Recent Advances in Nanodelivery Systems Based on Extracellular and Intracellular Reprogramming Strategies for Enhanced Therapy of Atherosclerosis

Introduction

Atherosclerosis (AS), a chronic inflammatory process affecting arterial vessels, serves as the principal driver of diverse cardiovascular pathologies,¹ characterized by the accumulation of lipid plaques on the arterial wall and accompanying endothelial dysfunction.² Due to its elevated morbidity and mortality rates, AS imposes a significant burden on both individuals and society, underscoring the critical need for effective therapeutic interventions.

In clinical practice, the primary therapeutic strategies for AS remain surgical plaque removal, implantation of stents, and pharmacological control.³ Statins remain the first-line pharmacological intervention, demonstrating efficacy in reducing low-density lipoprotein (LDL) through competitively inhibiting HMG-CoA reductase this intervention effectively suppresses de novo cholesterol biosynthesis.^4,5 Nevertheless, substantial therapeutic limitations persist, primarily attributable to complex pathophysiological disturbances across multiple cellular and molecular pathways that drive its pathogenesis. These abnormalities include the infiltration of excessive neutrophils (NEs),⁶ phenotypic switching of macrophages,⁷ the dysfunction of endothelial cells (ECs),⁸ dysregulated cellular expansion of vascular smooth muscle cells (VSMCs),⁹ the accumulation of lipid, and the overproduction of reactive oxygen species (ROS).^10–12 These dysregulated processes synergistically drive the development of a pathologically sophisticated atherosclerotic microenvironment (ASM) (Figure 1). Consequently, reprogramming strategies for the ASM including remodeling cellular phenotypes, inhibiting the proliferation of pathological cells, modulating inflammatory responses, and clearing lipids and ROS holds substantial therapeutic promise for the disease.

Figure 1 The complex inflammatory microenvironment of atherosclerosis. (A) Multiple cell types undergo changes during the atherosclerotic process, involving a vicious cycle driven by inflammatory factors, lipids, and ROS. (B) The inflammatory vicious cycle in ASM specifically includes: interstitialization of ECs, polarization of macrophages, transformation of NEs into NETs, phenotypic transformation of VSMCs, formation of lipid droplet, excessive ROS, and disruption of inflammatory factors.

Endothelial dysfunction, as a key driver of AS, is mainly attributed to the transformation of endothelial cells (ECs) into inflammatory endothelial cells and mesenchymal endothelial cells, which jointly promote the subsequent recruitment of large numbers of neutrophils (NEs) and the generation of neutrophil extracellular traps (NETs). The latter further exacerbates endothelial dysfunction, forming a self-perpetuating vicious cycle.^13,14 Consequently, reprogramming the endothelial cell phenotype or reversing the inflammatory state of endothelial cells represents a promising strategy for restoring endothelial barrier function.¹⁵ The overexpression of ICAM-1 on ECs, as well as p-selectin on NEs,¹⁶ enables their targeted interaction with ligand-modified nanodelivery systems (NDSs), allowing for specific therapeutic actions on these cells. Beyond neutrophil’s recruitment, significant monocyte’s infiltration occurs at inflammatory sites, followed by their polarization into pro-inflammatory M1 macrophages. These macrophages release substantial quantities of inflammatory factors and ROS. Furthermore, M1 Macrophage engulfment of oxidized low-density lipoprotein (ox-LDL) and conversion to foam cells, thereby promoting plaque formation.¹⁷ Accordingly, reprogramming monocytes or converting the M1 macrophage phenotype to the M2 is a promising strategy. At plaques site, inflammatory macrophages overexpress scavenger receptors (eg, SR-A, CD36), CD44, and other receptors on their surfaces.^18,19 Nanoparticles (NPs), through ligand modifications such as hyaluronic acid (HA) and folic acid, can enhance specifically targeting ability to macrophages.^20,21 Notably, VSMCs demonstrate proliferation and migration patterns resembling tumor cells, while the phagocytic uptake of lipids facilitates their transformation into foam cells, exacerbate endothelial damage and plaque’s formation.⁹ Thus, inhibiting abnormal proliferation of VSMCs or inducing their apoptosis is crucial for enhancing therapeutic efficacy. Previous studies have demonstrated that various nanomaterials, including iron oxide nanoparticles (Fe₃O₄ NPs),²² silver nanoparticles (Ag NPs),²³ etc, exhibit excellent capabilities in inducing apoptosis. These nanomaterials with appropriate modifying, can specifically target pathological cells such as inflammatory macrophages,²⁴ and VSMCs²⁵ at lesion sites, thereby inducing apoptosis and improving treatment of the disease. To sum up, inflammation within the ASM leads to morphological and functional alterations in key cells. Leveraging the precise targeting ability of NDSs could achieve better reprogramming strategies of ASM (Table 1).

Table 1 Nanodelivery Systems Based on Intracellular Reprogramming Strategies for Enhancing the Therapy of Atherosclerosis

Meanwhile, M1 macrophages and NEs release substantial amounts of inflammatory factors, which exacerbate the inflammatory response in the disease.⁵¹ Anti-inflammatory drugs, such as colchicine and inflammatory pathway inhibitors, can significantly suppress the expression of inflammatory factors. However, their clinical application is limited by the non-targeting biodistribution and systemic side effects.^52–54 Various optimized NDSs can target transport of anti-inflammatory drugs to the plaque site, thereby effectively modulating the inflammatory response. In addition, various inflammatory cells at lesion sites release considerable ROS and oxidize LDL to ox-LDL, which further contributes to endothelial damage, as well as the formation of foam cells.^55,56 Although natural enzymes such as superoxide dismutase (SOD) and catalase (CAT) can scavenge ROS at lesion sites, their poor stability and susceptibility to degradation have severely restricted their therapeutic potential.^11,57 Metal-organic framework (MOF),⁵⁸ silicon dioxide (SiO₂),⁵⁹ Prussian blue (PB) NPs, selenium (Se) NPs, and cerium oxide (CeO₂) have enzyme-like activity and demonstrate excellent ROS-clearing ability, bridging the gap of NDSs-based efficient reprogramming strategies of ROS.^60,61 More importantly, reprogramming lipid’s metabolism is of great research value for the treatment of AS, as increased dyslipidemia leads to the passage of lipids across the damaged endothelial barrier, and their uptake by inflammatory macrophages, driving the development of the disease. Statins could effectively inhibit cholesterol synthesis, nevertheless, the significant oral first-pass effect and high plasma protein binding rate limit their application.^62,63 Optimized NDSs enable lipid’s reprogramming strategies by targeting the delivery of statins to the lesion site. In addition, soybean polyunsaturated phosphatidylcholine (PL) NPs and NPs modified with liver X receptor (LXR) ligands can reprogram lipid metabolism by regulating the expression of ATP-binding cassette transporters ABCA1 and ABCG1 or by improving the lipid composition of high-density lipoprotein (HDL) to enhance the capacity of cholesterol efflux (CEC).^64–67 To sum up, NDSs significantly enhance extracellular-based reprogramming strategies for the therapy of AS through their anti-inflammatory properties, ROS-scavenging capabilities, and superior lipid-clearing potential (Table 2).

Table 2 Nanodelivery Systems Based on Extracellular Reprogramming Strategies for Enhancing the Therapy of Atherosclerosis

This paper provides the first comprehensive review of progress over the past five years on NDSs based on intracellular and extracellular reprogramming strategies to improve the treatment efficacy of AS. It delves into intracellular reprogramming strategies, including reversing the phenotype of ECs and inflammatory states, the reprogramming of monocyte, the repolarization of macrophage, inhibiting the recruitment of NEs, and suppressing the proliferation of VSMCs, as well as extracellular reprogramming strategies, such as regulating inflammatory factors, reprogramming lipid’s metabolism, and scavenging ROS. The review covers multiple aspects, including cellular phenotypes and molecular pathways, emphasizing the advantages of these strategies over traditional delivery systems in the treatment of AS. This review provides new insights and perspectives on improving ASM and advancing its treatment through intracellular and extracellular reprogramming strategies.

