The vascular endothelium serves as a barrier between the blood and the vascular wall, and it is responsible for maintaining physiological homeostasis of the vasculature under normal conditions (Khaddaj et al., 2017). During the development of AS, the vascular endothelium is impared by continually stimulation from various factors, including immune responses, pathogenic organisms, and hemodynamics dysfunctional (Topper and Gimbrone, 1999; Gimbronejr and García-Cardea, 2013). This leads to an increase in adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin and P-selectin. (Vanhoutte, 1997; Vanhoutte, 2009). These highly expressed adhesion molecules could be used as the potential targets of the nanoagents nanoagents (Figure 2) (Chan et al., 2018).

VCAM-1, also referred to as CD106, is a type I transmembrane protein with a molecular weight of 100–110 kDa. It typically consists of seven C2-type immunoglobulin structural domains and functions as a cell adhesion molecule. VCAM-1 is expressed in both early and advanced AS lesions, indicating its potential as a biomarker for vascular inflammation and endothelial cell dysfunction (Galkina and Ley, 2007; Fotis et al., 2012). Currently, antibodies to specific targeting peptides against VCAM-1 are usually modified on the surface of nanoparticles to target the inflammatory endothelium. Tsourkas et al. (2005) covalently attached VCAM-1 antibody onto iron oxide nanoparticles and labeled them with the near-infrared fluorescent dye Cy5.5, enabling detection of VCAM-1 expression level on endothelial cells and labeling of activated endothelial cells.

Sun et al. (2016) coupled a VCAM-1 targeting peptide and miRNA inhibitor (anti-miR-712) to a DNA vector with complementary sequences modified on the surface of gold nanospheres and selectively delivered anti-miR-712 to mouse aortic endothelial cells with elevated VCAM-1 expression to inhibit AS plaque formation. Distasio et al. (2020) synthesized a polyglutamic acid (PGA) coating (PGA-PEG-VHPK) containing polyethylene glycol (PEG) and VCAM-1 specific targeting peptide (VHPK), and mixed polyβ-amino ester (PBAE) and DNA plasmid (pDNA) solution to prepare nanoparticles (NP), and then a targeting coating was applied to obtain a VCAM-1 targeting nanoparticle (NP-VHPK) (Figure 3). The interaction between the NPs and the VCAM-1 receptor was studied by surface plasmon resonance and imaging (SPRi) under different physiological conditions. The findings revealed that transfection of inflammatory endothelial cells under static and dynamic flow conditions resulted in a significantly higher internalization rate of NP-VHPK compared to non-targeted controls (80% vs. 30% positive cells). In areas of AS plaque in the mouse aorta and aortic sinus, NP- VHPK rapidly bounded to inflammatory endothelial cells with high VCAM-1 expression. Additionally, the NP-VHPK could be localization in the thoracic region adjacent to the heart and the blood vessels in the atherosclerostic mouse model through jection. These results suggested that surface modification with specific antibodys, peptides or other targeting agents could significantly enhance targeting efficiency and bioavailability. In addition, VCAM-1, ICAM-1 and selectin, which are highly expressed in the inflammatory endothelium of AS lesions, may serve as potential targets for specifically targeting (Bhowmick et al., 2012; Park et al., 2008; Chan et al., 2018).

