\n30 and are described in detail in the Results section (Page 9 in the revised manuscript).\nFigure 4. AT release behavior from FA-P or FA-P@MC NPs under different conditions: a) high\nH O level (1.0 mM), b) physiological H O level (2.5 µM), and c) high H O level pre-treated\n2 2 2 2 2 2\nwith catalase (2000 U). Data are presented as mean ± SD (n = 3). Statistical significance was\nanalyzed using two-tailed Student’s t-test.\nComment 3: Please report assay kit details, n, mean ± SD, and units basis (per g tissue/protein).\nResponse: We sincerely appreciate the reviewer’s helpful suggestion. In response, we have now\nprovided full assay information, sample size, data presentation, and unit basis for the measurement\nof H O concentration in atherosclerotic plaques.\n2 2\nH O levels in plaque tissues were quantified using a commercially available Hydrogen Peroxide\n2 2\nContent Assay Kit (Solarbio, BC3595), following the manufacturer's protocol. Briefly, freshly\nisolated plaque tissues were homogenized in the supplied buffer on ice and centrifuged, and the\nresulting supernatants were used for analysis. Absorbance was measured at 415 nm using a UV–\nVis spectrophotometer (SHIMADZU), and H O concentrations were calculated based on a\n2 2\nstandard curve generated with known H O standards. The results are reported as mean ± SD (n =\n2 2\n6 individual plaque samples), yielding an H O level of 0.34 ± 0.05 mM, corresponding to\n2 2\napproximately 0.34 µmol per gram of wet tissue. These experimental details have now been added\nto the Methods section of the revised manuscript (Page 40 in the revised manuscript).\nComment 4: What is the start age/sex/strain and HFD duration, and whether a chow control was\nused or matched for energy. Statement on feeding logistics and include a confirmation statement\nthat all experimental groups (vehicle/treatment) received the same HFD and were housed under\nidentical conditions. Please add reproducibility details. Catalog/diet code and lot number, energy\ndensity, carbohydrate source (sucrose vs starch) and fiber type/amount, fatty-acid profile\n(SFA/MUFA/PUFA) and confirm whether sodium cholate is present, Cholesterol content also as\nmg/kg.\nResponse: We appreciate the reviewer’s thoughtful suggestions. In the revised manuscript,\nwe have now added detailed information on the animal model, diet formulation, and housing\nconditions (Page 37-38, and Page 46 in the revised manuscript). Male AopE-/- mice were used in\nthis study, and the high-fat diet (HFD) feeding was initiated when the animals were six weeks of\nage and continued for eight weeks. Thereafter, mice received the respective nanodrug treatment\nregimens while remaining on the HFD for an additional eight weeks. A standard chow-fed control\ngroup was included for comparison. All mice were housed in the same specific pathogen-free\nfacility and maintained under identical environmental parameters, including cage type,\ntemperature, humidity, handling procedures, and a 12-hour light/dark cycle. Treatment groups\nreceived the same HFD throughout the study, and food and water were provided ad libitum, with\ndiet replaced twice weekly.\nThe high-fat diet used in this study was sourced from SPF (Beijing) Biotechnology Co., Ltd.,\ncatalog number SFD004. The diet has an energy density of 4.7 kcal/g, with 41% of total kcal from\nfat, 42% from carbohydrate, and 17% from protein. Carbohydrates in the formulation are primarily\ncomposed of corn starch, sucrose, and maltodextrin, and the fiber component contains 31 g/kg\ncrude fiber based on 90% dry matter. The primary sources of fat are lard (42% saturated fatty acids,\n48% monounsaturated fatty acids, 10% polyunsaturated fatty acids) and soybean oil (15%\nsaturated fatty acids, 24% monounsaturated fatty acids, 61% polyunsaturated fatty acids). The diet\ncontains 0.2% cholesterol by weight (equivalent to 2 g/kg), and sodium cholate is not included in\nthe formulation. These details have now been added to the Methods section of the revised\nmanuscript, thereby improving the clarity and reproducibility of the experimental procedures.\nComment 5: Please add the spectra/normalised absorption and laser parameters for quantitative\ncomparison and a brief note on hemoglobin/water background and why 790 improves PA contrast\nwhile 808 remains efficient for FL.