Intracellular-Based Reprogramming Strategies

ECs-Based Reprogramming Strategies

Within the ASM, vascular shear and mechanical enlargement directly contribute to ECs damage.^88,89 Moreover, elevated levels of ox-LDL, ROS, and inflammatory factors at plaque sites contribute to mesenchymal transition of ECs.⁹⁰ NDSs have demonstrated the ability to reprogram the cells by reducing ROS production, enhancing endogenous antioxidant enzyme defenses, clearing lipids, mitigating shear stress, and increasing endothelial nitric oxide synthase (eNOS) activity. These mechanisms collectively inhibit or reverse endothelial mesenchymalization and restore endothelial barrier integrity.

Vascular cell adhesion molecule 1 (VCAM-1) is overexpressed on ECs,⁹¹ and it offers a wide range of perspectives for enhancing the targeting function of NDSs to ECs. Here, Xu et al prepared a ROS-responsive multifunctional NDSs (LFP/PCDPD) based on cyclodextrin loaded with a lipid-specific AIE fluorescent probe (LFP) and anti-inflammatory drug prednisolone (Pred) in its hydrophobic lumen, connected the hydrophilic and hydrophobic portions via poly(2-(methylthio) ethyl methacrylate (PMEMA), and modified the surface with dextran.²⁶ The NDSs effectively targeted ECs via VCAM-1 and CD44 receptors, and subsequently achieved ECs reprogramming strategy by scavenging ROS and lipids to effectively inhibit endothelial injury. Similarly, Dosta et al prepared a novel NDSs (VHPK-c-pBAE) by using targeted poly (β-amino ester) pBAE NPs, loaded with anti-microrna-712 (anti-miR-712) antibody and modified by VHPK peptide that could specifically target to ECs.²⁷ The NDSs selectively bound VCAM-1 on ECs, downregulating miR-712 while preserving tissue inhibitor of metalloproteinase 3 expression in inflamed endothelium. This dual action improved ECs function and therapeutic efficacy.

More importantly, by taking advantage of the recruiting effect of VCAM-1 overexpressed on the surface of ECs to NEs, the membrane of NEs could be used as a targeting material for ECs. Herein, Liu et al prepared a biomimetic NDSs (AM@ZIF@NM) using NEs membrane-coated zeolite imidazoline framework 8 (ZIF-8) to deliver microRNA-155 (anti-miRNA-155) antisense oligonucleotides (ASO).²⁸ The formulation could be recruited by VCAM-1 overexpressed on the surface of ECs, thereby specifically targeted the cells. Furthermore, it down-regulated the effects of miR-155 and the expression of its downstream target genes, C-C motif chemokine ligand (CCL2) and ICAM-1, effectively alleviating disease. It was shown that biomimetic NPs prepared through the plasma membrane of ECs had excellent targeting effects. Here, GAO et al designed a high-performance integrin-activating biomimetic (EC-BSAMnO₂) featuring reductively cleavable MnO₂, and core was fully encapsulated within ECs plasma membranes for enhanced biocompatibility.²⁹ The NPs effectively inhibited endothelial injury by reacting with considerable metabolites (H₂O₂ and H⁺) in the plaque and decomposing in situ to Mn²⁺, thereby activating integrin β₃ on the surface of ECs and inhibiting downstream integrin-coupled inflammatory signaling pathway.

Endothelial-mesenchymal transition (EndoMT) is a newly recognized process of cellular transdifferentiation in which ECs loosen their intercellular junctions, and endothelial junctions are reduced, while permeability is increased.⁹² Growing evidence supports EndoMT inhibition as a valuable therapeutic paradigm in inflammatory disease treatment. Here, Liu et al prepared a multifunctional NDSs (MNP@PA/BSA@NcadAb) using an N-cadherin antibody to modify melanin NPs.³⁰ By specifically targeting N-cadherin overexpressed on the surface, the NDSs modulated the downstream signaling of N-cadherin to reverse their EndoMT. Moreover, senescent ECs disrupt vascular endothelial integrity, accelerating vascular aging and increasing susceptibility to AS. Here, Xiao et al investigated that mesenchymal stem cell (MSCs)-derived extracellular vesicles (sEVs) enhanced AS therapeutic efficacy by preventing and attenuating senescence of ECs.⁹³ Results indicated MSCs-sEVs effectively suppressed senescent markers components while reversing oxidative damage, functional deficits and migratory capacity in aging ECs. It achieved reprogramming effects on senescent ECs, and stimulated angiogenesis via miR-146a/Src. MSCs-sEVs was expected to be used as an overlay material for enhancing the therapeutic effects for AS.

Monocyte-Based Reprogramming Strategies

The inflammatory properties of AS accelerate the production of bone marrow monocytes, and there is extravasation of circulating monocytes to sites of endovascular inflammation. The EndoMT leads to overexpression of P-selectin and E-selectin on the cell membrane, together with the release of adhesion factors, which recruit monocytes to accumulate at the AS lesion plaque.⁹⁴ Subsequently, Exposure to ox-LDL, ROS, and pro-inflammatory cytokines drives monocyte differentiation into inflammatory macrophages, amplifying the inflammatory cascade through positive feedback mechanisms.⁹⁵ In addition, anti-inflammatory cytokine-driven polarization of M2 macrophages promotes plaque microenvironment remodeling through resolution of inflammation and tissue repair mechanisms. Accordingly, targeted monocyte reprogramming emerges as a viable therapeutic paradigm for AS intervention.

Harnessing the inherent inflammatory chemotactic properties of monocytes for AS plaque targeting is a precise delivery strategy based on physiological mechanisms. Here, Yi et al prepared a positively charged NPs (BIN/PEI NPs) by self-assembly of MCP-1 synthesis inhibitor (bindarit) with polyethyleneimine, and further filled the BIN/PEI NPs into yeast-derived microcapsules (YCs) to obtain a biomimetic orally consumable NDSs (BIN/YCs).³¹ Upon oral administration of the NPs, they were distributed into the peripheral blood monocytes and transported to plaques via monocytes, thereby inhibiting release MCP-1, in turn inhibiting monocyte aggregation and chemotaxis, downregulating the inflammatory cascade response, and effectively preventing plaque formation. Furthermore, the monocyte-mediated delivery approach capitalizes on the marked increase in ROS that occurs during monocyte-to-macrophage differentiation within AS lesions, herein, Fu et al engineered citopril, an ROS-responsive prodrug, through thioketal-mediated conjugation of citral and captopril. This compound was subsequently self-assembled with amphiphilic polymers (PEG-DSPP) into nanomicelles (Citop-NMs).³² After intravenous injection, it was internalized into circulating monocytes and effectively accumulated within the plaque through inflammatory recruitment properties. Elevated levels of intracellular ROS triggered the pharmacological activation of Citopril, effectively reducing NLRP3-dependent IL-1β secretion, modulating the inflammatory response and preventing lipid deposition in plaques, and reducing the proportion of foam cells. Beyond suppressing monocyte recruitment, redirecting monocyte differentiation toward an M2 macrophage phenotype represents a promising therapeutic approach to stabilize vulnerable plaques. Nakashiro et al engineered pioglitazone-loaded nanoparticles (pioglitazone-NPs) using biodegradable poly (lactic-co-glycolic acid) (PLGA) polymers.³³ The NPs enhanced therapeutic outcomes by downregulating inducers of extracellular matrix metalloproteinases (MMP), reducing pro-inflammatory cytokine production in bone marrow-derived monocytes, and promoting their polarization into anti-inflammatory M2 macrophages.

Macrophage-Based Reprogramming Strategies

Macrophages serve as key immunomodulatory cells that critically orchestrate atherosclerotic plaque progression through their phenotypic plasticity and inflammatory mediator secretion.⁹⁶ M1 macrophages drive inflammatory pathogenesis through robust production of ROS and pro-inflammatory cytokines, while their M2 counterparts promote tissue homeostasis via anti-inflammatory mediators and reparative functions.⁹⁷ In early lesions, inflammatory macrophages have ability to remove apoptotic cells to avoid disease progression. During lesion progression, intimal macrophages actively engulf ox-LDL via scavenger receptors, transforming into lipid-laden foam cells that ultimately contribute to necrotic core formation through apoptotic cell death and defective efferocytosis.⁹⁸ Consequently, remodeling the inflammatory macrophage phenotype or induce apoptosis using NDSs is expected to improve the efficacy of AS.

