Nanomaterials to Resolve Atherosclerosis

Subscriber access provided by The University of North Carolina at Chapel Hill Libraries Perspective Nanomaterials to Resolve Atherosclerosis Erica B. Peters, and Melina R. Kibbe ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.0c00281 • Publication Date (Web): 18 May 2020 Downloaded from pubs.acs.org on May 22, 2020 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. 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However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Nanomaterials to Resolve Atherosclerosis Erica B. Peters1,3 and Melina R. Kibbe1-3 Erica B. Peters, Ph.D. and Melina R. Kibbe, M.D. 1Department 3Center of Surgery and 2Department of Biomedical Engineering for Nanotechnology in Drug Delivery University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA KEYWORDS. Nanomaterials, nanomedicine, atherosclerosis, cardiovascular disease, drug delivery ABSTRACT. Cardiovascular disease is the leading cause of death and disability in the world. Atherosclerosis, the buildup of fatty deposits in arteries, is a major underlying cause. Nanomedicine is an emerging treatment option to manage atherosclerotic plaque burden. Nanomaterials are critical to the success of nanomedicine therapies through their ability to enable targeted, controlled drug release. However, in order to move beyond slowing disease progression towards actively resolving atherosclerosis, nanocarriers must be designed to ensure that nanomaterials and therapeutics work in tandem, tailored to respond to the unique physiochemical properties of atherosclerotic lesions. This perspective serves to equip biomaterial scientists with the foundational knowledge needed to meet the challenge of designing ACS Paragon Plus Environment 1 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 2 of 66 such nanomaterials by reviewing the pathophysiology of atherosclerosis and highlighting design parameters that have shown success in targeted therapeutic delivery to atheromatous lesions. 1. Introduction Cardiovascular disease remains the leading cause of death and morbidity worldwide, and its impact is expected to rise with nearly half of all Americans afflicted by 2033, costing the nation over one trillion dollars in health care expenses and lost productivity.1 A major underlying cause of cardiovascular disease is the buildup of fatty deposits in arterial blood vessels, known as atherosclerosis.2,3 As the atherosclerotic plaque develops, blood flow is restricted and causes tissue ischemia, which can lead to coronary artery and peripheral artery disease. Additionally, the plaque can rupture leading to the formation of blood clots that occlude vessels, resulting in a potentially fatal heart attack or stroke. The most common treatments for advanced atherosclerosis include balloon angioplasty with and without stenting, endarterectomy, and bypass grafting. However, these surgical procedures are highly invasive and cause injury to the arterial site, leading to an exaggerated wound healing response—neointimal hyperplasia—that results in restenosis of the artery.4 Thus, there is a critical need for less invasive approaches to manage atherosclerosis. Nanomedicine is a promising treatment option to alleviate plaque burden.5,6 In this approach, biofunctionalized nanoparticles 1-100 µm in size are intravenously administered to target atherosclerotic lesions and locally deliver therapeutics.7 In the past few years, several landmark studies have taken the idea of atherosclerosis nanomedicine and translated it towards a feasible treatment option by engineering novel nanocarriers.8,9,10,11 ACS Paragon Plus Environment 2 Page 3 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Although these initial nanocarrier systems are compelling, significant work remains before atherosclerosis nanomedicine reaches the level of sophistication found in cancer nanomedicine. In particular, future nanocarriers should not only slow atherosclerosis progression, but also treat its underlying causes of impaired lipid metabolism and chronic inflammation, resulting in plaque regression and ideally disease resolution. This combinatorial therapeutic strategy requires the use of nanomaterials that can sense and respond to the atherosclerotic niche, enabling the release of multiple therapeutics in a spatiotemporal manner. Yet, due to the recent appearance of atherosclerosis nanomedicine, most biomaterials scientists are not equipped with the fundamental background in atherosclerosis pathophysiology or nanocarrier design to engineer nanomaterials that interact with the atheromatous environment. This perspective will review these topics as well as discuss emerging research in materials science that have the potential to advance atherosclerosis nanomedicine. 2. Atherosclerosis pathophysiology Atherosclerosis originates from the passive diffusion of circulating low density lipoproteins (LDL) through endothelial junctions into the vessel intima.12,13 The endothelium is activated to remove the accumulating LDL by expressing surface molecules that promote inflammatory cell adhesion.14 Tissue macrophages and endothelial cells produce reactive oxygen species (ROS), which oxidize LDL. Macrophages phagocytose oxidized LDL, transforming into foam cells that recruit vascular smooth muscle cells (VSMC). VSMC secrete matrix metalloproteinases 2 and 9 (MMP2/9) to migrate from the tunica media to the tunica intima where they produce collagen and elastin, forming a fibrous cap around the plaque and preventing its interaction with blood. Necrotic foam cells, lipoproteins, and VSMCs are not efficiently cleared from the plaque, causing additional inflammation and endothelial dysfunction.12,13,15,16 The ensuing sections ACS Paragon Plus Environment 3 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 66 provide more detail on the role of disturbed cholesterol homeostasis and immune imbalance in atherosclerosis. 2.1 Lipid metabolism during atherosclerosis During normal lipid metabolism, dietary fat is broken down by lipases to provide energy and cholesterol for cell repair and hormone synthesis. The resulting triglycerides and cholesterol are absorbed in the small intestine through enterocytes. The enterocytes enable transport of the nonpolar lipids to blood by combining them with apolipoprotein B-48, generating chylomicrons. The chylomicrons enter the blood through the lymphatic system where the triglycerides are hydrolyzed to free fatty acids by lipoprotein lipase and used as energy in muscle cells or stored as fat in adipose cells. The chylomicrons may also break into cholesterol-rich remnants that are taken up by liver hepatocytes through low density lipoprotein receptors (LDLR).17,18 In the liver, triglycerides combine with cholesterol via apolipoprotein B100 (ApoB-100), forming very low density lipoprotein (VLDL). Upon secretion into the bloodstream, the VLDL is hydrolyzed by lipoprotein lipase and hepatic triglyceride lipase, forming LDL. Both VLDL and LDL may acquire additional cholesteryl esters in the blood from high density lipoproteins (HDL) through cholesteryl ester transfer protein (CETP, Figure 1).18 Peripheral cells internalize LDL through LDLR-mediated endocytosis, releasing cholesterol from LDL to be utilized by the cell. The rise in free intracellular cholesterol leads to reduced LDLR expression, preventing further LDL uptake. As a result, the excess LDL remains in the circulation, passively diffusing between interendothelial junctions of blood vessels and amassing in the subendothelial space. The sites of greatest LDL accumulation are found in areas of disturbed blood flow, including arterial branch points and curved regions that do not receive the ACS Paragon Plus Environment 4 Page 5 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering atheroprotective benefits of laminar flow, leading to increased endothelial permeability.12,19,13 Additionally, oscillatory shear stress from disturbed blood flow activates the YAP/TAZ mechanotransduction signaling pathway in endothelial cells, leading to increased inflammatory gene expression.20 LDLs are retained in the arterial wall through ionic binding interactions of the positively charged arginine and lysine residues with negatively charged proteoglycans in the extracellular matrix of resident VSMC.21,22 In an effort to remove the accumulating LDL, the endothelium increases surface expression of vascular endothelial adhesion molecule (VCAM) and intracellular adhesion molecule (ICAM) as well as chemokine production to attract circulating immune cells. Additionally, the very high levels of cholesterol in the blood promotes an increase in circulating neutrophils via granulopoiesis in the bone marrow.23 Upon entering the vessel intima, neutrophils release the cationic azurocidin granule protein to attract monocytes and further increase endothelial VCAM and ICAM expression.24 The neutrophils also attempt to clear LDL by secreting myeloperoxidase, lipoxygenase, and ROS which oxidize the LDLs (oxLDL).15 The oxLDL reengages with endothelial cells through the lectin-like oxLDL receptor (LOX-1), activating NF-κB and AP-1 pathways that lead to increased IL-8, VCAM, and ICAM expression that further enhance monocyte infiltration. oxLDL can induce ROS from endothelial cells through NADPH oxidases (Nox) and promote cell death.25 oxLDL is also taken up by macrophages through scavenger receptors. Yet, rising intracellular levels of cholesterol do not initiate a negative feedback loop for scavenger receptor expression as with LDLR, allowing the macrophages to continue engulfing oxLDL until they transform into lipid-filled cells that have a foamy appearance, known as foam cells.19 The foam cells are unable to catabolize cholesterol— ACS Paragon Plus Environment 5 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 6 of 66 a process unique to liver hepatocytes—and will eventually die from the ensuing metabolic stress.26 To restore lipid homeostasis, reverse cholesterol transport is initiated through the release of cholesterol from macrophage foam cells to HDLs, which transport cholesterol to hepatocytes for final catabolism to bile acids and subsequent fecal excretion.26,27 Cholesterol efflux occurs through passive or active mechanisms. Passive mechanisms do not require adenosine triphosphate (ATP) for energy and are driven by the concentration gradient of free cholesterol. Passive cholesterol efflux from macrophages occurs via aqueous diffusion or scavenger receptor class B type 1 (SR-BI) pathways. SR-BI can “activate” free cholesterol to increase their efflux by modifying the domains to be more prone to oxidation by cholesterol oxidase, which enhances cholesterol desorption from the plasma membrane.28 Upon release, the cholesterol collides with an acceptor such as HDL and is quickly absorbed. The absorbed cholesterol is esterified in HDL by lecithin-cholesterol acyltransferase.26 Because cholesteryl esters are more hydrophobic than free cholesterol, they are retained in the hydrophobic core of HDL.29 Apolipoprotein E (ApoE) enhances cholesterol ester expansion in the HDL core.30 Active cholesterol efflux occurs through ATP-binding cassette A1 or G1 (ABCA1/G1), which are expressed on endosomes and use ATP to shuttle free cholesterol from the endoplasmic reticulum to the cell membrane. ABCA1 and ABCG1 differ in that ABCA1 can assist in HDL synthesis by transporting