Macrophages are typically located in the tissues and differentiated from the monocytes in the blood. Macrophages, as we foam cells drived from macrophage after phagocytized large amounts of lipids, play an important role in all stages of AS lesion development, from formation to plaque rupture (Wu et al., 2018; Moroni et al., 2019). By designing nanoparticles that carry lipid-lowering drugs, anticoagulant drugs, siRNA, DNA plasmids and other therapeutic agents to target macrophages, the development of AS can be effectively inhibited (Sato et al., 2014; Dipti et al., 2016). In AS lesions, macrophages adhere to and accumulate in the vessel wall through scavenger receptors on their surface, such as CD36, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and macrophage scavenger receptor-1 (MSR-1) (Reyk and Jessup, 1999; Murray and Wynn, 2011). These transmembrane glycoproteins exhibit a broad distribution in vivo and bind to diverse ligands. As a scavenger receptor, CD36 plays crucial roles in both lipid metabolism and the adhesion of anionic biomolecules. The findings suggest that CD36 plays a crucial role in lipid uptake and the development of atherogenesis (Febbraio et al., 2000; Kathryn and Mason, 2006), making it a common target for macrophage-directed therapy in AS lesions. For instance, the GHRPs protein family was employed to specifically target macrophages that exhibit high levels of CD36 expression in AS lesions (Demers et al., 2004). Nie et al. (2015) prepared liposome nanoparticles from phosphatidyl choline and KOdiA-PC (Figure 4). The liposomal nanoparticles were intravenously injected into low-density lipoprotein receptor (LDLR)-deficient mice, and it was found that these nanoparticles exhibited a remarkable ability to selectively target and bind to macrophages in AS lesions by binding specifically to CD36 receptors. The CD36-targeted nanoparticles were able to bind specifically to macrophages, resulting in a reduction of macrophage deposition within the AS lesion. This was achieved with small interfering RNA (siRNA) loaded into the nanoparticles, which effectively silenced CD36 expression in macrophages. LOX-1 plays a pivotal role in the pathogenesis of AS by acting as a major receptor of oxidized low-density lipoprotein (oxLDL), which contributes to endothelial cell dysfunction, monocyte adhesion, VSMCs migration and foam cell formation (Tian et al., 2019). Therefore, targeting LOX-1 may represent an effective therapeutic strategy for AS. In a study by Song et al. (2014), LOX-1 antibodies were conjugated onto the surface of superparamagnetic nanoparticles, resulting in higher binding affinity and increased uptake by RAW 264.7 macrophages compared to non-targeted modified nanoparticles. Following intravenous administration of the nanoparticle in ApoE^−/− mice, magnetic resonance imaging (MRI) revealed a significant enhancement signal at AS lesions, particularly in regions enriched with macrophage/foam cells. Additionally, MSR-1, an important scavenger receptor located on the surface of macrophages involved in oxLDL uptake by these cells (Winther et al., 2000). Modified nanoparticles with MSR-1 peptide-like ligands or antibodies on their surface could bind to macrophages in AS lesions, facilitating targeted delivery to these cells (Segers et al., 2013). In addition, the nanoparticles can specifically target macrophages through endocytosis mediated by glycosylated receptors such as mannose receptor (MR), galactose C-type lectin 1/2 (Mgl1/2, and folate receptor (FR) expressed on the surface of macrophages (Mohammadi et al., 2016; Alvarado-Vazquez et al., 2017; Daphne et al., 2019). The utilization of folic acid-modified radioactive material has been demonstrated to enable selective targeting of FR-β-positive macrophages for noninvasive imaging of vascular inflammation (Padilla et al., 2014).

During the development of AS, monocytes infiltrate the endothelium and differentiate into macrophages bytaking up oxidized lipids deposited within the arterial wall. These macrophages then secrete large amounts of inflammatory factors (e.g., TNF-α, IL-6, IL-1β, etc.), triggering a series of inflammatory responses that promote further progression of atherosclerosis. The production and release of inflammatory cytokines are effectively regulated by either introducing anti-inflammatory agents into the macrophage cytoplasm or by reducing the expression of inflammatory genes through RNA interference (siRNA) (Dinarello, 2010). Therefore, macrophages in AS lesions target not only inflammatory factors but also members of nuclear transcription factors (NF-κB) signaling pathways involved in promoting inflammatory transduction (Tornatore et al., 2012). For example, Wang et al. (2018) covalently conjugated antioxidants to the cyclic polysaccharide β-cyclodextrin (β-CD) to obtain antioxidant nanoparticles and found that the nanoparticles were internalized by macrophages and VSMCs efficiently and rapidly. Then, the inflammation and apoptosis of macrophage induced by inflammatory factorswere mitigated through the inhibition of pro-inflammatory factors such as TNF- α, IL-1β and MCP-1. Meanwhile, foam cell formation and the development of AS in ApoE ^−/− mice were effectively inhibited. Zhang et al. (2021) employed baicalein to target and inhibit the downstream signaling of MAPKs/NF-κB, effectively suppressing the production of pro-inflammatory factors such as IL-6, TNF-α and PAI-1.

The extracellular matrix (ECM) is a complex network of proteins and polysaccharide macromolecules secreted by cells in the extracellular mesenchyme, which interconnects tissue structures and orchestrates tissue development as well as cellular physiological activities. Studies have demonstrated the crucial role of ECM in the development of AS and the formation of fibrous cap (Adiguzel et al., 2009; Lee, 2014; Basatemur et al., 2019), as well as its potential contribution to plaque destabilization. Hence, ECM also could be used as a common target for the treatment of AS.