Polarizing M1 macrophages into M2 macrophages has the potential to reprogram the pro-inflammatory into an anti-inflammatory response, thereby improving the inflammatory microenvironment.^36,99 Studies demonstrate that combined ROS elimination and pro-inflammatory factor reduction reliably reprograms macrophages toward tissue-repairing M2 polarization.¹⁰⁰ Here, Wu et al prepared tannic acid (TA)-poloxamer NPs (TPNP) by self-assembly of TA and poloxamer to achieve macrophage reprogramming to M2 type.³⁴ It has been demonstrated that epigallocatechin gallate (EGCG) can inhibit inflammatory factors, NF-κB inflammatory pathway and glycolysis by scavenging ROS. Wan et al developed a co-delivery system comprising simvastatin and EGCG using liposomal NPs (SE-LNPs) prepared from distearoylphosphatidylcholine (DSPC).³⁵ The NPs were observed to reprogram M1 macrophages into M2 macrophages through superior ROS scavenging and anti-inflammatory capabilities. To optimize NDSs accumulation at inflammatory sites and enhance M1 macrophage specificity, Zhao et al devised a ROS-responsive bionic NDSs (KPF@MM-NPs) by enclosing dextran-g-PBMEO NPs loaded with the natural drug kaempferol (KPF) through macrophage membranes (MM).³⁶ The ROS-responsive NDSs demonstrated targeted plaque accumulation through oxidative stress-dependent activation, enabling selective macrophage delivery in lesions. This resulted in a reduction in key pro-inflammatory factors through regulate of the NF-κB pathway, thereby enabling macrophage reprogramming strategies.

The process of endocytosis, proliferation, accumulation, and absorb of ox-LDL by inflammatory macrophages at the lesion site exacerbates plaque development. Triggering oxidative stress, mitochondrial damage, and DNA destruction through the Fenton reaction, and ultimately activating the macrophage apoptotic pathway, can effectively arrest disease progression. Based on this, Mu et al used dopamine-modified hyaluronic acid (HD) loaded with stabilized iron (III)-TA NPs (HFTNPs) to specifically induce apoptosis in inflammatory macrophages.³⁷ The NDSs demonstrated selective targeting of inflammatory macrophages via HA-CD44 receptor interactions. This targeting strategy simultaneously elevated intracellular ROS levels, amplified iron-mediated Fenton reactions, and triggered significant apoptosis in pro-inflammatory macrophages. Consequently, this triple-action mechanism effectively suppressed inflammatory cytokine secretion and attenuated atherosclerotic plaque development.

NEs-Based Reprogramming Strategies

In the ASM, NEs undergo activation via ROS and pro-inflammatory cytokines, and their activation synergistically interacts with the calcium-dependent enzyme peptidyl arginine deiminase 4 (PAD-4) through histone citrullination. This interaction triggers cellular disintegration, resulting in the cellular contents release of cytoplasmic component,^101,102 and this cascade culminates in NETs formation, which accelerate plaque expansion.¹⁰³ Concurrently, NETs-derived histones and granular proteins directly induce VSMCs and ECs lysis, while neutrophil elastase-mediated extracellular matrix degradation synergistically compromises plaque structural integrity.¹⁰⁴ These findings position NDS-mediated NEs reprogramming strategies as a novel approach to address both ECs dysfunction and plaque vulnerability in AS, potentially overcoming current treatment limitations.

Due to recruitment of NEs is a typical feature of AS, inhibition of NEs recruitment by targeting NDSs is expected to block subsequent NETs-mediated endothelial damage and inflammatory responses. Here, Guo et al constructed a cyclodextrin-derived intrinsically bioactive NPs (LCD NPs),³⁸ which enhanced the therapeutic efficacy by inhibiting the recruitment of NEs and inflammatory macrophages, thereby preventing subsequent pro-inflammatory events. Moreover, the areas of the basement membrane that have undergone ECs detachment are characterized by a high concentration of type IV collagen. Here, Molinaro et al constructed targeted NDSs (GSK-Col IV NPs) that limit NET formation by utilizing poly(D,L-propenolactone) (PLA) as the central part and polyethylene glycolated phospholipid layer as the shell of type IV collagen-targeted NPs (Col-IV NPs) for the delivery of the PAD-4 enzyme inhibitor GSK484.³⁹ The NDSs effectively inhibited NEs infiltration and suppressed NETs-driven inflammatory response. Additionally, they reduced thrombus formation induced by NETs-associated superficial erosion while promoting endothelial repair. Notably, NEs elastase that catalyzes the formation of NETs, enhances immune cell recruitment, stimulates interferon-α release. More importantly, it degrades elastin and other matrix proteins in motile AS plaques, thereby accelerating the formation of vulnerable plaques.^105,106 Here, Yin et al constructed a plaque coupled targeting and NEs hitchhiking liposomes (cRGD-SVT-Lipo) by loading the NEs elastase-competitive inhibitor sivastigmine (SVT) into cRGD peptide-modified liposomes.⁴⁰ The NDSs used cRGD peptide to specifically recognize with integrin α_νβ₃ on the surface of NEs and being internalized into them. After reaching the plaque site with the inflammatory chemotactic effect of NEs, they effectively inhibited the elastase activity of intra- and extracellular NEs, reduced NETs formation, and ultimately alleviated the development of AS.

VSMCs-Based Reprogramming Strategies

VSMCs undergo significant phenotypic changes switching from contractile to synthetic phenotype in the inflammatory environment of AS, and the phenotype is distinguished by strengthened migration, multiplication, and secretion.^9,107 This is demonstrated by VSMCs migrating from the arterial wall’s medial layer, proliferating to form a fibrous protective cap abundant in extracellular matrix. This encapsulation process surrounds the plaque while simultaneously promoting expansion of the necrotic core.¹⁰⁸ Secondly, under the stimulation of ROS and inflammatory factors, VSMCs can be transformed into transformed into macrophage-like cells, which further release pro-inflammatory factors, extracellular matrix, and adhesion molecules, thereby recruiting a large number of inflammatory cells to accumulate at plaques, exacerbating the development of inflammation, and inducing plaque instability and calcification.^109,110 Consequently, reprogramming of the VSMCs phenotype holds promising future for optimizing the therapeutic effectiveness of AS.

VSMCs exhibit remarkable plasticity, transitioning from a quiescent, contractile state to a migratory, proliferative, and synthetic phenotype. It has been demonstrated that the peroxisome proliferators-activated receptors (PPARδ) agonist GW1516 inhibits VSMCs migration and apoptosis. Here, Huang et al developed a GW1516-loaded NDSs (GW1516@NP-OPN) modifying with anti osteopontin (OPN),²⁵ and they effectively suppressed VSMC migration and apoptosis, resulting in a significant reduction of lesion size in vivo.¹¹¹ Similarly, Gang et al delivered the PPARδ agonist GW0742 using anti-VCAM-1-modified nanocolloid,⁴¹ which effectively inhibited ox-LDL-induced apoptosis and migration of VSMCs. Furthermore, Profilin-1 (PFN1) is plentiful in VSMCs and is involved the reorganization of cytoskeletal polymerization.^112,113 Here, Zhang et al prepared a VSMCs-targeting NDSs (RAP@PFN1-CD-MNPs) by conjugating paramagnetic Fe₃O₄ NPs with pH-sensitive cyclodextrins (CDs) and coupled them with an antibody to PFN1 loaded with the anti-inflammatory drug (rapamycin) (Figure 2).⁴² The NDSs utilized the pH sensitivity of cyclodextrins to achieve responsive release in the plaque region and successfully inhibited the proliferation of vascular ECs. Moreover, RAP@PFN1-CD-MNPs could function as a biocompatible MRI contrast agent for AS lesion detection.