both free cholesterol and phospholipids to ApoA1 lipoproteins.31 Normally, reverse cholesterol transport processes restore cholesterol homeostasis. However, the continued oxidation of LDLs in atherosclerotic lesions leads to a chronic inflammatory response that weakens the ability of macrophage foam cells to engage in cholesterol efflux. For example, neutrophil myeloperoxidase chlorinates amino acid residues on ApoA1, impairing its ability to ACS Paragon Plus Environment 6 Page 7 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering act as a receptor of cholesterol from ABCA1.32 As well, excessive oxLDL uptake leads to cholesterol crystal production that destabilizes lysosomes and causes stress to the endoplasmic reticulum and mitochondria.16 Accordingly, mitochondrial ATP production is diminished, decreasing both ABCA1 and ABCG1-mediated cholesterol efflux. In addition, the hypoxic microenvironment within plaque significantly reduces ABCA1-mediated cholesterol efflux by decreasing its protein expression and altering its cellular localization.33 2.2 The role of the immune system in atherosclerosis progression To understand the interplay of lipid accumulation and the inflammatory response in atherosclerosis, it is helpful to conceptualize LDL-mediated activation of the innate immune response as analogous to pathogen-associated mechanisms. In fact, they are very similar. The same key players are involved: monocytes, neutrophils, macrophages, dendritic cells, B- and Tlymphocytes (Figure 2).15 Lipoproteins in LDL mimic damage-associated molecular patterns (DAMPs) that are recognized by pattern recognition receptors (PRRs) on immune cells.13 For instance, oxLDL interactions with PRR CD36 on macrophages triggers additional PRR signaling through toll-like receptor 2 and 4 (TLR2/4).34 This results in NF-κB activation that increases pro-inflammatory chemokine and cytokine production of tumor necrosis factor (TNF)-α, IL-1β, and IL-6, which recruit inflammatory cells to the plaque. Additionally, excess cholesterol in foam cells accumulates in the cell membrane to form an altered membrane structure with new lipid rafts that activate TLR4, leading to increased NF-kB signaling.35 Further, DAMPS are produced by stressed macrophage foam cells that are undergoing necrosis, activating neighboring cells and continuing the inflammatory response.15,24 ACS Paragon Plus Environment 7 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 8 of 66 Dendritic cells serve as the link between the innate and adaptive immune responses in atherosclerosis. Dendritic cells originate from lymphatic tissue and circulating monocytes that differentiate in the atherosclerotic lesion. Cellular debris in the necrotic core drives dendritic cells to an atherogenic phenotype by activating its TLR7 and TLR9 receptors.15,36,37 Upon activation, dendritic cells interact with CD4+ T-cells, resulting in increased IFN-γ levels that shifts T-cell phenotype away from tolerance to produce TNF-related apoptosis inducing ligand (TRAIL) that kills vascular VSMCs.38 Additionally, through CCL17 expression, dendritic cells recruit T cells to the plaque, and provide instructional cues through antigen priming that halts proliferation of atheroprotective Treg cells and stimulates atherogenic CD4+ T cells.39,40 B cells can have either pro- or anti-atherogenic effects depending on the subtype and antibody produced. While the exact mechanisms are not yet know, B1 cell production of natural IgM may reduce atherosclerosis progression by neutralizing oxLDL inflammatory properties and promoting efferocytosis—the clearance of dead cells by phagocytosis.41 Epigenetic and metabolic memory exacerbates immune activation during atherosclerosis. Monocytes in hematopoietic reservoirs and circulation are primed in patients with atherosclerosis from milieu in the systemic circulation caused by chronic inflammation and impaired cholesterol efflux.16,42 Thus, monocyte epigenetic memory is altered before recruitment to plaque lesions. Consequently, the primed monocyte response upon migration into the subendothelial space is stronger than in non-atherosclerotic patient monocytes exposed to the same stimuli, with increased production of pro-inflammatory cytokines.43,44 In an attempt to partially resolve the inflammation, VSMC are recruited to the plaque lesion from the underlying tunica media and produce collagen to form a fibrous cap—analogous to a protective scar—that prevents interactions of the plaque with blood. However, as the inflammation continues, the VSMC are ACS Paragon Plus Environment 8 Page 9 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering killed by cytotoxic T-cells. The corresponding decrease in collagen production, combined with the increase in matrix metalloproteinases from inflammatory cells, thins the protective fibrous cap. The result is vulnerable plaque that eventually ruptures to cause potentially fatal outcomes. 3. Design considerations for targeting nanoparticles to atherosclerotic plaque While an understanding of atherosclerosis pathophysiology provides numerous opportunities for therapeutic intervention, translational success will ultimately depend on the ability to restrict therapeutic effects to the plaque site, minimizing damage to surrounding tissues and organs. For example, systemic administration of synthetic liver X receptor agonists can reduce atherosclerotic plaque burden by enhancing cholesterol efflux yet causes liver steatosis.45,46 Targeted nanodrug delivery systems can modify in vivo drug kinetics and enable the drug to reach its diseased organ or cell of interest, reducing adverse effects and increasing both safety and efficacy. Even so, therapeutic efficacy is limited unless the physiological barriers for nanoparticle delivery to atherosclerotic sites are overcome. These barriers include rapid clearance through the reticuloendothelial systems or mononuclear phagocytic system, as well as the need for margination of the nanoparticle from the blood stream to the vessel wall and subsequent transendothelial migration to the plaque site.7,47,48 In this section, we will discuss design parameters that have proven successful for targeting nanodrug delivery systems to atherosclerotic plaque. To date, six classes of nanomaterials have been used in atherosclerosis nanomedicine: micelles, liposomes, polymeric nanoparticles, dendrimers, carbon nanotubes, and crystalline metals.47 In this perspective, we will focus on the bioresorbable classes of nanomaterials used for therapeutic delivery rather than diagnostic imaging of atherosclerotic lesions, which is covered in other reviews. 6,49,50 3.1 Nanoparticle size, shape, charge, and hydrophobicity ACS Paragon Plus Environment 9 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 10 of 66 The physical properties of nanoparticles are critical for avoiding rapid clearance upon intravenous injection. For example, nanoparticles less than 10 nm in diameter are rapidly cleared by passing through fenestrae on the glomerular capillary wall.7,51,52 Conversely, nanoparticles greater than 200 nm in diameter are restricted from escaping the liver and spleen by way of vasculature fenestrae. The trapped nanoparticles are quickly phagocytosed by resident macrophages, preventing their ability to re-enter the circulation and reach the target organ.53 During their circulation in the blood, nanoparticles are susceptible to opsonin protein adsorption—immunoglobulins and C3, C4, C5 complement proteins that facilitate nanoparticle recognition by macrophages for phagocytosis.54 Nanomaterials that are charged or have predominantly hydrophobic character more readily absorb opsonin proteins.55 Coating the nanoparticle with a hydrophilic polymer such as poly(ethylene glycol) (PEG) or zwitterionic polymers forms a hydrated shell that decreases opsonization, consequently reducing macrophage uptake and extending the nanocarrier circulation time.54,56 Regarding size, cylindrical or rod-shaped nanoparticles may offer benefits for targeting the diseased vasculature over spherical nanoparticles. For instance, elongated, worm-shaped polystyrene particles with aspect ratios of >20 significantly reduced macrophage phagocytic uptake over a period of 22 hours in comparison to spherical particles.57 This decrease in phagocytosis was attributed to low curvature along the length of the particle, inhibiting macrophage attachment.57 Similarly, worm-shaped filomicelles, derived from amphiphilic PEGpolyethylethylene or PEG-polycaprolactone nanoparticles with aspect ratios of 20-83, significantly reduced macrophage uptake in comparison to their spherical counterparts.58 Further, the filomicelles persisted in circulation one week after injection in comparison to spherical polymersomes, which were cleared within two days.58 Longer circulation times due to ACS Paragon Plus Environment 10 Page 11 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering reduced clearance from the mononuclear phagocytic system has the potential to improve nanoparticle targeting to the diseased site.59 An example of nanoparticles with high aspect ratios offering superior targeting to sites of cardiovascular disease was demonstrated by self-assembled peptide amphiphile nanofibers containing a collagen IV-binding peptide.60 The nanofibers showed superior binding to sites of vasculature injury in comparison to peptide amphiphile nanospheres of similar diameter and identical targeting sequence.60 Nanoparticle material properties are an emerging parameter for biodistribution and targeting, particularly elasticity and stiffness.59 Softer particles increase circulation time, as demonstrated in a study using 200-nm diameter PEG-hydrogels with soft (10 kPa) nanoparticles retained in the blood at concentrations approximately 35% higher than stiff (3000 kPa) nanoparticles two hours after injection.61 Additionally, zwitterionic nanogels composed of poly(carboxybetaine) displayed increased circulation time with decreasing gel stiffness.62 Specifically, nanogels with a bulk modulus of 180 kPa had a two-fold increase in circulation half-life—approximately 20 hours—in comparison to stiffer nanogels with a bulk modulus of 1350 kPa, as well as a significant decrease in spleen accumulation.62 The reason for the greater circulation time observed in soft versus stiff nanomaterials is not yet known, however, it has been proposed that soft nanomaterials have reduced phagocytic cell uptake due to their deformation in response to cytoskeletal forces exerted by the macrophages, preventing firm attachment.59 Additionally, the more elastic nanoparticles may be able to squeeze through venous slits in spleen in contrast to hard nanoparticles, which remain trapped and are subsequently cleared by resident macrophages.62 Despite the compelling evidence for nanoparticle elasticity affecting nanoparticle biodistribution, several caveats exist that prevent recommendations for the optimal elasticity to target ACS Paragon Plus Environment 11 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 12 of 66 atherosclerotic plaque. For example, a study showing intermediate stiffnesses of 35 and 136 kPa nanospheres formed from 170 nm diameter N,N-diethyl acrylamide and 2-hydroxyethyl methacrylate hydrogels have maximum phagocytic uptake..63 Also, the composition of the material must be considered in addition to the elasticity as phagocytic and biodistribution properties differ in polymeric nanoparticles of similar elasticity, yet unique chemical composition.61,62,63 Further, polymeric hydrogels have very different mechanical properties than self-assembled nanostructures, preventing extrapolation of results to micelles and peptide amphiphiles. In summary, systematic investigations are needed to understand how the physiochemical parameters of size, shape, hydrophobicity, and elasticity work in tandem, within an in vivo model of atherosclerosis, to determine the optimal conditions for plaque-targeting nanocarriers. 