Collagen, a major component of the ECM, influences the strength and integrity of the fibrous cap in the development of AS, and it regulates cellular responses through specific receptors and signaling pathways (Adiguzel et al., 2009). Collagen is commonly target in the ECM. Chan et al. (2010) have identified peptide sequence that exhibits high affinity for type IV collagen (Col IV) through phage display investigations. This protein is highly expressed at sites of vascular injury, and given that more than 50% of the lesion basement membrane is composed of Col IV (Kalluri, 2003). Therefore, modifying collagen-targeting peptides on the surface of nanoparticles is an effective strategy for targeting atherosclerotic plaques. As an illustration, Kamaly et al. (2016) used nanoprecipitation to induce the self-assembly of a double-block polymer to form a collagen IV-targeting nanoparticle (Col IV IL-10 NP22) containing the anti-inflammatory factor interleukin-10 (IL-10), and these NPs have anti-inflammatory effects on macrophages both in vivo and in vitro. The particles have the ability to prevent vulnerable plaque formation by increasing the thickness of fibrous cap and reducing necrotic cores. Meyers et al. (2017) immobilized collagen-targeting peptides onto the surface of gold nanoparticles, resulting in the production of a collagen IV-targeting gold nanoparticle (T-AuNP) that specifically bonded to injured vascular sites.

In addition to collagen, other proteins in the ECM, polysaccharides, and other macromolecules observed on the surface or inside the plaque, which are associated with the progression of AS (Bini, 1989; Hultgardh-Nilsson et al., 2015; O'Brien et al., 2005), also serveas targets for the diagnosis and treatment of AS. Yoo et al. (2016) developed one kind of amphiphilic microparticles (PAMs) that incorporate fibrin-binding peptides, enabling targeted imaging of AS plaques using both MRI and NIRF techniques. Dipti et al. (2017) designed an emulsion agent with ω-3-fatty acid flaxseed oil coated with 17-βE and modified CREKA targeting peptide on its surface to specifically bind to fibrin clots. And, in vivo experiments demonstrated that the pro-inflammatory factors could be downregulated by the agent in ApoE^−/− miceand the lesion area also be reduced while possessing good biocompatibility.

Vascular smooth muscle cells (VSMCs) are the predominant cellular constituents of the vessel wall, responsible for maintaining vascular tone and structural integrity. Normal VSMCs are primarily located in the media layer of blood vessels and predominantly express contractile components such as smooth muscle myosin heavy chain and α-smooth muscle actin. In contrast, in AS lesions, the intima also harbors VSMCs, which exhibit a high proliferation index and predominantly synthesize extracellular mesenchyme, proteases and cytokines, while expressing limited contractile components (Br, 2004). Studies have demonstrated that VSMCs undergo a phenotypic switch from contractile to synthetic in response to various factors, including injury and lipid exposure (Pidkovka et al., 2007). Subsequently, the contractile capacity of VSMCs diminishes and they become responsive to various stimuli such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), etc., which are released in response to vascular injury. This leads to the migration and proliferation of VSMCs, thereby promoting further development of AS.Studies have indicated that the proliferation and migration of VSMCs are closely linked to their expression of pluripotency factors, such as Oct4/Pou5f1 and KLF4 (Cherepanova et al., 2016; Majesky et al., 2017). Therefore, regulating the expression of these factors may offer a promising approach for treating atherosclerosis. Currently, there is a dearth of research on the treatment of AS with VSMCs. However, given their pivotal role in the pathogenesis and progression of AS as well as their distinctive phenotypic characteristics from normal VSMCs, targeting VSMCs may hold promise for therapeutic intervention in AS. Zhang et al. (2020) combined the VSMC- targeting profilin-1 antibody (PFN1) with the anti-inflammatory drug rapamycin (RAP) and loaded it on a complex of superparamagnetic iron oxide and pH-sensitive cyclodextrin to create an integrated therapeutic and MRI imaging nanoparticle (RAP@PFN1-CD-MNPs) (Figure 5). The PFN1-CD-MNPs were demonstrated to effectively bind to VSMCs for targeted localization of AS lesions, followed by rapid drugrelease in their micro-acidic environment, thereby mitigating the progression of atherosclerosis.