Figure 2 Preparation of RAP@PFN1-CD-MNPs and its mechanism of enhancing the therapy of AS by reprogramming strategy of ECs (A) Schematic diagram of synthesis principle of [email protected] NPs. (B) TEM images of NPs (scale bar = 10 nm). (C) Magnetic properties of NPs. (D) FTIR spectra of NPs. Uptake of NPs in VSMCs. Fluorescence imaging (E) and quantification (scale bar = 5 μm) (F) of the ability of PFN1-CD-MNPs to bind to VSMCs in vitro. NIRF of aorta at 2 months after treatment (G) and quantification (H). General ORO staining (I) and quantification (J). Animals in the control group were administered saline, *P < 0.05, **P < 0.01, ***P < 0.001. Reproducted from Zhang S, Xu W, Gao P, Chen W, Zhou Q. Construction of dual nanomedicines for the imaging and alleviation of atherosclerosis. Artif Cells Nanomed Biotechnol. Dec 2020;48(1):169–179.42 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).

Given the phenotypic transformation of VSMCs and its high degree of plasticity, the use of targeted NDSs to modulate its phenotype or intervene in its differentiation is expected to attenuate plaque development. Herein, Chen et al used MCP-1 peptide-modified nucleic acid micelles (miR-145 micelles) to target and meddle in the phenotypic transition of VSMCs.⁴³ This micelle specifically binds to C-C chemokine receptor-2, which was upregulated in VSMCs surface, and enhances the expression of cardiac myosin, calponin, and α-smooth muscle actin by upregulating contractile genes. At the same time, Krüppel-like factor-4/5 and ETS domain-containing protein-1 were downregulated, thereby inhibiting VSMCs-derived plaque propagating cell types, maintaining the normal contractile state of VSMCs, and attenuating the development of AS. Recent studies have shown that an extensive majority of macrophages and foam cells in AS stem from VSMCs differentiation, which is positively correlated with markers of dedifferentiation and Glp1r expression (eg, increased Klf4).¹¹⁴ Maiseyeu et al prepared a theranostics NPs (GLP NP) by utilizing synthesized protease-resistant and “activatable” NPs conveying the GLP-1R agonist liraglutide (GlpNP) and incorporated gadolinium chelates (MRI imaging agents).⁴⁴ The NPs were capable of effectively preventing the transition of VSMCs to macrophage-like phenotype.

Multiple Cells-Based Reprogramming Strategies

AS progression involves a sophisticated interplay of diverse cell populations and complex signaling networks.^115,116 Under inflammatory conditions, elevated levels of ROS, inflammatory factors, and injury signals induce ECs damage. This damage triggers the secretion of substantial amounts of adhesion factors, which recruit large numbers of monocytes and NEs. Monocytes differentiate into a significant population of inflammatory macrophages, while NEs are stimulated to form NETs. These NETs not only aggravate endothelial dysfunction but also potentiate inflammatory cascades, thereby accelerating AS progression.^117–120 In summary, in the ASM, various cells interact through complex cross-talk mechanisms, forming a network that collectively drives inflammation and plaque progression. Consequently, reprogramming cells and the associated network of contacts in ASM has significant implications for AS therapy.

In the initial phase of AS, ECs injury triggers the recruitment of pro-inflammatory macrophages that release abundant cytokines, creating a vicious cycle of endothelial dysfunction. Therefore, simultaneous targeting and reprogramming of ECs and macrophages can achieve suppression of inflammation while repairing endothelial damage. Here, our group prepared a pH-responsive dual-targeting NDDS (BC@CS/cRGD NPs) by combining baicalin (BC) with chondroitin sulphate (CS) through an amidation reaction followed by conjugation with the cRGD peptide targeting moiety.⁴⁵ This NDSs could effectively target ECs and inflammatory macrophages through the specific binding of the cRGD peptide to the α_Vβ₃ protein receptor, thereby effectively inhibiting inflammation and repairing endothelial damage. In addition, removal of large numbers of inflammatory macrophages from AS plaques, inhibition of foam cell formation, and repair of ECs are important for the treatment of the disease. Herein, Li et al constructed a bimodal propellant NDSs CD-LA-Au-aV sensitive to ROS and near-infrared lasers by covalent binding and self-assembly of β-cyclodextrin (β-CD) with L-arginine (L-Arg), followed by the immobilization of gold NPs.⁴⁶ The NDSs could remove inflammatory macrophages through the excellent photothermal conversion properties of Au, thereby inhibiting inflammation along with foam cell formation, while promoting cholesterol removal from foam cells and reducing ROS to achieve endothelial repair. To sum up, the NDSs could reprogram multiple cells and components of ASM in multiple ways, thereby synergistically and comprehensively enhancing the therapeutic efficacy in a non-pharmacological strategy.

Macrophages and foam cells play pivotal roles in AS plaque formation and the associated inflammatory cascade. Therapeutic strategies that simultaneously modulate macrophage polarization and enhance foam cells CEC may provide a comprehensive approach for mitigating AS progression. An et al developed an endogenous H₂S-based gas therapy utilizing multienzyme-mimicking nanozymes (^LYP−1 LIP@HS) using hollow mesoporous prussian blue (HMPB) loaded with the H₂S donor S-allyl-L-cysteine (SAC), and to enhance the targeting specificity and achieve controlled release kinetics of the HS NP, further the LYP-1 peptide-functionalized lipid membrane was modified on its surface.⁴⁸ The NDSs unlocked the polymerase-like activity of HMPB after cavitation induced by low-intensity focused ultrasound (LIFU), while promoting the release of the endogenous H₂S donor SAC. Notably, the enzyme-catalysed generation of endogenous H₂S plays multiple roles by upregulating ATP-binding cassette transporter A1 in foam cells, enhancing cholesterol efflux, and facilitating pro-inflammatory M1 polarization to anti-inflammatory M2 phenotype, thereby effectively delaying disease. In addition, the crosstalk between T cells and macrophages constitutes a pivotal regulatory axis driving inflammatory pathogenesis in this disease, and inhibition of antigen presentation from macrophages to T cells not only modulates the number and type of T cell subsets, but also the levels of inflammatory factors in ASM, which in turn remodels the macrophage phenotype. Here, Zhou et al designed a pH-responsive and charge reversible NPs (ASPA NPs) through synthesizing short hairpin RNA targeting mouse sialic-acid-binding immunoglobulin-like lectin-1 (Siglec-1) loaded and polyethyleneimine-acetylsalicylic acid (PEI-ASA) coated Au-PEI NPs (Figure 3).⁴⁹ The NPs blocked CD8⁺T/NKT cell-macrophage interactions by silencing Siglec-1, thereby inhibiting CD8⁺T/NKT cell infiltration and inhibited Th 17 cell differentiation, as well as activating intracellular macrophage peroxisome proliferator-activated receptor α (PPAR-α) and γ (PPAR-γ) to facilitate cholesterol efflux, thus attenuating foam cell formation. In addition, reduced lipid antigen presentation by macrophages led to decreased IL-6 and IL-1β levels in lesions. To sum up, the NDSs multifaceted and remodeling of multiple cells in ASM effectively delayed AS.

Figure 3 Preparation of ASPA NPs and their mechanism of action through remodeling of various cells to enhance the therapy of AS. (A) Schematic diagram of the construction of ASPA NPs. (B) TEM image of Au-PEI NPs (scale bar = 50 nm). (C) Inflammatory factor expression levels in RAW264.7 cells after different treatments. (D) ASPA NPs inhibits the formation of foam cells (scale bar = 100 μm). (E) Representative photographs of ORO-stained enface aortas from mice after different treatments. Data are shown as mean ± SD. #P < 0.05, ##P < 0.01, ox-LDL group vs Con group; test group vs ox-LDL group, *P < 0.05, **P < 0.01, ***P < 0.001. Reproducted from Zhou Y, Wang S, Liang X et al. Turning Hot into Cold: Immune Microenvironment Reshaping for Atherosclerosis Attenuation Based on pH-Responsive shSiglec-1 Delivery System. ACS Nano. Jul 26 2022;16(7):10,517–10,533.49 Copyright 2022, American Chemical Society.