\nResponse: We thank the reviewer for this constructive suggestion. In the revised manuscript,\nwe have now added the normalized absorption spectrum of the probe together with the corresponding\nlaser parameters used for both photoacoustic and fluorescence imaging. For fluorescence imaging,\nthe laser power was 50 mW cm-2; for PA measurements, pulse energy = 20 mJ cm-2, frequency =\nduration of pulsed laser = 7 ns. The probe exhibits a strong broad absorption band across 750–820\nnm, with a pronounced maximum near 790 nm (Figure 5 below). Based on this absorption profile,\n790 nm was selected for PA imaging. Endogenous absorbers such as hemoglobin exhibit reduced\nabsorption in this spectral region compared to their strong visible-band absorption, and water\nshows low absorption at this wavelength before its absorption increases at longer wavelengths.\nFor fluorescence imaging, 808 nm light was employed as the excitation source because the probe\nhas strong absorption at this wavelength and can be efficiently excited using the widely available\ncommercial 808 nm laser. This wavelength selection ensures sufficient excitation efficiency while\nmaintaining compatibility with commonly available NIR laser. The updated Methods section now\nincludes the normalized absorption (Supplementary Fig. 25), and laser parameters, along with a\nbrief explanation of wavelength considerations (Page 44 in the revised manuscript).\n)\nd\ne\nz\nila 1.0\nm\n0.8\nr\no\nN 0.6\n(\nn o 0.4\nitp\n0.2\nr\no\ns b 0.0\nA 600 700 800 900\nWavelength (nm)\nFigure 5. Normalized absorption spectrum of FA-P@MC.\nComment 6: The authors provide preclinical feasibility data but clinical justification for CAD\nscreening remains limited.\nResponse: We sincerely appreciate the reviewer's comments on the gap between preclinical\nfeasibility and clinical application. Following the reviewer’s valuable suggestion, we have\ncarefully moderated our wording related to clinical translation. Particularly, in the Discussion\nsection (Page 35 in the revised manuscript), we now clearly clarify that while our findings present\na promising proof-of-concept in animal models, translating this platform to the clinic will require\nextensive future studies to comprehensively evaluate its efficacy, safety, and overall feasibility in\nhumans.\nComment 7: The revisions significantly strengthen the mechanistic claims, although caution is\nneeded in overstating direct causal relationships.\nResponse: We sincerely thank the reviewer for the positive assessment of our revised data and for\nthe kind reminder regarding the cautious interpretation of causal relationships. According to the\nreviewer’s suggestion, in the revised Discussion, we have carefully refined our interpretation to\ndescribe the observed biological outcomes as interconnected and collectively contributive effects\nof our nanoplatform, while also recognizing that other concurrent mechanisms may also contribute\nto the overall therapeutic benefits (Page 36 in the revised manuscript).\nComment 8: Similar to above mentioned: Provide quantitative biodistribution for major organs for\nFA-P@MC vs FA-P@M, add selectivity evidence in plaques: colocalisation of NP signal with PS\nand foam-cell markers (CD68, PLIN2/Oil-Red O. To confirm PS mediated targetting, include a\nsmall peptide control to block or show competition. Need to clarify whether liver uptake differs\nbetween groups. Also note to temper language for foam cell specific as PS is not unique to foam\ncells.\nResponse: We sincerely thank the reviewer for the insightful comments, which have helped us\nstrengthen the experimental evidence regarding nanoparticle targeting.\nWe have now included quantitative biodistribution data for major organs (heart, liver, spleen, lung,\nkidneys, and carotid plaques) in the revised manuscript (Figure 6 below and new Supplementary\nFig. 53). The results confirm that FA-P@MC exhibits significantly higher accumulation in\natherosclerotic plaques compared to FA-P@M (about 2-fold increase, p < 0.0001). No significant\ndifferences in AT signals were detected in other major organs, including the liver, spleen, or\nkidneys, between the FA-P@MC and FA-P@M groups. Both FA-P@MC and FA-P@M exhibited\nobvious liver uptake, consistent with the common clearance of nanoparticles via the\nreticuloendothelial system (RES). These data support the enhanced plaque-targeting advantage\nconferred by the PS-binding peptide modification.\nTo further assess targeting to PS-exposing foam cells, we performed immunofluorescence co-\nlocalization analysis in plaque sections. As shown in Figure 7 (new Supplementary Fig. 56),\nCY5-labeled FA-P@MC signals exhibited strong co-localization with PS (Sigma-Aldrich, 05-719,\n1:200), CD68 (Abcam, ab283654, 1:200), and PLIN2 (Huabio, ET1704-17, 1:200). Quantitative\ncolocalization analysis indicated a higher degree of overlap for FA-P@MC compared to the non-\ntargeted FA-P@M group, consistent with preferential accumulation in foam cells within plaques.