Platelets, anucleated blood cells produced by megakaryocytes in the bone marrow and lungs, possess coagulation and hemostatic functions that are essential for maintaining physiological homeostasis and repairing vascular damage Carsten (2018). In addition, platelets could adhere to various cells and factors, thereby participating in a series of cascading inflammatory responses that promote the formation of AS. Studies have also found an elevation in platelet adhesion to the vessel wall at the site of AS lesion. (Lievens and Hundelshausen, 2011; Christian and Steffen, 2012; Eduardo et al., 2013). Therefore, platelets also could be a potential target for targeting AS. For example, Jacobin-Valat et al. (2015) prepared a multifunctional superparamagnetic iron oxide nanoparticle (VUSPIO) that were surface-modified with a recombinant antibody (rIgG4 TEG4), enabling them to bind to activated platelets. Bachelet-Violette et al. (2014) modified superparamagnetic iron oxide nanoparticles with a sulfated polysaccharide that could specifically bind to P-selectin. The functionallized nanoparticles exhibited specific binding affinity towards activated platelets in the bloodstream, rendering them suitable for MRI imaging of AS lesions. However, nanoparticles targeting P-selectin may be potentially limited due to the expression of P-selectin on inflammatory endothelial cells, which challenges the specificity of these nanoparticles binding to platelets. Therefore, further investigations are warranted to validate the reliability of platelets as a viable target for both diagnosis and treatment of AS.

Non-coding RNAs (ncRNAs) are emerging as crucial regulators of cellular function and disease progression (Poller et al., 2018; Yang et al., 2020), with a key role in cardiovascular disease (Liu et al., 2018; Tang et al., 2018; Liu et al., 2020; Mengdie et al., 2020). Among ncRNAs, micrRNAs (miRNAs) are one of the most extensively studied and widely researched with a length ranging from 20 to 25 nucleotides. MiRNAs participate in a diverse range of physiological processes and pathological conditions primarily by exerting post-transcriptional repression on their target mRNAs, thereby impeding the translation of proteins encoded by the genetic information carried within these transcripts. (Aryal and Suárez, 2019; Mori et al., 2019). In the AS lesions, miRNAs generally regulate the expression of cell adhesion molecules to modulate inflammatory responses through two distinct mechanisms, transcriptional regulation of the pro-inflammatory NF-κB signaling pathway and direct targeting. Different miRNAs are responsible for regulating different pro-inflammatory NF-κB signaling pathways Zhong et al. (2018). Additionally, miRNAs have multiple functions in regulating vascular cell homeostasis, participating in lipoprotein secretion and metabolism, and immune responses, and play a key role in the progression, modulation, and regulation of each stage of AS (Feinberg and Moore, 2016; Shoeibi, 2020). MiRNAs have emerged as a novel class of therapeutic targets for AS disease in recent years (Yang et al., 2017; Wang et al., 2020; Zong et al., 2021). Apelin is an endogenous ligand for the angiotensin II type 1 receptor-related protein APJ, which exhibits a wide distribution in various organs and tissues of both animals and humans. Apelin exerts various physiological effects on the cardiovascular, immune, nervous and other systems by binding to the APJ receptor. It is involved in pathophysiological processes such as inflammatory, oxidative stress, hormone secretion regulation, vascular tone maintenance and myocardial contractility regulation (Mughal and O’Rourke, 2018). It also exhibits antagonistic effects with the renin-angiotensin system and participates in the regulation of the stress response. Therefore, APJ receptor agonists or antagonists are anticipated to emerge as novel targets for the diagnosis and treatment of AS by nanoagents.

ROS is a collective term for a class of small reactive molecules that are continuously produced and utilized in all living systems. They play a key role in maintaining self-homeostasis of vascular tissue and regulating a variety of cellular functions (El-Mohtadi et al., 2018). ROS is primarily comprised of superoxide, hydroxyl radicals, hydrogen peroxide (H₂O₂), peroxynitrite and hypochlorite (Yuliya et al., 2015; Nowak et al., 2017). ROS levels are a key factor in the pathogenesis of AS and angiogenesis, as cellular physiological activities are intricately linked to ROS concentrations (El-Mohtadi et al., 2018) (Figure 7).

High ROS levels lead to an increase in oxidized lipoproteins (Ox-LDL), endothelial dysfunction, DNA damage, leukocyte migration and differentiation, proliferation of VSMCs, and elevated MMPs. According to the ROS high levels environment in AS diseased tissues, researchers have explored various ROS-responsive drug carriers. These carriers are typically constructed using polymers containing sulfur, selenium or tellurium, phenylboronic acid ester and co-administered photosensitizer ROS sensitive structures.