3.2 Passive vs. active targeting To date, successful strategies for localizing nanoparticles to atherosclerotic plaque utilize passive or active targeting.48,50 Passive targeting relies on the body’s unique biophysical characteristics of the disease to localize nanoparticles. In the case of atherosclerosis, passive targeting utilizes the enhanced vascular permeability and retention (EPR) effect on vessel endothelium that is caused by the chronic, local inflammation, and also found in plaque microvasculature.64,65 Rapid angiogenesis from the vasa vasorum during plaque progression gives rise to these microvessels, preventing pericyte recruitment and the associated vessel stability.5,66 In late-stage atherosclerosis, passive targeting strategies take advantage of the increased blood velocity at narrowed points of highly occluded vessels, which leads to higher local shear stress.67 ACS Paragon Plus Environment 12 Page 13 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering In contrast, active targeting alters the surface chemistry of nanoparticles to incorporate ligands to biochemical cues overexpressed or unique to the plaque site.48 These molecules include proteins expressed on inflamed endothelium, macrophage foam cells, and oxidized lipids that are targeted by way of antibodies, oligonucleotides, and peptides.48,50,68,69 Examples of passive and active targeting of nanoparticles to atherosclerotic plaque are discussed below and summarized in Tables 1-2 and Figure 3. 3.2.1 Passively targeting nanoparticles to atherosclerotic plaque The first evidence of using EPR for nanoparticle targeting to atherosclerotic lesions was demonstrated by the Langer group in 2014.64 The group utilized both in vitro and in vivo models to show that enhanced endothelium permeability led to increased accumulation of 70 nm lipidpolymer hybrid nanospheres comprised of gold and poly(lactic-co-glycolic acid) (PLGA) cores, coated with PEGylated lipid 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC). Specifically, a microfluidic chip model of confluent human endothelial cells—treated with TNFα and shear stress to enhance permeability—showed significantly greater nanoparticle accumulation when compared to untreated controls. This observation was corroborated in vivo by intravenously injecting Cy5.5 dye-labeled nanoparticles into a rabbit atherosclerosis model. Using near infrared fluorescent imaging, the atherosclerotic aortas showed significant nanoparticle accumulation in contrast to aortas from normal rabbits (Figure 3A). Moreover, TEM imaging showed the nanoparticles localized to subcellular regions of macrophages that were in close proximity to luminal endothelium and plaque microvessels.64 Coating nanoparticles with DNA also promotes uptake by aortic macrophages, as shown by Zhang et al. using poly(thymine)-coated iron oxide nanoparticles.70 Despite these promising results that use the EPR effect for targeting plaque, an important limitation is the long circulation time required ACS Paragon Plus Environment 13 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 14 of 66 to accumulate nanoparticles at therapeutic levels. Also, the targeting is not specific, as the spleen and liver contain large fenestrae that also allow nanoparticle accumulation.65 In 2012 Ingber’s group demonstrated that shear stress could be used to passively target vulnerable plaques in advanced atherosclerosis.67 The shear stress near stenotic blood vessels is >1000 dyne/cm2, over an order of magnitude higher than ~70 dyne/cm2 in normal blood vessels.67,71,72 The shear-targeting nanoparticles are comprised of PLGA nanospheres, 180 ± 70 nm in diameter. Through a spray-drying technique, the PLGA nanoparticles are deposited as 1-5 µm diameter microparticles—a similar size as platelets. These microparticles break apart to constituent nanoparticles when exposed to stenotic shear stress, which exerts forces on the microparticles that exceeds the noncovalent attractive forces holding the nanoparticles together (Figure 3B). Using a mouse arterial thrombus model, the PLGA microparticles localized near the occluded vessels a few minutes after injection. Further, when the nanoparticles were coated with tissue plasminogen activator—an enzyme that breaks down fibrin clots—the occlusion time for the blood vessel was significantly reduced.67 This approach, however, is limited to advanced plaques with thrombosis, and whether therapeutic released at the stenosis site can infiltrate into the plaque to promote regression remains unknown. 3.2.2 Actively targeting nanoparticles to atherosclerotic plaque Active targeting overcomes limitations of passive targeting by localizing nanoparticles to the plaque in a shorter amount of time, with greater specificity. Also, due to the unique molecular profiles displayed at each stage, atherosclerosis can be targeted at specific time courses of disease progression.48,49,50 At present, nanoparticles containing ligands for endothelial cells, macrophages, monocytes, platelets, extracellular matrix proteins collagen and fibrin, as well as ACS Paragon Plus Environment 14 Page 15 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering oxLDL have successfully targeted atherosclerotic lesions. 9,11,73,74,75,76,77 The majority of studies utilize peptides for guiding nanoparticles to plaque. Peptides have several key benefits that make it an attractive alternative to antibodies. Peptides are smaller than antibodies, enabling a higher density of peptides to attach to nanoparticles, thus increasing the chances for successful binding to the target ligand or structure. Peptides also fit into shallow and hydrophobic binding pockets more effectively than antibodies. Additionally, the targeting peptides may also impart therapeutic effects by competing for binding sites with molecules that drive atherosclerosis progression.50 As a result of lipid accumulation, the endothelium surrounding the developing plaque increases expression of inflammatory biomarkers. The biomarkers include leukocyte adhesion receptors E-selectin, ICAM-1, VCAM-1, and platelet endothelial cell adhesion molecule-1 (PECAM1).48,78 Developing nanoparticles that target these adhesion receptors may provide therapeutic benefits by competing with leukocyte binding, thus reducing leukocyte extravasation to atheroma, and accordingly, the local inflammatory response. In 2014, the Tirrell group demonstrated the effectiveness of actively targeting nanoparticles to endothelium through spherical, PEGylated self-assembled micelles 17 ± 2 nm in diameter, containing a high affinity peptide for VCAM-1 receptor, VHPKQHR. The VCAM-1 targeting micelles bound to earlyand mid-stage atherosclerotic plaques in ApoE-/- mice within 24 hours, and displayed significantly greater binding than PEG micelle controls.79 Similarly, Calin et al. showed that attaching VHPKQHR to PEGylated liposomes 128 ± 19 nm in diameter significantly enhanced binding to aortic plaque in ApoE-/- mice (Figure 4A).73 Still, shear stress caused by blood flow produces drag forces that limit spherical nanoparticle binding to vascular endothelium.80 Additionally, chronic shear stress induces actin stress fibers in endothelial cells that lead to ACS Paragon Plus Environment 15 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 16 of 66 reduced nanocarrier uptake.81 Tethering ligands to nanoparticles for multiple, rather than single, leukocyte adhesion receptors may address these shortcomings by better mimicking leukocyte binding dynamics with engagement of multiple endothelial receptors. For example, polystyrene nanoparticles containing antibodies against ICAM-1, PECAM-1, and VCAM-1 (235 ± 6.8 nm diameter) showed enhanced endothelial internalization in contrast to nanoparticles containing ICAM-1 and PECAM-1 antibodies (272 ± 17 nm diameter), potentially due to the activation of two distinct uptake pathways for clathrin-mediated endocytosis (VCAM-1) and cell adhesion molecule (CAM)-mediated endocytosis (ICAM-1 and PECAM-1).82 Given their role as key propagators of atherosclerosis, macrophages are a common target for localizing nanoparticles to atheroma.13 In 2017, Beldman and colleagues hypothesized that macrophage binding to hyaluronan in the matrix of atherosclerotic lesions could be utilized as a targeting strategy.83,84 The group engineered hyaluronan nanoparticles 90 nm in diameter that preferentially targeted activated macrophages in vitro and in vivo, with significantly greater nanoparticle uptake seen in plaque macrophages than bone marrow or spleen macrophages in ApoE-/- mice. Additionally, the hyaluronan nanoparticles showed better uptake in early vs. late atherosclerotic lesions, and stabilized plaque by a 30% decrease in macrophage number and a 30-40% increase in collagen content.84 A possible mechanism for these effects is that the hyaluronan nanoparticles compete with oxLDL for binding to macrophages, reducing the number of apoptotic/necrotic foam cells. HDL mimics are also a popular choice for targeting plaque macrophages due to the binding interactions of ApoA1—the major component of HDL—with ABCA1 transporter, expressed on the surface of foam cells.11,31,85,86,87 Moreover, nanoparticles containing ApoA1-mimetic peptides may impart therapeutic effects by decreasing plasma cholesterol levels.86,87 Recently, ACS Paragon Plus Environment 16 Page 17 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering we showed that peptide amphiphiles containing 4F, an ApoA1 peptide mimic, can self-assemble into high-aspect ratio nanofibers and target plaque within 24 hours of intravenous injection in low density lipoprotein receptor knockout (LDLR KO) mice (Figure 4B).11,88 In addition to macrophages, platelets also play an important role in atherosclerosis. Platelets bind to activated endothelium and exposed basement membrane proteins collagen, fibrin, and fibronectin.89 As well, platelets recruit inflammatory cells, including monocytes and T cells, to the developing plaque.90 Currently, the most promising technique for targeting platelets in atherosclerotic lesions involves coating nanoparticles with the platelet cell membrane.76,91 In this approach, platelets are isolated from peripheral blood via differential centrifugation before undergoing a series of freeze/thaw cycles. The resulting platelet membranes are coated onto polymeric nanoparticles through sonication.76 In 2018, the Zhang group demonstrated the utility of this approach using platelet membrane-coated PLGA nanoparticles to target atherosclerotic lesions in ApoE-/- mice (Figure 5A).76 The multivalent binding interactions of platelet membranes was confirmed by colocalization of the nanoparticles to activated endothelium, macrophages, and collagen type IV. Furthermore, the platelet-PLGA nanoparticles were shown to bind to activated endothelium in atherosclerosis-prone