Accumulating evidence demonstrates that cellular senescence promotes AS progression across all disease stages and exacerbates cardiovascular pathogenesis. Emerging evidence indicates that CD9 critically regulates both cellular senescence induction and atherosclerotic plaque development via activation of the PI3K-AKT-mTOR-p53 signaling cascade. Here, Pham et al prepared a multifunctional NDSs (CD9-HMSN@RSV) utilizing CD9 antibody-modified, HA-coated mesoporous silica NPs, to deliver the anti-aging drug Rosuvastatin (RSV).⁴⁷ This NDSs could effectively target senescent foam-like macrophages and senescent ECs. In vitro and in vivo studies explanation that these NDSs could productively remove senescent macrophages and ECs, further reduce ROS levels and inhibiting HDL oxidation, TNF-α and IL-6 production. In addition, inhibiting endothelial cell senescence while reprogramming the macrophage phenotype is expected to yield better therapeutic results. Herein, Peng et al developed osteopontin (OPN)-functionalized nanoliposomes, designated CZALO, which encapsulated both L-Arg and cerium-zirconium oxide nanoparticles (CZ NPs).⁵⁰ On the one hand, the NDSs could achieve anti-inflammatory and antioxidant effects by reprogramming the macrophage phenotype through multi-enzyme mimetic activity, including SOD, CAT, peroxidase, and glutathione peroxidase. On the other hand, nitric oxide synthase overexpressed in macrophages selectively catalyze the in-situ conversion of L-Arg to NO, which modulates the release of senescence-associated secretory phenotype (SASP) factors from ECs, enhances lysosomal activity, overcomes cell-cycle blockade, and mitigates DNA damage in aged ECs. To sum up, the NDSs-mediated antioxidant and anti-aging therapeutic “killing two birds with one stone” strategy showed good anti-AS efficacy in vitro and in vivo.

Extracellular-Based Reprogramming Strategies

Inflammatory Response-Based Reprogramming Strategies

Clinical and epidemiological studies confirm that inflammation is pivotal in the development of AS. In its early stages, ox-LDL causes endothelial damage and subsequent mobilization and infiltration of monocytic and T cells. Circulating monocytes undergo macrophage differentiation, acquiring enhanced expression of innate immune receptors such as scavenger receptors and Toll-like receptors. Toll-like receptors further transmit activation signals, triggering the secretion of pro-inflammatory cytokines, matrix-degrading proteases, and vascular mediators that collectively establish a pro-inflammatory niche.⁵¹ During initial inflammatory responses, the dynamic interplay of inflammatory mediators orchestrates immune cell differentiation pathways and phenotypic commitment through concentration-dependent signaling gradients. For example, pro-inflammatory cytokines including TNF-α and IL-1β drive both endothelial-to-mesenchymal transition and monocyte polarization toward pro-inflammatory macrophage phenotypes.¹²¹ Subsequently, these activated immune cells and stromal cells secrete substantial quantities of inflammatory factors to exacerbate the inflammatory response and plaque growth.¹²² More importantly, inflammatory factors can also exacerbate plaque development by advancing apoptosis and necrotic tissue formation. In contrast, various anti-inflammatory factors can not only attenuate the inflammatory response induced by pro-inflammatory factors, but also induce the shift of immune cells to an anti-inflammatory phenotype, IL-10 and TGF-β can reverse mesenchymalisation of ECs and achieve macrophage repolarization.¹²³ These immune cells with anti-inflammatory phenotypes can also secrete various anti-inflammatory factors to improve the inflammatory microenvironment and inhibit plaque growth.¹²⁴ Therefore, the use of multifunctional NDSs to remodel the inflammatory response at the AS plaque site not only improves the inflammatory microenvironment, but also reprograms various immune cells into anti-inflammatory phenotypes to achieve enhanced therapeutic effects.

Although natural polyphenols exhibit potent anti-inflammatory properties, their clinical application is significantly constrained by poor bioavailability and chemical instability. NDSs with optimized physicochemical characteristics may effectively address these limitations. Here, Anghelache et al loaded protocatechuic acid (PCA) into dextran-modified iron oxide shells to obtain a core-shell magnetic NPs (MNP-Dex/PCA).⁶⁸ The magnetic NPs enabled disease visualization to ensure localized release at the plaque site, which in turn significantly reduced the release of multiple pro-inflammatory factors by down-regulating the levels of ERK1/2 and p38-alpha mitogen-activated protein kinases (MAPKs), coupled with suppression of NF-κB transcriptional activity. Interestingly, the combination of statins and other bioactive drugs into a single polymeric nanocarrier promises enhanced synergistic therapeutic outcomes through complementary pathways. Here, Leal et al prepared a pleiotropic nanoplatform (PLGA/miRNA NPs) by co-loading atorvastatin (ATOR) with miRNA-124a into PLGA NPs.⁶⁹ The nanoplatform was able to regulate inflammation-related genes simultaneously, and statins’ pleiotropic anti-inflammatory properties enabled targeted modulation of inflamed vascular endothelium, representing a therapeutic breakthrough in endothelial dysfunction management, utilizing complementary pathways to achieve synergistic therapeutic outcomes.

Increasing evidence suggests that delivery of anti-inflammatory factors such as IL-10 to ASM is expected to reprogram the inflammatory response and achieve enhanced therapeutic effects. To enhance IL-10 accumulation at plaque sites, Kim et al prepared plaque-localized NDS (IL10-NC) using cRGD-conjugated pluronic-based nanocarriers (NC) to deliver IL-10.⁷⁰ Compared with free IL-10, IL-10-NC significantly improves its pharmacokinetic profile, thereby effectively increasing IL-10 accumulation at plaque sites, enabling an inflammatory response reprogramming strategy. The development from exogenous cytokine delivery to endogenous gene therapy can achieve the extension from short-term effects to long-term modulation. Herein, Distasio et al engineered a DNA NPs (NP-VHPK) utilizing VHPK peptide-conjugated branched poly (β-amino ester) for targeted delivery of IL-10 encoding plasmid DNA.⁷¹ The NPs achieves ECs-specific targeting via VHPK peptide-VCAM-1 molecular recognition, followed by robust IL-10 transgene expression post-internalization that potently attenuates inflammatory signaling.

ROS-Based Reprogramming Strategies

Numerous studies have shown that ROS, consisting of H₂O₂, O₂^·-, OH-, etc, is indispensable for the development of AS.¹²⁵ Elevated ROS levels trigger NF-κB pathway activation, amplifying pro-inflammatory signaling cascades. This oxidative stress-mediated response enhances leukocyte-endothelial adhesion through upregulated cell adhesion molecules, ultimately inducing endothelial dysfunction and vascular injury.^8,126 Therefore, therapeutic modulation of the inflammatory milieu through ROS scavenging may represent a promising strategy for attenuating disease progression.^95,127

Contemporary antioxidant therapeutics primarily utilize ascorbic acid (VC), polyunsaturated fatty acids, and various polyphenolic compounds as core therapeutic agents.^128–130 However, there are three major limitations of these antioxidants: firstly, short biological half-life and susceptibility to rapid hepatic and renal clearance; secondly, lack of plaque site targeting and inefficient delivery; and most importantly, limited ROS scavenging ability of single antioxidants.^131,132 Here, Lu et al constructed a novel natural antioxidant (VC@cLAVs) by loading VC into cross-linked vesicles of lipoic acid (LA).⁷² This synergistic formulation substantially prolonged the plasma circulation time of the natural antioxidant while enhancing its redox activity. The coupled LA/DHLA and VC/DHA cycling systems created an amplified antioxidant effect, demonstrating superior ROS neutralization capacity. However, the NDSs still have the problem of insufficient targeting, in order upgrade the therapeutic strategy from “passive removal” to “active regulation”, Luo et al prepared a ROS-sensitive NPs (LLC NPs) by using heparin-lipoic acid coupling (LMWH-LA) as an amphiphilic shell for encapsulating curcumin (Cur) (Figure 4).⁷³ The NPs target P-selectin on ECs at the plaque site, competitively blocking the migration of inflammatory cells to ECs and reducing ROS production from cellular sources. Subsequently, the NPs were oxidatively triggered to convert from hydrophilic to hydrophobic, accelerating the release of Cur, thereby removing excess ROS from the plaque.