The acidic cellular microenvironment at the site of inflammation is a well-established fact. Macrophages present at the AS plaque site phagocytosing large amount of Ox-LDL, leading to significant accumulation of lactic acid and further aggravating the local acidity (Parathath et al., 2013; Zhao and Herrington, 2016). Katariina et al. (2015) utilized two pH-sensitive dyes to determine the pH levels within atherosclerotic plaques, revealing that human atherosclerotic plaques exhibited a pH range of 6.5–8.5 while rabbit was 5.5–7.5. The pH of lysosomes in macrophages was measured at 4.7–4.8. The weakly acidic microenvironment (pH 6.0–6.8) of atherosclerotic lesions and the acidic environment (pH below 5.0) of macrophages lysosomes (Ohkuma and Poole, 1978) can be exploited for precise atherogenic response therapy. Currently, various types of pH-responsive nanoagents have been designed and constructed for the treatment of AS, including those initiated by covalent bonds, intermolecular forces, and physical structures.

Many enzymes are involved in the formation process of AS, such as matrix metalloproteinases (MMPs), hyaluronidases, and cathepsins, which also could be used as the stimulators and targets for drug delivery and controlled release in atherosclerosis treatment. At present, MMPs and hyaluronidase are the most extensively researched stimulators and targets, of which MMP13, MMP2, and MMP9 are three common target metallo-mechanism proteases. Additionally, cathepsin B has significantly higher activity in unstable plaques than in stable plaques and may be used as both a target and a stimulator of AS (Tawakol et al., 2016).

Vessels are exposed to a diverse array of hemodynamic forces, including fluid shear stress, hydrostatic pressure, pulsatile blood pressure and tensile stresses induced by circulating blood flow (Chiu and Chien, 2011). As the AS plaque continues to develop, luminal narrowing occurs and there is an increase in fluid shear and wall shear stress (WSS) at the site of the lesion. The normal range of WSS in vessels is 1–10 dyn cm^-2, whereas in vulnerable plaque vessels, it increases to a range between 31.90 and 136.09 dyn cm^-2 with significantly higher values observed at the proximal end (approximately 180 dyn cm^-2) compared to the distal end (approximately 100 dyn cm^-2) (Korin, 2012; Chen et al., 2017). This abnormal shear stress could act as a stimulating and targeting factor, triggering local drug delivery and released to the target lesion plaque lesion. Currently, the majority of micro-nanoparticles utilized for drug delivery via fluid shear stimulation are designed based on platelets structure mimicry, with their drug release primarily attributed to carrier deformation or degradation under high shear conditions. For example, Margaret et al. (2012) developed liposomal nanoparticles with a lentilshaped that can undergo deformation under high shear stress, facilitating the release of loaded drugs. Korin (2012) prepared a microscale aggregation of nanoparticles (SA-NTS) that can be activated by shear stress, mimicking the structure of platelets, and loaded the thrombolytic drug tissue-type fibrinogen activator (tPA) into SA-NTS. The SA-NTS released drug to the obstructed vessel using the high shear stress caused by vascular stenosis. In vitro experiments revealed that under abnormally high fluid shear stress, SA-NTS disintegrated into individual nanofractions. When SA-NTS loaded with tPA were injected intravenously in mice, these shear-activated nanotherapeutic drugs induced rapid clot lysis in a model of mesenteric injury, restored normal hemodynamics, and increased survival in pulmonary embolism mouse model.

Currently, researchers have developed a range of exogenous stimuli-responsive nanoagents, primarily composed of inorganic nanoparticles commonly used for in vitro diagnosis of AS diseases. These agents are activated by various exogenous stimuli such as light, ultrasoundand magnetic fields. These nanoagentsare activated in response to specific exogenous stimuli for imaging and detected by corresponding devices. Some inorganic nanoagentsare capable of imaging along with the diagnostic integration, such as gold nanoparticles (Kharlamov et al., 2015), ions nanoparticles (Wang et al., 2013), and copper sulfide (Gao et al., 2018) nanoparticles. In addition, the integration of these exogenous stimuli-responsive inorganic nanoparticles with organic counterparts can enable drug delivery while simultaneously fulfilling the diagnostic imaging function of inorganic nanoparticles, thereby achieving multiple potential functionalities.

Multi-stimuli-responsive nanoagents utilize multiple endogenous or exogenous stimuli to target the lesion site for intelligent therapy, and the multi-stimulus response enhances the performance of nanoagents with superior targeting and higher drug delivery efficiency. There is a limited number of studies on multi-responsive nanotherapeutic agents targeting AS, and the majority of multi-responsive nanoplatforms are constructed based on light response, such as light/ROS response (Jager et al., 2016), light/pH response (Hong et al., 2018), and light/enzyme response (Shon et al., 2013). Other nanoagents that respond to multiple stimuli, such as those combining pH, shear stress, and ROS (Dou et al., 2017; Peters et al., 2019), hold great potential for treating AS.