regions, indicating their potential to target atherosclerosis at early and advanced disease stages.76 In 2019, Song et al. extended the platelet-PLGA nanocarrier technology to deliver rapamycin, an immunosuppressant, to slow plaque progression.91 Still, platelet membrane coatings have several limitations that may hinder clinical translation including non-uniform coating, batch to batch variability, scalability, and the potential to denature endogenous membrane proteins which could elicit an autoimmune response.92 ACS Paragon Plus Environment 17 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 18 of 66 In atheroprone regions of disturbed flow, the extracellular matrix of the basement membrane undergoes remodeling, shifting the composition from collagen IV and laminin towards fibronectin, fibrinogen, and thrombospondin.93,94 Targeting these exposed vascular basement membrane proteins has shown promise in guiding nanoparticles to atheroma.9,10,77 For instance, in 2009 Tirrell’s group developed multifunctional micelles, self-assembled from lipopeptides containing CREKA, a fibrin-targeting peptide.77 The CREKA micelles showed approximately 40-fold increase in binding to the aorta of atherosclerotic ApoE-/- mice in comparison to nontargeted micelles, based on fluorescence intensity (Figure 5B). The binding was observed near fibrous caps of plaque, underneath the endothelial layer, which has the highest rupture risk.77 As well, in 2015, research from Farokhzad and Tabas showed that a collagen IV-targeting peptide, KLWVLPK, enhanced PLGA-PEG nanoparticles targeting to atherosclerotic lesions by two-fold in LDLR KO mice.9 However, this targeting strategy may have disadvantages in comparison to directly targeting plaque components, as the nanocarrier cargo must diffuse from the vascular endothelium to its drug targets within atheroma. In summary, several nanomaterial formulations are capable of homing to atherosclerotic plaque in animal models. These nanomaterials employ both passive and active targeting, with a broad range of biological targets in the atherosclerotic lesion. While the results are promising, we are limited in accurate comparisons of targeting approaches due to the lack of consistency in animal models, as well as dosage and circulation time. For active targeting, future studies should incorporate more quantitative characterization of nanoparticle binding to target ligands through techniques such as surface plasmon resonance. Comparing the trans-endothelial penetrating efficiency of nanocarriers would also aid in optimizing drug delivery. A recent study by Sindhwani et al. indicates passive nanoparticle transport to tumors relies on active extravasation ACS Paragon Plus Environment 18 Page 19 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering through endothelium rather than passive diffusion via the EPR effect. These findings may apply to nanoparticle transport in atherosclerotic lesions.95 Further analysis is also needed on nanoparticle biodistribution, pharmacokinetics, long-term safety, and varying dosages to determine the therapeutic index. 4. Nanomaterials to treat atherosclerosis At present, several types of nanocarriers can deliver therapeutic payloads to atherosclerotic lesions, resulting in decreased lesion size or enhanced stability of vulnerable plaques.6,96 Nanomaterial design has evolved to meet the unique needs of each therapeutic approach, employing bioresponsive systems that sense and respond to exogenous triggers as well as endogenous stimuli within the atherosclerotic niche.97 Additionally, nanocarriers can be tailored to target the different stages of atherosclerosis, with therapeutic approaches ranging from preventative measures, such as restoring vascular integrity to reduce LDL penetration, to clearing advanced plaques using thermal ablation.49,98 Targeting shear stress forces on the vascular lumen is an example of treatments for early stages of atherosclerosis. These treatments examine molecular mechanisms in endothelial cells that are activated in response to laminar or disturbed blood flow, such as endothelial transcription factor EB (TFEB) and focal TLR4, respectively, to reduce endothelial activation and corresponding monocyte attachment. 99,100 An emerging approach is to instruct the diseased atherosclerotic niche to self-repair.9,101,102 The appeal of this method is the possibility of requiring fewer injections to reach a longer-lasting therapeutic effect, and protection against future challenges from cholesterol buildup. In this section, we summarize those nanocarrier designs proven successful in treating atherosclerosis by delivering therapeutics that address underlying causes of impaired lipid metabolism and chronic inflammation through instructing niche cells towards self-repair. ACS Paragon Plus Environment 19 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 20 of 66 4.1 Regulating lipid metabolism Cholesterol content is 20-fold higher in atherosclerotic vs. healthy aortas, which increases the likelihood of cardiovascular events.103 To reduce the amount of cholesterol in vessels, macrophage foam cells release stored cholesterol into the circulation, which bind to HDL for transport back to the liver for fecal elimination (Figure 1).26,27 This reverse cholesterol transport process is compromised during atherosclerosis due to reduced cholesterol efflux from foam cells.16,32,33 Given this role of cholesterol in atherogenesis, a key focus in nanocarrier design has been to deliver therapeutics that regulate lipid metabolism. Although treatments such as statins or proprotein convertase subtilisin-kexin type 9 (PCKS9) inhibitor therapies are a common approach to manage lipid blood levels, they do not promote the reversal of atherosclerosis by clearing existing plaque lesions.104,105 For example, statins are hydroxymethylglutaryl-coenzyme A (HMG-CoA) inhibitors, which prevent the liver from producing cholesterol and instead allow more lipid absorption from the blood stream. However, statins reduce cholesterol efflux in macrophages by decreasing ABCA1 and ABCG1 expression, and thus are not ideal for reducing plaque burden.106,107 Therapeutics targeting Liver X Receptors α and β (LXRα/β) enhance cholesterol efflux from foam cells through increased expression of ABCA1 and ABCG1 cholesterol transporters.103 LXR signaling also mediates potent anti-inflammatory effects by transrepressing the TLR4-LPS signaling pathway.108 Yet, LXR agonists have different effects in different tissues. Namely, plaque macrophages are activated by LXR agonists to increase ABCA1/G1 expression for cholesterol efflux to lipoprotein acceptors, ApoA1 and HDL, and LDLR expression is reduced via targeted degradation. In contrast, liver hepatocytes do not pump free intracellular cholesterol back out to the circulation. Instead, cholesterol is processed for conversion to bile acids and subsequent ACS Paragon Plus Environment 20 Page 21 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering elimination in feces, or triglyceride production and secretion is increased via lipogenesis pathways. These parallel, yet antagonistic effects on hepatocyte cholesterol levels by LXR agonists have prevented their translation to the clinic due to concerns of hyperlipidemia. Encapsulating LXR agonists in nanoparticles that target plaque reduces the risk of liver toxicity. For instance, in 2017 the Farokhzad group demonstrated that collagen IV-targeting PEG-PLA lipid-polymer nanoparticles delivered LXR agonist GW3965 to plaque of LDLR KO mice, increasing macrophage ABCA1 gene expression while reducing macrophage content by 30% in comparison to GW3965 alone after five weeks of treatment (Table 2, Figure 6).10 Further the targeted nanocarriers did not cause significant increases in plasma or hepatic triglyceride and cholesterol levels in comparison to free GW3965.10 As the composition of nanomaterials have shown potential for anti-inflammatory effects, a limitation of this study is the lack of a lipidpolymer nanoparticle control, which would clarify whether the reduction in macrophage number was due to the LXR agonist or the nanomaterial alone.8 In 2018, Guo et al. also demonstrated the potential of targeted nanocarriers to retrofit LXR agonists through synthetic HDL nanodiscs containing an ApoA1-mimetic peptide 22A to deliver T0901317 to the plaque of ApoE-/- mice.109 These nanoparticles increased ABCA1 and ABCG1 gene expression within atherosclerotic plaques to a similar extent as T0901317 alone, four hours after injection. After six weeks of HDL-LXR agonist treatment, a 40.8% reduction in plaque area was seen in the aortic root in comparison to saline controls. No significant plaque reduction was found using the LXR agonist alone. Additionally, unlike the LXR agonist, the HDL-LXR agonist nanoparticles did not increase triglyceride levels in comparison to saline controls.109 Recently, we established that peptide amphiphiles containing the ApoA1-mimetic peptide 4F could self-assemble into nanofibers that encapsulated LXR agonist GW3965 within the ACS Paragon Plus Environment 21 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 66 hydrophobic core (ApoA1-LXR PA).11,88 These nanofibers selectively targeted aortic plaque and demonstrated potential to promote cholesterol efflux from macrophages in vitro.11 After eight weeks of treatment, the ApoA1-LXR PA nanofibers significantly reduced plaque burden in comparison to saline controls to a similar extent as GW3965. The ApoA1-LXR PAs decreased liver toxicity in comparison to LXR agonist alone by no increase in aspartate aminotransferase blood levels, an indicator of liver toxicity, in comparison to saline treatment in male mice.88 Taken together, these findings show the use of atheroma-targeting nanomaterials substantially improve the safety and therapeutic outcomes for restoring lipid metabolism in foam cells. 