Figure 4 Preparation process of LLC NPs and their enhancement of the therapy of AS by scavenging ROS. (A) Schematic diagram of self-assembly and ROS response of LLC NPs (B) TEM images of LLC NPs (scale bar = 100 nm). (C) Flow cytometry was used to quantify the levels of intracellular ROS in RAW264.7 cells following various treatments (scale bar = 10 μm). (D–F) Inflammatory cytokines TNF-α (D), IL-6 (E), CRP (F) secreted by RAW264.7 cells treated with different formulation. (G) Image of ORO-stained enface aortas. Data are shown as mean ± SD, n = 5. *P < 0.05, **p < 0.01. Reproduction from Luo X, Zhang M, Dai W et al. Targeted nanoparticles triggered by plaque microenvironment for atherosclerosis treatment through cascade effects of reactive oxygen species scavenging and anti-inflammation. J Nanobiotechnology. Jul 27 2024;22(1):440. © The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License.73

Notably, a variety of inorganic nanomaterials possess CAT and SOD-like enzyme that can productively scavenge ROS, including CeO₂, manganese oxide (MnO_x), PB analogues and black phosphorus nanosheets (BPNSs).¹³³ Among them, CeO₂ NPs mimic the dual enzyme activities of SOD and CAT through the Ce³⁺/Ce⁴⁺ redox cycle.¹³⁴ In addition, MnO_x catalyzes ROS’s catabolism using its Mn²⁺/Mn³⁺/Mn⁴⁺ interconversion.^135,136 Similarly, PB analogues scavenging ROS by redox of iron ions Fe²⁺/Fe³⁺.¹³⁷ Functionalizing nanozymes as drug loading and delivery platforms is not only conducive to the improvement of their multi-enzyme activity to reduce toxic side effects, but also to enhance ROS’s scavenging.¹³⁸ Here, Fu et al innovatively constructed a biomimetic delivery system (PCZ@PB NCs) by self-assembling zoledronic acid with cerium ions into cerium-like-MOF nanomaterials (CZ NCs) and encapsulating them through platelet membranes.⁷⁴ This study not only enhanced the multi-enzyme activity of CeO₂, the formulation demonstrated selective biodistribution to atherosclerotic plaques, where it effectively attenuated local oxidative stress through ROS downregulation. To optimize plaque targeting and therapeutic performance of nanozymatic delivery systems Wu et al prepared a NEs hitchhiking nanoplatform (MPB NPs) by using bacterial mimetic membranes (mycoderm) encapsulating Prussian blue nanoparticles (PB NPs).⁷⁵ The NPs can be specifically recognized and efficiently internalized by NEs. Using the inflammatory chemotaxis mechanism, they can accurately accumulate at the AS plaque site. In addition, the NPs have the dual function of effectively scavenging intra and extracellular ROS and inhibiting NET formation. This multimodal action ultimately contributes to their anti-atherosclerotic efficacy. In addition, Tao et al obtained a type of nano-NDSs (BPNSs@PEG-S2P/R) by preparing a two-dimensional BPNSs and modifying its surface with ROS-responsive targeting materials for the delivery of Resolvin D1.⁷⁶ These nanosheets exhibit high ROS’s scavenging rates due to their excellent specific surface area. Importantly, their layered structure facilitates their uptake by macrophages, allowing them to accumulate within plaques of AS, thereby effectively reducing expression levels of inflammatory factor and achieving the reprogramming of inflammatory macrophage.

Lipids-Based Reprogramming Strategies

Lipids are divided into three main categories: total cholesterol, HDL, and LDL. AS lesions typically manifest lipids metabolic dysregulation, exhibiting elevated LDL concentrations, reduced HDL levels, and overall cholesterol homeostasis disruption.¹³⁹ When LDL penetrates damaged ECs, it is oxidized in response to ROS to form ox-LDL, which has the characteristics of a high degree of AS. Modified LDL particles undergo extensive macrophage internalization primarily through scavenger receptor-mediated endocytosis, such as CD36, LOX-1, and SR-A. This receptor-dependent uptake drives foam cell transformation through intracellular cholesterol ester accumulation, while simultaneously activating NF-κB dependent proinflammatory signaling cascades-collectively accelerating plaque development. Incorporating lipids reprogramming strategies into the design of NDSs is therefore essential to reduce lipid deposition, particularly the retention of LDL in the arterial wall. This can be achieved by a dual mechanism: reducing cellular uptake of cholesterol and enhancing cellular efflux of cholesterol. This approach has great therapeutic potential to improve the treatment of AS.

Studies show that statins have ability to decrease lipid uptake by M1 macrophages and increase the antioxidant capacity of LDL. Here, Rakshit et al prepared a simvastatin (STAT) nano-liposomal formulation (LIPOSTAT).⁸¹ When administered intravenously, it reduced serum cholesterol and LDL receptor expression in hepatocytes. It was found that oxidized phosphatidylcholines (PCs) on the surface of LDL promote its binding to macrophage CD36 receptors. To this end, Zhang et al prepared an NDSs (L-Enano) and successfully synthesized EGCG-loaded NPs (Enano) by using EGCG, PC, (+)α-tocopheryl acetate, and surfactants as the main raw materials and modified the surface of the nanocapsules with KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octen-diene) PC (KOdiA-PC)) to modify it.⁷⁷ The NDSs competitively blocked oxLDL internalization in macrophages via KOdiA-PC’s high-affinity binding to the CD36 scavenger receptor, effectively disrupting foam cell formation.

Studies have shown that facilitating reverse cholesterol transport from macrophages can inhibit foam cell formation. Ligands for LXR or LXR agonists can upregulate the ATP-binding cassette transporter proteins ABCA1 and ABCG1 on macrophages, thereby enhancing cholesterol excretion.^140,141 To this end, studies have utilized PLGA-b-PEG NPs to target delivery of LXR to macrophages on ABCA1 and ABCG1 as way to increase cholesterol efflux.¹⁴² Second, when excess LDL penetrates the arterial wall, it causes cholesterol crystals (CCs) to accumulate within the plaque and induces inflammation in a various cells through multiple pathways.¹⁴³ Kim et al engineered core-shell NPs through host-guest complexation of simvastatin within methyl-β-cyclodextrin cavities, enveloped by a phospholipid bilayer shell, creating a cargo-switching NDSs (CSNP) capable of controlled drug release.⁷⁸ The CSNP demonstrated excellent extracellular and intracellular CCs and cholesterol scavenging ability, effectively reducing cholesterol levels in AS. To further enhance the plaque targeting of NDSs, Chen et al constructed a target-responsive nanoplatform, LPLCH by using a covalent cholesterol group (PLC) of poly-ε-lysine coupled to a pH-responsive imine bond, loaded with an LXR agonist, and surface-modified with oxidized hyaluronic acid (oxHA).⁷⁹ The nanoplatform could first be activated by the interaction between HA and CD44 targeting foam cells, and triggering a surface charge shift during lysosomal immune escape at acidic pH 5.5, effectively removing CCs from lysosomes by carrying them. Moreover, the NDSs could promote lipid efflux by targeting up-regulation of ABCA1 and ABCG1 at the site of plaques, they synergistically achieved dual-track reverse cholesterol transporter. In addition, HDL can interact with ABCA1, ABCG1, and scavenger receptor B1 (SR-BI), to facilitate macrophage-specific cholesterol efflux from foam cells through enhanced reverse cholesterol transport (RCT) pathways, promoting lipid clearance from plaque microenvironments.^139,144 Here, Kudinov et al utilized PL NPs containing PL to reprogram HDL and improve the CEC.⁶⁴ Likewise, Guo et al prepared a cholesterol efflux-promoting NDSs (sHDL-T1317) using phospholipid recombinant apoA-1 peptide (22A)-derived HDL NPs (sHDL) delivering the LXR agonist T1317.⁶⁶ The NDSs significantly enhanced ABCA1 and ABCG1 transporter expression in macrophages and increased cholesterol efflux. More importantly, inhibiting cholesterol uptake while promoting cholesterol efflux would enable a better lipid reprogramming strategy. Here, a multifunctional NDSs (mDNP-LXR-L-siRNA) was prepared by optimized mannose-functionalized dendritic NDSs (mDNP) co-coating with reduce LDL uptake SR-A siRNA and LXR-L.⁸⁰ This delivery strategy achieved aortic cholesterol-lowering function through a synergistic multi-pathway approach.