\nWe also conducted a competitive blocking experiment in which free CLIKKPF peptide was\ninjected prior to FA-P@MC administration. Pre-injection of the peptide reduced plaque\naccumulation of FA-P@MC to levels comparable to the non-targeted FA-P@M, whereas plaque\nuptake remained high in the non-blocked FA-P@MC group (Figure 8 below and new\nSupplementary Fig. 51). This result suggested that PS-binding peptide interactions contributed to\nthe plaque-targeting effect.\nWhile foam cells show high expression of PS, we fully agree with the reviewer that PS is not\nunique to foam cells. According to the reviewer’s suggestion, we have moderated language\nregarding “foam cell specificity” in the revised manuscript, clarifying that our strategy exploits the\nhigh density of PS exposure on foam cells within atherosclerotic plaques rather than absolute\nspecificity.\nPBS\n40\nns Free AT\nFA-P@M\ng 30 FA-P@MC\n/%\np＜ 0.0001\nD 20\nI\n10 ns\nns\nFigure 6. Quantitative analysis of AT content in aorta and main organs of ApoE-/- mice at 12 h post\nadministration of different formulations. Data are expressed as mean ± SD (n = 3). Statistical\nsignificance was analyzed using one-way ANOVA.\nFigure 7. a) Confocal microscopy images of aortic root sections from mice injected with different\nCy5-labeled nanoformulations. The foam cells were stained using PS antibody, CD68 antibody,\nand PLIN2 antibody. The plaque lesion is outlined by a white dotted line. Scale bars: 20 μm. b)\nThe relative fluorescence intensity of Cy5+CD68+PLIN2+PS+ foam cells in different groups. Data\nare expressed as mean ± SD (n = 3). Statistical significance was analyzed using two-tailed\nStudent’s t-test.\nFigure 8. a) Representative NIR-II FL images of carotid plaques at various time points following\ni.v. injection of PBS, FA-P@MC, or CLIKKPF + FA-P@MC. Scale bars: 1 cm. b) Quantitative\nanalysis of NIR-II FL intensity in carotid plaques across different treatment groups (n = 3 mice per\ngroup).\nComment 9: It is recommended to perform a functional cholesterol efflux assay with appropriate\nApoA-I (ABCA1-dependent) and HDL (SR-BI/ABCG1). Also report % efflux (BODIPY]-\ncholesterol) normalized to cellular cholesterol, with n/stats. It is best to show dependence on\ntransporters by knockdown and also confirm in primary macrophages (BMDM or human\nmonocyte-derived) for translatability.\nResponse: We thank the reviewer for this valuable suggestion. Following the recommendation,\nwe performed functional cholesterol efflux assays in foam cells using ApoA-I (ABCA1-dependent)\nand HDL (SR-BI/ABCG1-associated) as acceptors, with BODIPY-labeled cholesterol. The percent\nof cholesterol efflux was calculated as (fluorescence in the supernatant/the sum of the fluorescence\nin the medium and the fluorescence remaining in the cells) × 100%. The data show that FA-P@MC\nincreases cholesterol efflux to both ApoA-I and HDL compared with the relevant controls,\nsupporting with engagement of these transporters in promoting efflux.\nAdditionally, we employed established pharmacological inhibitors to evaluate the contribution of\nABCA1 and SR-BI to cholesterol efflux in foam cells. Inhibition of ABCA1 (using Probucol)\nmarkedly reduced the FA-P@MC–enhanced efflux to ApoA-I, while inhibition of SR-BI (using\nBLT-1) reduced efflux to HDL (Figure 9c,d). These findings are consistent with involvement of\nthese transport pathways under our experimental conditions. To provide additional support for\nrelevance in primary cells, parallel studies were conducted in foam cells induced by bone marrow–\nderived macrophages (BMDMs), where FA-P@MC similarly increased cholesterol efflux to both\nacceptors (Figure 10a,b). And inhibition of ABCA1 or SR-BI in BMDMs attenuated the FA-\nP@MC–enhanced efflux, consistent with the participation of these pathways without suggesting\nexclusivity. The new results have been incorporated into the revised manuscript and are described\nin detail on Page 15, as well as in new Supplementary Fig. 36 and 37.\nFigure 9. Cholesterol efflux to a) ApoA-I (10 μg/mL) or b) HDL (40 μg/mL) from BODIPY-\ncholesterol loaded foam cells in the indicated groups. Cellular cholesterol efflux to c) ApoA-I or\nd) HDL from BODIPY-cholesterol loaded foam cells with corresponding cholesterol transporter\ninhibitors. Data are expressed as mean ± SD (n = 3). Statistical significance was analyzed using\none-way ANOVA.\nFigure 10. Cellular cholesterol efflux to a) ApoA-I or b) HDL from BODIPY-cholesterol loaded\nBMDM cells with corresponding cholesterol transporter inhibitors in the different groups. Data\nare expressed as mean ± SD (n = 3). Statistical significance was analyzed using one-way ANOVA."}]

Decision letter