4.2 Resolving local inflammation Lipid metabolism and inflammation during atherosclerosis are intricately intertwined, as oxLDL is treated as a foreign substance, triggering the innate and adaptive immune responses to clear the invading pathogen. However, unlike invading pathogens, the oxLDL is never cleared and instead accumulates overtime. This results in ongoing inflammation that intensifies mitochondrial stress, reduces ATP production, and thus reduces ABCA1/G1 expression, leading to impaired cholesterol efflux. ROS and protease secretion are also increased, thinning fibrous caps that eventually leads to plaque rupture.110,111 A hypothesis gaining momentum among atherosclerosis researchers is interrupting the inflammatory response at early checkpoints of the immune cascade can more quickly cause resolution to occur.112,113 In support of this hypothesis, delivery of pro-resolving lipids and protein mediators such as lipoxins, and IL-10 to atherosclerotic lesions have shown benefits of reducing inflammatory cell accumulation and promoting their migration out of plaque, increasing phagocytosis of dead cells within the necrotic core, and repairing damaged tissue.114,115 Nonetheless, delivery of pro-resolving mediators is limited by a short half-life, risk for broad ACS Paragon Plus Environment 22 Page 23 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering immunosuppression, and the need for continuous injections to reach therapeutic levels.116 These limitations may be overcome using targeted, nanoparticle delivery aimed at re-educating atheroma macrophages and dendritic cells—regulators of innate and adaptive immunity, respectively—to dampen their inflammatory activation in response to oxLDL. For example, in 2016 Nakashiro et al. succeeded in shifting atheroma macrophages towards a non-inflammatory M2 phenotype via pioglitazone-loaded PLGA nanoparticles.102 Pioglitazone, a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, binds to PPARγ and forms a heterodimer with nuclear receptor retinoid X receptor to regulate downstream gene expression of monocyte differentiation towards alternative M2 macrophages.117 Yet, systemic administration of pioglitazone using oral delivery is limited by increases in kidney epithelial Na+ channels, which leads to water retention that exacerbates congestive heart failure. Incorporating pioglitazone into PLGA nanoparticles and administering via intravenous injection improved targeted delivery as circulating monocytes phagocytosed the nanoparticles prior to migrating to the plaque and undergoing macrophage differentiation (Table 2). A single intravenous dose of the pioglitazone nanoparticles to ApoE-/- mice skewed circulating monocytes towards a less inflammatory phenotype, without affecting overall monocyte count, by two days after treatment. After four weeks of treatment, the pioglitazone nanoparticles decreased macrophage accumulation in the plaque and aortic root, resulting in a lower number of buried fibrous caps—a risk factor for plaque destabilization and rupture in contrast to free drug and nanoparticle controls. As well, macrophage proteinase activity was suppressed and gene expression of M2 polarization markers Arg1 and IL-10 increased. Lastly, the authors found no increase in expression of kidney epithelial Na+ channels after 4 weeks of treatment, in contrast to the orallyadministered drug.102 ACS Paragon Plus Environment 23 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 66 Fredman et al. demonstrated another example of promoting resolution by the delivery of proresolving peptide Ac2-26 to plaque macrophages via collagen IV-targeting diblock PLGA-PEG nanoparticles (Table 2). Ac2-26 is derived from annexin A1 protein and binds to N-formyl peptide receptor 2 (FPR2/ALX) on monocytes to dampen the inflammatory response to oxLDLs. Due to its rapid clearance and risk of broad immunosuppression, the authors proposed that targeted delivery of Ac2-26 via nanocarriers could decrease rapid clearance and the risk for broad immunosuppression. After five weeks of treatment in LDLR KO mice, the collagen IVAc2-26 nanoparticles reduced macrophage ROS in response to oxLDL and collagenase activity while restoring efferocytosis function to clear necrotic debris, leading to an 80% reduction of plaque in the brachiocephalic arteries in comparison to collagen IV-scrambled Ac2-26 nanoparticle controls.9 Dendritic cells are also key to regulating the atherosclerotic inflammatory response as they uptake oxLDL and present ApoB-100 antigens to T cells. The antigen-specific response can promote inflammation by activating TH1 cells. Yet, if dendritic cell maturation is suppressed through decreased CD80/CD86 expression, their contact with naïve T cells will prompt differentiation towards Treg cells that reduce inflammation.118,119 The Scott group has pioneered nanoparticle technologies that promote dendritic cell-mediated resolution of atherosclerosis.101,120 In 2019 they developed a nanocarrier system that targets dendritic cells within plaque for co-delivery of immunomodulator 1,25-dihydroxyvitamin D3 (aVD) and ApoB100-derived antigen peptide P210 (Table 2, Figure 7). The nanocarrier system consisted of PEG-b-poly(propylene sulfide) (PPS) polymersomes containing a CD11c-derived peptide, P-D2, that preferentially binds to the dendritic cell surface. aVD induces tolerogenic dendritic cells by reducing NF-kB, MHC-II, and CD80/CD86 expression.118 Following eight weeks of treatment ACS Paragon Plus Environment 24 Page 25 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering in ApoE-/- mice, the P-D2/aVD/P210 nanoparticles significantly increased Foxp3+ Treg cells in the CD4+ T cell populations in contrast to free aVD and aVD nanoparticles. The resulting therapeutic benefits were reduced macrophage number in aortic plaque by 34% and 54% in comparison to the free aVD and aVD nanoparticles, respectively, as well as decreased arterial stiffness and IL-6 production.101 In summary, nanomaterials are essential to suppressing the immune response in the atheroclerotic niche by improving therapeutic uptake to circulating monocytes, resident macrophages and dendritic cells that induces a pro-resolving phenotype. Still, more standardization is needed in quantifying effects of nanoparticles on plaque reduction, particularly the need for nanoparticle-only and drug antagonist controls to delineate whether therapeutics effects are due to the material vs. drug. There is also considerable potential in tailoring cuttingedge nanotechnologies to reduce inflammation by interactions with the intracellular space. 121,122,123,124 For example, efforts to develop self-assembled organelles may be directed to engineer stress granules, which forms in response to cellular stress, protecting proteins and nucleic acids from degradation as well as sequestering harmful proteins.123 5. Conclusions and future perspectives While nanomaterials have shown potential for treating atherosclerosis, challenges remain for their clinical translation. A major issue is the lack of transfer in efficacy between proof-ofconcept pre-clinical studies to human clinical trials. This may be due to animal models used in studies that do not accurately represent the disease processes in humans. Yet, due to disease progression taking several years to decades to develop, it is difficult to study human plaque formation. One possible solution is to employ organ-on-chip models of atheroma. These microfluidic devices replicate diseased tissue architecture by culturing human cells that comprise ACS Paragon Plus Environment 25 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 26 of 66 the atherosclerotic niche, and can be perfused to mimic the physiological forces of shear stress found in atheroprone regions.125 Relatedly, prior to drug loading, all nanocarriers should be screened for their interactions with macrophages, a key cell regulator of atherosclerosis progression. The tests should inform how the nanomaterial impacts macrophage cell viability, proliferation, oxidative stress, lipid handling (uptake and efflux), and response to inflammatory stimulus challenge.126 Another limitation to clinical translation is the need for repeated nanoparticle injections to observe an effect on plaque prevention, stabilization or regression. In terms of nanoparticle design, developing oral formulations would offer a noninvasive delivery route. As well, engineering nanomaterials that dynamically assemble in response to stresses within the atheroma niche, creating intracellular “delivery depots” would be innovative. Even so, rethinking targets for atherosclerosis treatment may be a better approach to reducing the number of doses needed. For example, instead of targeting a single causative inflammatory pathway, we should focus on tipping the balance back to normal immune resolution by restoring the expression of proresolving proteins and mediators that include IL-10, annexin A1, lipoxins, and resolvins.13 Another restorative approach is targeting macrophage immunometabolism, which aims to “reboot” the lipid metabolism of macrophages by targeting metabolic pathways and intermediates as well as epigenetic marks that are altered during the transformation of macrophages to foam cells.16 Lastly, approaches that block mechanosensitive transcription factors in macrophages, which are activated from increasing plaque stiffness, should be explored as a means to improve responsiveness to therapeutics.127 ACS Paragon Plus Environment 26 Page 27 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering FIGURES Figure 1. Depiction of reverse cholesterol transport. The free cholesterol (FC) in peripheral foam cells is shuttled to the plasma membrane by ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) for cholesterol efflux. Apolipoprotein A1 (ApoA1) expressed on nascent HDL binds to ABCA1 for direct loading of FC. HDL esterifies the FC via lecithincholesterol acyltransferase (LCAT) to form cholesterol esters (CE). Apolipoprotein E (ApoE) enhances CE expansion in the HDL core. ABCG1 promotes FC desorption from the plasma membrane for subsequent loading onto mature HDL. HDLs transport the effluxed FC to the liver for uptake by scavenger receptor B1 (SR-B1). Cholesterol esterase transfer protein (CETP) can exchange CE from HDL with triglycerides (TG) from VLDL/LDL particles. Red blood cells (RBCs) may also aid in reverse cholesterol transport of FC from HDL and VLDL/LDL. The FC is catabolized within the hepatocyte to bile acids and transported by ABCG5 and ABCG8 for fecal elimination. Illustration by David Schumick, BS, CMI. Reprinted with the permission of the Cleveland Clinic Center for Medical Art & Photography © 2012. All Rights Reserved. ACS Paragon Plus Environment 27 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 28 of 66 Figure 2. Innate and adaptive immunity in atherosclerosis. Oxidized LDL (oxLDL) activates scavenger receptors on macrophages to initiate pro-inflammatory chemokine production, which stimulates endothelial cells to expression adhesion molecules that recruit monocytes and T cells. Endothelial cells are also activated by very low density lipoproteins (VLDL) containing apolipoprotein-CIII (Apo-CIII), which act through toll-like receptor 2 (TLR2). Recruited monocytes differentiate into macrophages that phagocytose the oxLDL, turning into lipid-laden foam cells. The foam cells attempt to release cholesterol through ABCA1 and ABCG1 transporters that promote cholesterol loading onto HDL for reverse cholesterol transport to the liver through the vasa vasorum. Cholesterol efflux from foam cells is impaired by the inflammatory microenvironment, and leads to cell death. Cellular debris in the necrotic core is taken up by antigen-presenting cells, such as dendritic cells, which interact with T cells to promote a TH1/CD4+ phenotype that produces IFN-γ, exacerbating the inflammatory response. Treg cells attempt to dampen inflammation by secreting TGF-β and IL-10 that shift macrophage phenotype from pro-inflammatory to a pro-resolving state. B cells also aid in dampening inflammation with the B1 cell-generated IgM antibodies that neutralize the pro-inflammatory effects of oxLDL. Reproduced with permission from 128. Copyright 2011, Springer Nature. ACS Paragon Plus Environment 28 Page 29 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Figure 3: Passively targeting nanoparticles to atherosclerotic plaque. (A-B) Example of enhanced permeability and retention (EPR) targeting. (A) schematic of PEGylated lipid-coated PLGA-gold nanoparticles that showed enhanced localization to atherosclerotic rabbit aortas (B, right aorta) versus controls (B, left aorta) using near infrared imaging. ***indicates p < 0.0001. Reproduced with permission from64. Copyright 2014, National Academy of Sciences. (C-D) Example of shear-targeting nanoparticles to thrombus. (C) Illustration depicting the sequence of thrombus formation (top and top