Multiple Components-Based Reprogramming Strategies

In ASM, multiple extracellular components, including inflammatory factors, ROS, and lipids, interact in a complex manner, creating a vicious cycle that exacerbates the inflammatory response and plaque formation.^145–148 Consequently, a simultaneous reprogramming strategy targeting multiple components holds promise for increasing the AS therapy.

The deterioration of AS is characterized by overexpressed ROS and lipid deposition.¹⁴⁹ Consequently, simultaneous modulation of ROS and lipid levels at the disease site is critical for improving efficacy. Here, Ma et al developed a therapeutic nanomicelles (TPCDP@PMM) for lipid and inflammation reprogramming.⁸² In addition, they first bridged LXR-L with β-cyclodextrin (β-CD) and loaded Pred onto β-CD via host-guest interactions to construct LCDP complexes, then encapsulated LCDPs and Π-conjugated polymer-based photoacoustic probes with ROS-responsive polymers (poly (2-methylthioethanol methacrylate)) to form PLCDP@PM NPs. Finally, oxHA was utilized to encapsulate the NPs and cross-link the MMP-9-sensitive peptide (MMP9 Pep) on their surface to obtain multifunctional NDSs (PLCDP@PMH).⁸³ The NDSs effectively inhibited inflammatory response and plaque formation through M1 macrophage reprogramming strategy that synergized with LXR-mediated lipid reprogramming strategy, showing excellent therapeutic effects.

The phytochemical curcumin mediates cholesterol clearance from lipid-laden macrophages through SCAR-B1/SR-B1 receptor upregulation, while concurrently attenuating pro-inflammatory signaling via NF-κB and STAT-3 pathway suppression.^150,151 Pillai et al obtained NDSs (Cur-Bio PLGA NPs) using polyethylene glycol delivery of Cur and biopiperidine.⁸⁴ The NDSs significantly increased the bioavailability of Cur through the bioaugmentation of bioperine, effectively down-regulating inflammatory pathway-related genes to achieve the inflammatory reprogramming strategy. Meanwhile, it upregulated SCAR-B1 expression and inhibited the c-Jun N-terminal kinase pathway (JNK) to remove cholesterol accumulated in macrophages and reduced foam cells formation. Docosahexaenoic acid (DHA), which demonstrates potent anti-inflammatory effects. Here, Chong et al developed an injectable liposomal DHA for the treatment of AS.⁸⁵ The NDSs could be effectively taken up by macrophages after reaching the plaque site, exerting anti-inflammatory and antioxidant effects. Moreover, the liposomal DHA formulation reduction in macrophage infiltration, decrease in plaque lipid content, increase in fibrillar collagen, and improving the stability of plaques.

Accumulating evidence indicates that molecular hydrogen (H₂) can scavenging ROS and suppressing NF-κB mediated production of proinflammatory cytokines. Herein, Hu et al designed a distinct tetrapod needle-like palladium-hydrogen nanozyme (TN-PdHs) by adding Fe during Pd nano-enzyme synthesis and inserting H₂ into the Pd nano-enzyme lattice.⁸⁶ Interestingly, the specific spiky morphology of TN-PdHs triggers a strong autophagic response in macrophages, synergizing excellent ROS scavenging and anti-inflammatory capabilities to mitigate AS progression. Studies have shown that CCs exacerbate disease progression in AS by inducing inflammation and promoting plaque destabilization; therefore, depletion of CCs is expected to achieve dual regulation of inflammation and lipids. Herein, Luo et al prepared a phospholipid-based HDL-like nanoparticle (miNano) (Figure 5).⁸⁷ The preparation enhanced cholesterol efflux by inhibiting the TLR4-NF-kB pathway, dissolved CCs in AS plaques, prevented foam cells formation, and concurrently inhibited inflammatory responses for effective disease treatment.

Figure 5 Preparation of TN-PdH@Ms and its enhancement of AS therapy by scavenging ROS and modulating inflammatory factors. (A) The preparation process of TN-PdH@Ms. (B) TEM image of TN-Pds (scale bar = 100 nm). (C) H2O2-scavenging performance of TN-PdHs. (D) O2•−-scavenging capability of TN-Pds and TN-PdHs. (E) LPO inhibition performance of TN-Pds and TN-PdHs. Inflammatory factor levels of (F) TNF-α, (G) IL-1β, and (H) IL-6 in LPS-induced RAW264.7 cells after varied treatments, including control, LPS, LPS + low dose TN-Pds (25 ppm), LPS + high dose TN-Pds (50 ppm), LPS + low dose TN-PdHs (25 ppm), and LPS + high dose TN-PdHs (50 ppm). (I) Representative photographs of enface ORO-stained aortas from each mouse after varied treatments. (J) Quantitative analysis of the lesion areas in the tested aortas. Reproduction form Hu R, Dai C, Dong C et al. Living Macrophage-Delivered Tetrapod PdH Nanoenzyme for Targeted Atherosclerosis Management by ROS Scavenging, Hydrogen Anti-inflammation, and Autophagy Activation. ACS Nano. Oct 25 2022;16(10):15,959–15,976.86 Copyright 2022, American Chemical Society.

Discussion

The activation of various inflammatory cells and the accumulation of inflammatory mediators in ASM play an important role in disease progression and plaque stability. Undeniably, intracellular and extracellular reprogramming strategy solves the challenges of multidrug resistance and inferior specificity in AS treatment to some extent.¹⁵² NDSs offer substantial advantages for intracellular and extracellular reprogramming caused by their superior drug-loading capacity, target-specific modifications, ability to protect enzymatic stabilization, and intrinsic properties such as lipid-lowering and ROS scavenging capabilities. By reprogramming the interactions among various cells and components within the ASM, NDSs can further optimize the ASM and significantly enhance the therapeutic efficacy of AS.¹⁵³