middle), followed by shear-activated microparticles targeting thrombus (bottom middle) and subsequent dissociation into nanoparticles due to local shear stress to release tissue plasminogen activator (tPA) that reduces thrombus (bottom). (D) Quantitative results demonstrating the therapeutic potential of shear-activated nanoparticles containing tPA (tPA SA-NT) significantly (p < 0.0005) delay vessel occlusion after FeCl injury in comparison to controls of soluble tPA (free tPA), bare SA-NT, tPA-coated NPs that were artificially dissociated from SA-NTs before injection (dispersed tPA-NPs), and heat-fused NP microaggregates with tPA coating that do not dissociate (fused SA-NT). Reproduced with permission from 67. Copyright 2012, AAAS. ACS Paragon Plus Environment 29 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 30 of 66 Figure 4. Actively targeting nanoparticles to atherosclerotic plaque through inflamed endothelium and oxLDL. (A-B) Targeting inflamed endothelium through VCAM-1. (A) Schematic of PEGylated liposomes containing a CCR2 agonist and VCAM-1-targeting peptide. (B) Binding of fluorescently labelled liposomes to ApoE-/- or C57BL/6 mice aortas, where (a) is the liposome containing VCAM-1 recognizing peptide (VP-TSL), (b) is the nontargeted liposome (TSL), and (c) is the PBS control. Binding was quantified by radiant efficiency using an in vivo imaging system. ∗p < 0.05 versus TSL and PBS. Reproduced with permission from 73. Copyright 2015, Elsevier. (C-D) Targeting plaque through apolipoprotein A1 (ApoA1)-mimetic peptides. (C) Chemical structure and schematic of peptide amphiphiles (PAs) containing ApoA1-mimetic peptide 4F. self-assembled into a supramolecular nanofiber. The PAs are coassembled with diluent PA in aqueous solution to promote self-assembly into a nanofiber. (D) Representative images of aortic roots from LDLR KO mice depicting PA binding (red) of ApoA1 PAs and ApoA1 PAs containing a liver x receptor agonist (LXR-ApoA1 PA). No binding is observed in PAs containing a scrambled 4F peptide sequence (Scr PA). Reproduced with permission from 11. Copyright 2018, John Wiley and Sons. ACS Paragon Plus Environment 30 Page 31 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Figure 5. Actively targeting nanoparticles to atherosclerotic plaque through platelets and exposed vascular basement membrane. (A-C) Targeting plaque through platelet membrane- ACS Paragon Plus Environment 31 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 32 of 66 coated PLGA nanoparticles (PNPs). (A) Depiction of PNPs targeting to plaque through binding interactions of platelet surface markers to activated endothelium, collagen, and foam cells. (B) Analysis of fluorescently-tagged nanoparticle binding to aortic arches from ApoE-/- (ApoE KO) or wild type (WT) mice. PEG-NP are PLGA nanoparticles coated with PEG, RBCNPs are PLGA nanoparticles coated with red blood cell membranes. (C) Quantification of nanoparticle binding in (B). ****p < 0.0001. Reproduced with permission from 76. Copyright 2018, American Chemical Society. (D-F) Targeting micelles to plaque through fibrin-binding peptide CREKA. (D) 3D structure illustration of a self-assembled micelle nanoparticle comprised of DSPE lipid tail, poly(ethylene glycol) spacer, and polar head group (X) containing carboxyfluorescein-CREKA (FAM-CREKA), Cy7, and hirulog, an anticoagulant therapeutic. (E) Fluorescent imaging of the aortic tree in ApoE-/- mice depicting (left to right) FAM-labeled, non-targeted micelles; FAM-CREKA micelles, FAM-CREKA micelles injected after unlabeled CREKA micelles, and FAM-CREKA micelles injected after unlabeled, non-targeted micelles. (F) Quantification of binding from (E). p<0.05 for FAM-CREKA micelles versus FAM-labeled non-targeted micelles and FAM-CREKA + unlabeled CREKA micelles. Reproduced with permission from 77. Copyright 2009, National Academy of Sciences. ACS Paragon Plus Environment 32 Page 33 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Figure 6: Treating atherosclerosis by enhancing lipid metabolism. (A-D) Delivery of LXR agonists to atherosclerotic plaque. (A) Schematic of collagen-IV targeting nanoparticle containing fluorescently labeled GW3965 (GW-BODIPY) incorporated into the fluorescent PLA-Cy5.5 core. (B) Representative aortic root sections from low density receptor knockout mice stained for CD68+ cells by immunohistochemistry. Mice were treated with saline (PBS), free GW3965 (Free GW), PLA nanoparticles containing GW3965 (GW-NPs), and collagen IVtargeting PLA nanoparticles with GW3965 (Col IV-GW-NPs) twice per week for five weeks. (C) Quantification of CD68+ cells within plaque lesion area. (D) Analysis of hepatic triglyceride levels from total lipid homogenate. PBS, n = 8; free GW, n = 6; GW–NPs, n = 9; Col IV–GW– NPs, n = 7. *p<0.05, **p<0.01, data mean ± SEM. Reproduced with permission from ref. 10. Copyright 2017, John Wiley and Sons. ACS Paragon Plus Environment 33 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 34 of 66 Figure 7: Treating atherosclerosis by suppressing dendritic cell maturation in the atherosclerotic niche. (A-D) (A) Illustration of dendritic cell-targeting nanoparticle composed of poly(ethylene glycol)‐b‐poly(propylene sulfide) polymersome for co-delivery of immunomodulator 1,25-dihydroxyvitamin D3 (aVD) and ApoB-100-derived antigen peptide P210. A CD11c-derived peptide, P-D2, was attached to the nanoparticle surface via a PEG spacer, and a palmitoleic acid lipid tail to target dendritic cells. (B) Representative flow cytometry dot plots indicating an increase in the number of Foxp3+CD25+ Treg cells gated on CD45+CD3+ live cells in spleen of ApoE−/− mice following treatment with aVD or aVD+ P210 polymersomes. (C-D) Representative images and quantification of Oil Red O staining showing a reduction in plaque area upon treatment with polymersomes loaded with aVD or aVD+ P210. Reproduced with permission from ref. 94. Copyright 2019, John Wiley and Sons. * ACS Paragon Plus Environment 34 Page 35 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ACS Biomaterials Science & Engineering TABLES Table 1: Summary of approaches for targeting nanoparticles to atherosclerotic plaque Targeting mechanism Nanomaterial Animal model Key findings Ref. Gold and PLGA cores coated Four-month-old New Zealand white rabbits with PEG2000 and Cy5.5-labeled fed a high fat diet of 4.7-4.85% palm oil, DSPC 0.15-3% cholesterol for 6 months. Balloon angioplasty injury occurred at 2 and 6  Diameter: 70 nm weeks after diet initiation. Injections used the marginal ear vein. Increased PLGA aggregates of 3.8 ± 1.6 µm Arterial thrombus induced in 3 to 4-weekshear stress in diameter, comprised of old C57BL/6 mice by placing a 10% FeCl3 near stenotic nanoparticles 180 ± 70 nm in filter paper over vessels for 5 minutes. blood vessels diameter Fluorescently labeled PLGA aggregates coated with tissue plasminogen activator were injected retro-orbitally. One hour after injection, atherosclerotic aortas showed significant nanoparticle uptake in comparison to controls via NIRF imaging 61 PLGA aggregates localized to sites of thrombus and reduced its size within 5 minutes of injection, as shown by intravital fluorescence microscopy. 64 Inflamed endothelium After 24 hours of injection:  Co-localization of Cy7 labeled micelles with VCAM-1 antibody in aortic tree sections  No tissue damage found in the heart, lung, liver, spleen, intestine, kidney, or bladder 75 EPR effect in atherosclerotic endothelium and plaque microvessels Self-assembled spherical micelles containing VCAM-1 targeting peptide CVHPKQHR conjugated to DSPE-PEG2000maleimide   Four-week-old female ApoE-/- mice were fed a TD88137 high fat diet. Early and late atherosclerotic lesions were developed after 10 or 16 weeks of the diet, respectively. Micelles injected through tail-vein. Diameter: 17 ± 2 nm CMC: 14 µM Liposomes composed of PEG- Seven-week-old male ApoE-/- mice fed a Liposomes targeting VCAM-1 69 DSPE, DOPE, and DOPA high fat diet of 1% cholesterol for 6 weeks. showed significant binding to aortic containing CVHPKQHR peptide C57BL/6 mice fed a chow diet were used as regions of ApoE-/- mice. No a control. Mice were perfused with fluorescent liposomes using abdominal ACS Paragon Plus Environment 35 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 and encapsulating antagonist Macrophages CCR2 aortic ligature for 20 minutes. The aorta was immediately excised for analysis using IVIS Imaging System 200.  Diameter: 128 ± 19 nm Hyaluronan nanoparticles, Eight-week-old ApoE-/- mice fed a formed from amine- TD.88137 high fat diet for 6 or 12 weeks to functionalized oligomeric develop early and advanced atherosclerotic hyaluronan, 66-99 kDa, reacted lesions, respectively. Nanoparticles were with cholanic ester. injected at 25 mg/kg through tail-vein.   Diameter: 100 ± 17 nm Zeta potential: -31.3 ± 2.6 mV Peptide amphiphiles (PAs) comprised of a palmitic acid tail, beta sheet sequence, and 4F, an ApoA1-mimetic peptide: C16V2A2E2GDWFKAFYDKVAEKFKEAF. Self-assembled in aqueous solutions to form nanofibers Page 36 of 66 binding of liposomes was observed in the aorta of C57BL/6 mice. After 24 hours of injection:   80 Early lesions showed targeting to aortic macrophages as 6-fold higher than splenic macrophages, 40-fold higher than bone marrow macrophages Early lesions showed ~4-fold increase in targeting vs. advanced lesions Four-week-old LDLR KO mice were fed a After 24 hours of injection TD.88137 high fat diet for 14 weeks to  Demonstrated targeting to develop atherosclerotic lesions. atherosclerotic plaque in the Mice received intravenous injections of PA aortic root  No targeting seen in scrambled nanofibers dissolved in PBS at 6 mg/kg. 4F PA controls 11, 84 upon co-assembly with diluent PA (C16-V2A2E2)   Diameter: 8-10 µm Length: up to 1 µm ACS Paragon Plus Environment 36 Page 37 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ACS Biomaterials Science & Engineering Platelets Exposed vascular basement membrane PLGA nanoparticles coated with ApoE-/- mice received a high fat diet for 4 After 24 hours of injection platelet membranes. months. DiD-labeled nanoparticles injected  Targeting to atherosclerotic via tail-vein at 0.5 mg.  Diameter: ~140 nm plaque in the aortic root in both early and late-stage lesions  Zeta potential ~-30 mV  Colocalization to endothelium (ICAM-1), macrophages (CD68), and collagen IV antibodies  No targeting seen with PEGPLGA or bare PLGA nanoparticles, or in wild type mice controls Self-assembled spherical ApoE-/- mice fed a TD88137 high fat diet for After 3 hours of injection micelles containing fibrin 6 months. Micelles were injected via tail  ~40-fold increase in the targeting peptide CREKA vein at 1 mM, 100 µL. fluorescence intensity of aortas conjugated to caroxyfluorescein treated with CREKA vs. nonand DSPE-PEG2000-maleimide targeted micelles  Diameter: 17 ± 1 nm  No binding of CREKA micelles  Circulation half-life: 130 to aortas of wild type BALB/c minutes mice PLGA-PEG nanoparticles Eight to 10-week-old male LDLR KO mice After 5 days of injection containing maleimide conjugated fed a TD.88137 high fat diet for 12 weeks to collagen IV-binding peptide before intravenous injection with 200 µL of  Two-fold increase in fluorescence within the aortic KLWVLPK, Ac2-26 pro- nanoparticles containing 10 µg of Ac2-26 root from targeted vs. nonresolving peptide, and PLGA peptide (4% w/w). targeted nanoparticles containing Alexa 647 70:30 biodistribution of cadaverine. targeted nanoparticles in aortic  Diameter: ~80 nm root vs. spleen and liver. Opposite biodistribution seen  Zeta potential: ~-20 mV with non-targeted nanoparticles. 72 73 9 ApoE-/-: apolipoprotein E knockout; CMC: critical micelle concentration; CCR2: CC chemokine receptor 2; DiD: 1,1′-dioctadecyl3,3,3′,3′-tetramethylindodicarbocyanine; DOPA: 1,2-dioleoyl-sn-glycero-3-phosphatidic acid; DOPE: 1,2-dioleoyl-sn-glycero-3- ACS Paragon Plus Environment 37 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Page 38 of 66 phosphoethanolamine; DSPC:1,2-dioctadecanoyl-sn-glycero-3-phosphocholine; DSPE: 1,2-distearoyl-sn-glycero-3phosphoethanolamine; EPR: enhanced permeability and retention; ICAM-1: intercellular adhesion molecule 1; IVIS: in vivo imaging system; NIRF: near infrared imaging; LDLR KO: low density receptor knockout; PA: peptide amphiphile; PEG: poly(ethylene glycol); PLGA: poly(lactic-co-glycolic acid); RES: reticuloendothelial system; TD.88137: diet to induce atherosclerosis comprised of 20% fat, 0.2% cholesterol, and 34% high sucrose ACS Paragon Plus Environment 38 Page 39 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ACS Biomaterials Science & Engineering Table 2: Summary of therapeutic nanomaterials to treat atherosclerosis Therapeutic approach Nanomaterial Regulating lipid metabolism Collagen IV-targeting PLA-PEG lipid-polymer LDLR KO mice fed HFD  nanoparticles containing LXR agonist GW3965 for 14 weeks, then switched to chow diet  Collagen IV-targeting peptide KLWVLPK during intravenous  0.7:1 weight ratio of PLA:DPSE-mPEG1000,  injections of 8 mg/kg 7:3 molar ratio of lipids in the surface layer. 20 GW3965, 2x weekly for 5 wt% GW3965. weeks.  Hydrodynamic size: 83.9 ± 2.6 nm, zeta potential: 1.8 ± 0.8 mV  65% drug release by 48 hours in PBS at 37°C  ↓ CD68+ macrophages by 30% in atheroma using GW3965 nanoparticles vs. PBS and free drug. ↑ ABCA1 expression in CD68+ atherosclerotic lesions cells using targeted vs. untargeted GW3965 nanoparticles, free drug, and saline control. ↓ CYP7A1 & FASN gene expression in the liver using targeted vs. untargeted GW3965 nanoparticles 10 Plaque-targeting synthetic HDL (sHDL) ApoE-/- mice fed a HFD  nanoparticles containing LXR agonist T0901317 for 14 weeks, then switched to chow diet  ApoA1-mimetic peptide 22A: during intraperitoneal PVLDLFRELLNELLEALKQKLK injections 3x weekly for 6   22A, DMPC, POPC, and T0901317 combined weeks. sHDL at 30 mg/kg at 3, 3, 3, and 0.15 mg/ml, respectively containing 1.5 mg/kg  Hydrodynamic size: 12.1 ± 0.8 nm, discoidalT0901317. shaped ↓ Plaque in whole aorta and aortic root by 15 ± 1.7% and 40.8%, respectively, vs. PBS control In contrast to free drug, using sHDL-T0901317 nanoparticles caused no increase in liver triglyceride levels or SREBP1, FASN, ABCA1, & SCD1 gene expression. 97 Selective targeting of nanocarrier to plaque lesions Treatments with ApoA1-LXR PA caused ~10% plaque reduction in mice aortic root vs. PBS 84  Animal model Key findings 80% drug release by 8 hours in PBS at 37°C Plaque-targeting nanofibers composed of self- LDLR KO mice fed HFD assembled peptide amphiphiles (PAs) to serve as for 14 weeks, continued nanocarriers for delivery of LXR agonist GW3965 HFD during intravenous injections of 6 mg/kg  ApoA1 mimetic peptide 4F ApoA1-LXR PA, 2 (DWFKAFYDKVAEKFKEAF) covalently   Ref. ACS Paragon Plus Environment 39 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47    Resolving local inflammation bound to a PA backbone composed of palmitic acid and the beta-sheet forming peptide sequence V2A2E2. GW3965 incorporated during PA selfassembly through hydrophobic core at 1:1 weight ratio (ApoA1-LXR PA) α-helical secondary structure of co-assembled nanofiber Fiber diameter: 10.3 ± 2.2 nm, median length: 712 nm Pioglitazone-loaded PLGA nanoparticles  PLGA MW=20 kDa  Average diameter = 247 nm  Emulsion solvent diffusion method used Page 40 of 66 mg/mL, 2x weekly for 8 weeks.  ApoE-/- mice fed a HFD for 4 weeks and infused with angiotensin II during treatments. Weekly intravenous injections of pioglitazone-PLGA nanoparticles for 4 weeks at 7 mg/kg. Equivalent oral dose of pioglitazone at 1 mg/kg served as a control. Treatments with pioglitazone-PLGA 95 nanoparticles:  Significantly decreased the number of buried fibrous plaques while increasing fibrous cap thickness, without increasing in gene expression of kidney epithelial Na+ channels vs. oral controls  Suppressed proteinase activity and increased M2 polarization of plaque macrophages via Arg1 and IL-10 gene expression vs. control nanoparticles Treatment with Col IV-Ac2-26 9 nanoparticles  Significantly increased thickness of protective collagen layer, decreased collagenase activity, suppressed oxidative stress and caused >70-fold increase in IL10 gene expression within the atherosclerotic lesions vs. free Ac2-26, Col IV-scrambled Ac2- Collagen IV-targeting PLA-PEG lipid-polymer Male LDLR KO mice fed nanoparticles containing annexin A1-derived HFD for 12 weeks, peptide Ac2-26 injections of 10 µg/kg Ac2-26, 1x weekly for 5  Collagen IV-targeting peptide KLWVLPK weeks.  Ac2-26 peptide: AMVSEFLKQAWFIENEEQEYVQTVK In contrast to free GW3965, treatments with ApoA1-LXR PA in male mice did not cause an increase in liver toxicity based on AST blood levels ACS Paragon Plus Environment 40 Page 41 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ACS Biomaterials Science & Engineering  87% PLGA-PEG–COOH, 5% PLGA-PEG– Col IV peptide, 4% PLGA–Alexa 647, and 4% Ac2-26 (w/w).  Hydrodynamic size: ~80 nm, zeta potential: 20 mV >95% drug release by 5 days Polymersome nanospheres self-assembled from PEG-b-PPS copolymers  Immobilized onto the nanoparticle surface are P-D2 and ApoB-100 P210 peptides, which targets DCs via CD11c and induces tolerogenic DC response, respectively  P-D2 peptide: GGVTLTYQFAAGPRDK P210 peptide: KTTKQSFDLSVKAQY-KKKNKHK  aVD incorporated within PPS to reduce inflammation via suppression of NF-κB signaling  4% molar ratio P-D2 peptide to copolymer, 25% mass fraction of PEG average diameter: 143.6 nm, zeta potential: -5.32 ± 1.24 mV  ApoE-/- mice fed a HFD for 4 weeks. Weekly intravenous injections for 8 weeks of 12.5 µg P210/100 ng aVD/1.5 mg polymer/injection 26 nanoparticles, and vehicle controls Decreased plaque necrosis by 80% vs. Col IV-scrambled Ac226 nanoparticles Treatment with polymersomes containing aVD/P-D2/P210:  Suppressed DC maturation and increased Foxp3+ T regulatory cells in the atheroma  Lowered elastic modulus in aortic arch vs. control by 4.8fold  Decreased CD68+ macrophages within atheroma by 55% vs. control Reduced atherosclerotic lesion formation vs. control by 1.5-fold 94 ABCA1= ATP-binding cassette transporter A1; ApoA1= apolipoprotein A1; ApoE-/-= apolipoprotein E knockout; Arg1=arginase 1; AST= aspartate aminotransferase; aVD= 1,25‐dihydroxyvitamin D3; Col IV=collagen IV; CYP7A1= cholesterol 7-hydroxylase; DC=dendritic cell; DMPC= 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DSPE = 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine; FASN= fatty acid synthase; HFD= high fat diet; IL-10=interleukin 10; LDLR KO= low density lipoprotein receptor knockout; LXR= liver X receptor; mPEG1000=methoxy(polyethylene glycol) of molecular weight 1000 kDa; PA= peptide amphiphile; NF-κB=Nuclear Factor kappa-light-chain-enhancer of activated B cells; PBS=phosphate buffered saline; PEG= poly(ethylene glycol); PLA=poly(D,L‐lactide); POPC= 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; PPS=poly(propylene sulfide); SCD1= stearoylCoA desaturase-1; SREBP1= sterol regulatory element-binding protein 1; ACS Paragon Plus Environment 41 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 42 of 66 AUTHOR CONTRIBUTIONS EBP performed the review of the literature and prepared the draft of the manuscript. MRK critically revised the manuscript. Both EBP and MRK gave approval to the final version of the manuscript. FUNDING SOURCES E.B.P. was supported by the American Heart Association Postdoctoral Fellowship 18POST33960499. ABBREVIATIONS ABCA1/G1, ATP-binding cassette A1 or G1; AP-1, activator protein 1; ApoA1, apolipoprotein A1; ApoB-100, apolipoprotein B100; ApoE, apolipoprotein E; ApoE-/-, apolipoprotein E knockout; Arg1, arginase 1; ATP, adenosine triphosphate; aVD, 1,25-dihydroxyvitamin D3; CCL17, chemokine ligand 17; DAMPs, damage-associated molecular patterns; DNA, deoxyribonucleic acid; DSPC, 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine; E-selectin, endothelial-leukocyte adhesion molecule 1; EPR, enhanced vascular permeability and retention; Foxp3; forkhead box p3; FPR2/ALX, N-formyl peptide receptor 2; HDL, high density lipoproteins; HMG-CoA, hydroxymethylglutaryl-coenzyme A; ICAM, intracellular adhesion molecule; IFN-γ, interferon-gamma; IgM, immunoglobulin M; IL-1β, interleukin 1 beta; IL-6, interleukin 6; IL-8, interleukin 8; IL-10, interleukin 10; LDL, low density lipoproteins; LDLR, low density lipoprotein receptors; LDLR KO, low density lipoprotein receptor knockout; lectinlike oxLDL receptor (LOX-1); LPS, lipopolysaccharide; LXR, liver X receptor agonist; MMP2/9, matrix metalloproteinases 2 and 9; NADPH, the reduced form of Nicotinamide ACS Paragon Plus Environment 42 Page 43 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering adenine dinucleotide phosphate; NF-κB, nuclear factor kappa-B; Nox, NADPH oxidase; oxLDL, oxidized low density lipoproteins; PA, peptide amphiphile; PCKS9, proprotein convertase subtilisin-kexin type 9; PECAM, platelet endothelial cell adhesion molecule; PEG, poly(ethylene glycol); PLA, poly(D,L-lactide); PLGA, poly(lactic-co-glycolic acid); PPARγ, peroxisome proliferator-activated receptor-γ; PPS, poly(propylene sulfide); PRRs, pattern recognition receptors; ROS, reactive oxygen species; SR-BI, scavenger receptor class B type 1; TAZ, transcriptional coactivator with PDZ-binding motif; TEM, transmission electron microscopy; TH1, T helper type 1 cells; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand; Treg, regulatory T cells; VCAM, vascular endothelial adhesion molecule; VLDL, very low density lipoprotein; VSMC, vascular smooth muscle cells; YAP, Yes-associated protein. ACS Paragon Plus Environment 43 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 44 of 66 REFERENCES (1) Benjamin, E. J.; Virani, S. S.; Callaway, C. W.; Chamberlain, A. M.; Chang, A. R.; Cheng, S.; Chiuve, S. E.; Cushman, M.; Delling, F. N.; Deo, R.; De Ferranti, S. D.; Ferguson, J. F.; Fornage, M.; Gillespie, C.; Isasi, C. R.; Jiménez, M. C.; Jordan, L. C.; Judd, S. E.; Lackland, D.; Lichtman, J. H.; Lisabeth, L.; Liu, S.; Longenecker, C. T.; Lutsey, P. L.; MacKey, J. S.; Matchar, D. B.; Matsushita, K.; Mussolino, M. E.; Nasir, K.; O’Flaherty, M.; Palaniappan, L. P.; Pandey, A.; Pandey, D. K.; Reeves, M. J.; Ritchey, M. D.; Rodriguez, C. J.; Roth, G. A.; Rosamond, W. D.; Sampson, U. K. A.; Satou, G. M.; Shah, S. H.; Spartano, N. L.; Tirschwell, D. L.; Tsao, C. W.; Voeks, J. H.; Willey, J. Z.; Wilkins, J. T.; Wu, J. H. Y.; Alger, H. M.; Wong, S. S.; Muntner, P. Heart Disease and Stroke Statistics - 2018 Update: A Report from the American Heart Association. Circulation 2018, 137 (12), E67–E492. https://doi.org/10.1161/CIR.0000000000000558. (2) Lusis, A. J. Atherosclerosis. Nature. 2000, pp 233–241. https://doi.org/10.1038/35025203. (3) Frostegård, J. Immunity, Atherosclerosis and Cardiovascular Disease. BMC Medicine. 