However, significant challenges remain for the practical application of NDSs in AS therapy. Plaque formation is primarily driven by a complex interplay of immune and inflammatory responses. ASM is a highly complex and dynamic process: it involves cell-cell, cell-molecule, and molecule-molecule interactions.¹⁵⁴ It should be emphasized that NPs are merely discrete carrier units with particle sizes ranging from 1 to 100 nm, while NDSs integrate surface modifications, targeting ligands, and controlled-release modules on this basis to form a fully functional delivery system. The two are related as “components” and “systems”.¹⁵⁵ Current research predominantly focuses on isolated aspects of NDSs’ effects on the ASM, often overlooking the complex network of interactions within the microenvironment. This narrow focus frequently results in suboptimal therapeutic outcomes.¹⁵⁶ Furthermore, enzymes, inflammatory factors, and extracellular matrix components in the ASM can alter the surface properties of NPs, including surface zeta potential, hydrophilicity or hydrophobicity, functional groups, and ligand morphology, thereby affecting their binding efficiency to target cells.¹⁵⁷ Additionally, after intravenous injection, NPs must traverse the endothelial barrier. Larger NPs tend to accumulate in the bloodstream, imposing additional challenges for AS treatment.¹⁵⁸ In addition, compositional and structural changes in the ECM, such as alterations in collagen and glycosaminoglycans, increase the viscosity of the microenvironment, thereby reducing the targeting efficiency of NPs. The physicochemical properties of ASM, such as a weakly acidic environment and a low concentration of oxygen, also affect the stability and release rate of the drug.¹⁵⁹ During systemic circulation, specific enzymes, such as MMP, degrade the carrier materials of NPs, including poly (lactic acid) (PLA), poly (glycolic acid) (PGA), and collagen. Proteolytic cleavage of the delivery vehicle triggers untimely payload liberation, reducing therapeutic in simulated physiological conditions.¹⁶⁰ Furthermore, premature release of NPs loaded with apoptosis-inducing or antiproliferative drugs can result in insufficient cell specificity, causing unintended damage to healthy cells. Therefore, delivery strategies must be optimized to improve the safety and specificity of NDSs.¹⁶¹ This includes exploring more specific biomarkers such as angiogenesis targets, apoptosis/necrosis targets, etc. Developing superior targeted ligands for existing biomarkers, such as VCAM-1 antibodies and RGD peptides. Avoiding the clearance by immune system through invisible coatings and biomimetic nanoparticles. Reducing interference from blood rheology through shape’s optimization, promoting drug’s accumulation at plaque sites through charge’s regulation, microenvironment-responsive design, and targeted ligand’s modification. At the same time, NPs exhibit more complex biotoxicity and toxicological mechanisms compared to traditional drug formulations. Certain NPs, such as metal-based NPs (eg, Ag NPs, Fe₃O₄NPs, and zinc NPs [Zn NPs]), can damage the endothelial barrier, trigger inflammatory responses, and exacerbate the progression of AS. Therefore, when designing NDSs, it is important to consider whether NPs exacerbate the risk of AS.¹⁶²

Although NPs could enhance therapeutic efficacy, difficulties with their safety and efficacy assessment technologies have made it difficult to move them into clinical translation.^163,164 For example, the complex in vivo behavior of nanomaterials, including poor stability, unpredictable biodistribution, accelerated blood clearance, and low permeability through physiological barriers, often leads to diminished efficacy in human’s trials compared to animal’s studies. More importantly, the immunostimulatory and immunosuppressive properties of NPs pose a series of unique challenges. Their interaction with the immune system can sometimes cause adverse reactions, such as inflammation triggered by lipid or surfactant components, which may impair therapeutic efficacy. Conversely, immunosuppressive NPs may inadvertently suppress local immune responses, compromising the body’s ability to fight infection. A comprehensive assessment of immunological safety is critical for successful clinical translation. In addition, insufficient understanding of the pathogenesis of severe cardiovascular diseases, the classification of macrophage’s phenotype in the microenvironment, and the actual clinical relevance of specific cell’s states further limits the application of NDSs based on intracellular reprogramming strategies in clinical practice. Pharmacological and pathological differences in nanoparticle’s behavior, both within cells and between animals’ models and clinical patients, contribute to low clinical translation rates.¹⁶⁵ Finally, the preparation process for nanomaterials is complex and significantly more expensive than conventional treatment’s regimens, which often results in poor patient’s compliance, posing another obstacle to the clinical translation of nanomedicine. Consequently, further research is needed to optimize the performance and conduct comprehensive risk assessments of NDSs. This includes optimizing the targeting of nanoparticles in complex biological environments, establishing standardized methods of toxicity assessment, and addressing factors that limit large-scale production and regulatory approval.

Studies have shown that abnormalities in glycolytic flux during the development of AS accelerate its progression, and promote endothelial dysfunction.^166,167 Glycolysis-mediated has been established as a key regulator of phenotypic switching of macrophage in AS.¹⁶⁸ Enhanced glycolytic activity also drives the proliferation and migration of VSMCs. Consequently, reducing ECs’s dysfunction by inhibiting glycolysis and inhibiting polarization of macrophage and proliferation of VSMCs may stand for a creative therapeutic approach for AS. In addition, dysregulated lipid’s metabolism serves as a pathogenic cornerstone of AS, and it has implications for the design of NDSs’ targeting therapies.¹⁶⁹ To sum up, the complex metabolic lattice in ASM requires further investigation to advance the integration of metabolic reprogramming strategies with AS. More importantly, the metalloproteinase system, comprising MMP and their endogenous tissue inhibitors (TIMPs), exhibits dualistic regulation of advanced plaque pathophysiology. On one hand, their beneficial effects include promoting VSMCs’ growth and survival, thereby contributing to plaque’s stabilization. On the other hand, excessive MMP’s activity and persistent monocyte/macrophage’s accumulation lead to the degradation of extracellular matrix, which promotes plaque’s destabilization and rupture.¹⁷⁰ Accordingly, the controlled regulation of MMP is important for the treatment of AS. Other studies have showed that CXCR1 and CXCR2 chemokine receptors are overexpressed on the surface of various cell types. These cell surface receptors critically mediate the trafficking of leukocyte and NEs to inflamed tissues through chemotactic signaling cascades, thereby amplifying pathological inflammation and accelerating pathogenesis of the disease.¹⁷¹ Therefore, targeting and inhibiting chemokine receptors on cell’s surfaces by optimized NDSs to remodel the cellular phenotype and inhibit inflammatory cell’s recruitment has potential research value for the treatment of AS. Previously, we prepared NDSs with hitchhiking strategy of a circulating monocyte for the treatment of myocardial ischemia/reperfusion injury (MI/RI).¹⁷² Our results demonstrated that these NDSs could efficiently target lesion sites by leveraging the inflammatory homing and plasticity properties of monocytes. Furthermore, the NDSs’ reprogrammed MNs into anti-inflammatory macrophages, thereby exerting therapeutic effects. This approach provides valuable insights for designing reprogramming strategies targeting monocytes in AS, given the similarities in the inflammatory microenvironments of these two diseases. In summary, several critical considerations remain for NDSs-based modulation of ASM, including optimization of its physicochemical characteristics, biological complexity of heterogeneous plaque components, and risk-benefit assessment of potential off-target effects and long-term biocompatibility. However, the enhancement of drug delivery efficiency and the improvement of the inflammatory microenvironment in AS through NDSs represent a highly promising research direction. Our ongoing research focuses on pioneering next-generation NDSs’ engineered to precisely target and remodel the ASM. These efforts aim to develop multifunctional platforms for direct plaque’s regression, and synergistic adjuvants to enhance conventional therapies, ultimately advancing precision medicine for the management of AS.

Conclusion

This review summarizes recent advances in NDSs for enhancing the treatment of AS based on intracellular and extracellular reprogramming strategies. Among intracellular reprogramming approaches, we highlight various NDSs specifically targeting plaque-resident endothelial cells, monocytes, inflammatory macrophages, NEs, and VSMCs within plaque sites. These strategies aim to reverse endothelial-mesenchymal transition and inflammatory states, reprogram the differentiation pathways of monocyte, promote the repolarization of inflammatory macrophages into anti-inflammatory macrophages, suppress infiltration of NEs and NETs-driven inflammatory responses, and inhibit proliferation and migration of VSMCs. In extracellular reprogramming strategies, we focused on regulating inflammatory mediators, ROS, and lipids within the ASM using various NDSs. This achieves extracellular reprogramming by reshaping pro-inflammatory responses into anti-inflammatory ones, scavenging ROS, and remodeling the fate of lipid. More importantly, we examined how optimized multifunctional NDSs simultaneously reprogram multiple cell types or components within plaques of AS to achieve enhanced therapeutic outcomes. Finally, we explored the advantages and potential of NDSs based on intracellular and extracellular reprogramming strategies for improving treatment efficacy of the disease, including restoring endothelial barriers, suppressing inflammation, clearing lipids, and enhancing plaque’s stability. Additionally, we address challenges and critical issues in the clinical translation of novel NDSs. In summary, future research should prioritize elucidating the relationship between structure and function of NDSs while deepening understanding of its microenvironment of the disease. This will guide the design of superior NDSs capable of regulating multiple inflammatory cells and mediators at the disease site, thereby comprehensively improving its microenvironment. This review aims to provide guidance for designing NDSs based on intracellular and extracellular reprogramming strategies.