2013. https://doi.org/10.1186/1741-7015-11-117. (4) Allaire, E.; Clowes, A. W. The Intimal Hyperplastic Response. Annals of Thoracic Surgery. 1997. https://doi.org/10.1016/S0003-4975(97)00960-0. (5) Lobatto, M. E.; Fuster, V.; Fayad, Z. A.; Mulder, W. J. M. Perspectives and Opportunities for Nanomedicine in the Management of Atherosclerosis. Nature Reviews Drug Discovery. 2011, pp 835–852. https://doi.org/10.1038/nrd3578. (6) Chan, C. K. W.; Zhang, L.; Cheng, C. K.; Yang, H.; Huang, Y.; Tian, X. Y.; Choi, C. H. J. Recent Advances in Managing Atherosclerosis via Nanomedicine. Small. 2018. https://doi.org/10.1002/smll.201702793. ACS Paragon Plus Environment 44 Page 45 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering (7) Morgan, C. E.; Wasserman, M. A.; Kibbe, M. R. Targeted Nanotherapies for the Treatment of Surgical Diseases. Ann. Surg. 2016, 263 (5), 900–907. https://doi.org/10.1097/SLA.0000000000001605. (8) Lewis, D. R.; Petersen, L. K.; York, A. W.; Zablocki, K. R.; Joseph, L. B.; Kholodovych, V.; Prud’homme, R. K.; Uhrich, K. E.; Moghe, P. V. Sugar-Based Amphiphilic Nanoparticles Arrest Atherosclerosis in Vivo. Proc. Natl. Acad. Sci. 2015, 112 (9), 2693– 2698. https://doi.org/10.1073/pnas.1424594112. (9) Fredman, G.; Kamaly, N.; Spolitu, S.; Milton, J.; Ghorpade, D.; Chiasson, R.; Kuriakose, G.; Perretti, M.; Farokhzad, O.; Tabas, I. Targeted Nanoparticles Containing the Proresolving Peptide Ac2-26 Protect against Advanced Atherosclerosis in Hypercholesterolemic Mice. Sci. Transl. Med. 2015, 7 (275), 275ra20-275ra20. https://doi.org/10.1126/scitranslmed.aaa1065. (10) Yu, M.; Amengual, J.; Menon, A.; Kamaly, N.; Zhou, F.; Xu, X.; Saw, P. E.; Lee, S. J.; Si, K.; Ortega, C. A.; Choi, W. Il; Lee, I. H.; Bdour, Y.; Shi, J.; Mahmoudi, M.; Jon, S.; Fisher, E. A.; Farokhzad, O. C. Targeted Nanotherapeutics Encapsulating Liver X Receptor Agonist GW3965 Enhance Antiatherogenic Effects without Adverse Effects on Hepatic Lipid Metabolism in Ldlr-/- Mice. Advanced Healthcare Materials. 2017. https://doi.org/10.1002/adhm.201700313. (11) So, M. M.; Mansukhani, N. A.; Peters, E. B.; Albaghdadi, M. S.; Zheng, W.; Rubert Pérez, C. M.; Kibbe, M. R.; Stupp, S. I. Peptide Amphiphile Nanostructures for Targeting of Atherosclerotic Plaque and Drug Delivery. Adv. Biosyst. 2018, 2 (3), 1700123. https://doi.org/10.1002/adbi.201700123. (12) Ross, R. Atherosclerosis--an Inflammatory Disease. N. Engl. J. Med. 1999, 340 (2), 115– ACS Paragon Plus Environment 45 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 46 of 66 126. https://doi.org/10.1056/NEJM199901143400207. (13) Tabas, I.; García-Cardeña, G.; Owens, G. K. Recent Insights into the Cellular Biology of Atherosclerosis. Journal of Cell Biology. 2015, pp 13–22. https://doi.org/10.1083/jcb.201412052. (14) Kim, J. A.; Montagnani, M.; Chandrasekran, S.; Quon, M. J. Role of Lipotoxicity in Endothelial Dysfunction. Heart Failure Clinics. 2012. https://doi.org/10.1016/j.hfc.2012.06.012. (15) Weber, C.; Noels, H. Atherosclerosis: Current Pathogenesis and Therapeutic Options. Nature Medicine. 2011, pp 1410–1422. https://doi.org/10.1038/nm.2538. (16) Koelwyn, G. J.; Corr, E. M.; Erbay, E.; Moore, K. J. Regulation of Macrophage Immunometabolism in Atherosclerosis. Nature Immunology. 2018, pp 526–537. https://doi.org/10.1038/s41590-018-0113-3. (17) Olson, R. E. Discovery of the Lipoproteins, Their Role in Fat Transport and Their Significance as Risk Factors. J Nutr 1998, 128 (2 Suppl), 439S-443S. https://doi.org/10.1093/jn/128.2.439S. (18) Reiner, Ž. Hypertriglyceridaemia and Risk of Coronary Artery Disease. Nature Reviews Cardiology. 2017, pp 401–411. https://doi.org/10.1038/nrcardio.2017.31. (19) Truskey, G. a; Yuan, F.; Katz, D. F. Transport Phenomena in Biological Systems. Proc. Natl. Acad. Sci. 2004, No. 4, 816. https://doi.org/10.1016/j.bandc.2015.07.005. (20) Wang, L.; Luo, J.-Y.; Li, B.; Tian, X. Y.; Chen, L.-J.; Huang, Y.; Liu, J.; Deng, D.; Lau, C. W.; Wan, S.; Ai, D.; Mak, K.-L. K.; Tong, K. K.; Kwan, K. M.; Wang, N.; Chiu, J.-J.; Zhu, Y.; Huang, Y. Integrin-YAP/TAZ-JNK Cascade Mediates Atheroprotective Effect of Unidirectional Shear Flow. Nature 2016, 540 (7634), 579–582. ACS Paragon Plus Environment 46 Page 47 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering https://doi.org/10.1038/nature20602. (21) Skålén, K.; Gustafsson, M.; Knutsen Rydberg, E.; Hultén, L. M.; Wiklund, O.; Innerarity, T. L.; Boren, J. Subendothelial Retention of Atherogenic Lipoproteins in Early Atherosclerosis. Nature 2002, 417 (6890), 750–754. https://doi.org/10.1038/nature00804. (22) Fogelstrand, P.; Borén, J. Retention of Atherogenic Lipoproteins in the Artery Wall and Its Role in Atherogenesis. Nutrition, Metabolism and Cardiovascular Diseases. 2012, pp 1–7. https://doi.org/10.1016/j.numecd.2011.09.007. (23) Drechsler, M.; Megens, R. T. A.; Van Zandvoort, M.; Weber, C.; Soehnlein, O. Hyperlipidemia-Triggered Neutrophilia Promotes Early Atherosclerosis. Circulation 2010, 122 (18), 1837–1845. https://doi.org/10.1161/CIRCULATIONAHA.110.961714. (24) Soehnlein, O.; Lindbom, L. Phagocyte Partnership during the Onset and Resolution of Inflammation. Nature Reviews Immunology. 2010, pp 427–439. https://doi.org/10.1038/nri2779. (25) Valente, A. J.; Irimpen, A. M.; Siebenlist, U.; Chandrasekar, B. OxLDL Induces Endothelial Dysfunction and Death via TRAF3IP2: Inhibition by HDL3 and AMPK Activators. Free Radic. Biol. Med. 2014, 70, 117–128. https://doi.org/10.1016/j.freeradbiomed.2014.02.014. (26) Phillips, M. C. Molecular Mechanisms of Cellular Cholesterol Efflux. Journal of Biological Chemistry. 2014, pp 24020–24029. https://doi.org/10.1074/jbc.R114.583658. (27) Tall, A. R.; Yvan-Charvet, L.; Terasaka, N.; Pagler, T.; Wang, N. HDL, ABC Transporters, and Cholesterol Efflux: Implications for the Treatment of Atherosclerosis. Cell Metabolism. 2008, pp 365–375. https://doi.org/10.1016/j.cmet.2008.03.001. (28) de la Llera-Moya M. Scavenger Receptor BI (SR-BI) Mediates Free Cholesterol Flux Independently of HDL Tethering to the Cell Surface. J. Lipid Res. 1999, 40 (3), 575–580. ACS Paragon Plus Environment 47 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 (29) Page 48 of 66 Czarnecka, H.; Yokoyama, S. Regulation of Cellular Cholesterol Efflux by Lecithin:Cholesterol Acyltransferase Reaction through Nonspecific Lipid Exchange. J. Biol. Chem. 1996, 271 (4), 2023–2028. https://doi.org/10.1074/jbc.271.4.2023. (30) Mahley, R. W.; Huang, Y.; Weisgraber, K. H. Putting Cholesterol in Its Place: ApoE and Reverse Cholesterol Transport. Journal of Clinical Investigation. 2006, pp 1226–1229. https://doi.org/10.1172/JCI28632. (31) Pownall, H. J.; Massey, J. B.; Sparrow, J. T.; Gotto, A. M. Lipid-Protein Interactions and Lipoprotein Reassembly. New Compr. Biochem. 1987, 14 (C), 95–127. https://doi.org/10.1016/S0167-7306(08)60197-0. (32) Shao, B.; Oda, M. N.; Bergt, C.; Fu, X.; Green, P. S.; Brot, N.; Oram, J. F.; Heinecke, J. W. Myeloperoxidase Impairs ABCA1-Dependent Cholesterol Efflux through Methionine Oxidation and Site-Specific Tyrosine Chlorination of Apolipoprotein A-I. J. Biol. Chem. 2006, 281 (14), 9001–9004. https://doi.org/10.1074/jbc.C600011200. (33) Parathath, S.; Mick, S. L.; Feig, J. E.; Joaquin, V.; Grauer, L.; Habiel, D. M.; Gassmann, M.; Gardner, L. B.; Fisher, E. A. Hypoxia Is Present in Murine Atherosclerotic Plaques and Has Multiple Adverse Effects on Macrophage Lipid Metabolism. Circ. Res. 2011, 109 (10), 1141–1152. https://doi.org/10.1161/CIRCRESAHA.111.246363. (34) Seimon, T. A.; Nadolski, M. J.; Liao, X.; Magallon, J.; Nguyen, M.; Feric, N. T.; Koschinsky, M. L.; Harkewicz, R.; Witztum, J. L.; Tsimikas, S.; Golenbock, D.; Moore, K. J.; Tabas, I. Atherogenic Lipids and Lipoproteins Trigger CD36-TLR2-Dependent Apoptosis in Macrophages Undergoing Endoplasmic Reticulum Stress. Cell Metab. 2010, 12 (5), 467–482. https://doi.org/10.1016/j.cmet.2010.09.010. (35) Wong, S. W.; Kwon, M. J.; Choi, A. M. K.; Kim, H. P.; Nakahira, K.; Hwang, D. H. Fatty ACS Paragon Plus Environment 48 Page 49 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-Dependent Manner. J. Biol. Chem. 2009, 284 (40), 27384–27392. https://doi.org/10.1074/jbc.M109.044065. (36) Niessner, A.; Shin, M. S.; Pryshchep, O.; Goronzy, J. J.; Chaikof, E. L.; Weyand, C. M. Synergistic Proinflammatory Effects of the Antiviral Cytokine Interferon-α and Toll-like Receptor 4 Ligands in the Atherosclerotic Plaque. Circulation 2007, 116 (18), 2043–2052. https://doi.org/10.1161/CIRCULATIONAHA.107.697789. (37) Barrat, F. J.; Meeker, T.; Gregorio, J.; Chan, J. H.; Uematsu, S.; Akira, S.; Chang, B.; Duramad, O.; Coffman, R. L. Nucleic Acids of Mammalian Origin Can Act as Endogenous Ligands for Toll-like Receptors and May Promote Systemic Lupus Erythematosus. J. Exp. Med. 2005, 202 (8), 1131–1139. https://doi.org/10.1084/jem.20050914. (38) Niessner, A.; Sato, K.; Chaikof, E. L.; Colmegna, I.; Goronzy, J. J.; Weyand, C. M. Pathogen-Sensing Plasmacytoid Dendritic Cells Stimulate Cytotoxic T-Cell Function in the Atherosclerotic Plaque through Interferon-α. Circulation 2006, 114 (23), 2482–2489. https://doi.org/10.1161/CIRCULATIONAHA.106.642801. (39) Zernecke, A. Distinct Functions of Specialized Dendritic Cell Subsets in Atherosclerosis and the Road Ahead. Scientifica (Cairo). 2014, 2014, 1–7. https://doi.org/10.1155/2014/952625. (40) Weber, C.; Meiler, S.; Döring, Y.; Koch, M.; Drechsler, M.; Megens, R. T. A.; Rowinska, Z.; Bidzhekov, K.; Fecher, C.; Ribechini, E.; Van Zandvoort, M. A. M. J.; Binder, C. J.; Jelinek, I.; Hristov, M.; Boon, L.; Jung, S.; Korn, T.; Lutz, M. B.; Förster, I.; Zenke, M.; Hieronymus, T.; Junt, T.; Zernecke, A. CCL17-Expressing Dendritic Cells Drive ACS Paragon Plus Environment 49 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 50 of 66 Atherosclerosis by Restraining Regulatory T Cell Homeostasis in Mice. J. Clin. Invest. 2011, 121 (7), 2898–2910. https://doi.org/10.1172/JCI44925. (41) Tsiantoulas, D.; Diehl, C. J.; Witztum, J. L.; Binder, C. J. B Cells and Humoral Immunity in Atherosclerosis. Circulation Research. 2014, pp 1743–1756. https://doi.org/10.1161/CIRCRESAHA.113.301145. (42) Murphy, A. J.; Tall, A. R. Disordered Haematopoiesis and Athero-Thrombosis. European Heart Journal. 2016, pp 1113–1121. https://doi.org/10.1093/eurheartj/ehv718. (43) Bekkering, S.; Quintin, J.; Joosten, L. A. B.; Van Der Meer, J. W. M.; Netea, M. G.; Riksen, N. P. Oxidized Low-Density Lipoprotein Induces Long-Term Proinflammatory Cytokine Production and Foam Cell Formation via Epigenetic Reprogramming of Monocytes. Arterioscler. Thromb. Vasc. Biol. 2014, 34 (8), 1731–1738. https://doi.org/10.1161/ATVBAHA.114.303887. (44) Shirai, T.; Nazarewicz, R. R.; Wallis, B. B.; Yanes, R. E.; Watanabe, R.; Hilhorst, M.; Tian, L.; Harrison, D. G.; Giacomini, J. C.; Assimes, T. L.; Goronzy, J. J.; Weyand, C. M. The Glycolytic Enzyme PKM2 Bridges Metabolic and Inflammatory Dysfunction in Coronary Artery Disease. J. Exp. Med. 2016, 213 (3), 337–354. https://doi.org/10.1084/jem.20150900. (45) Lund, E. G.; Menke, J. G.; Sparrow, C. P. Liver X Receptor Agonists as Potential Therapeutic Agents for Dyslipidemia and Atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 2003, pp 1169–1177. https://doi.org/10.1161/01.ATV.0000056743.42348.59. (46) Kirchgessner, T. G.; Sleph, P.; Ostrowski, J.; Lupisella, J.; Ryan, C. S.; Liu, X.; Fernando, G.; Grimm, D.; Shipkova, P.; Zhang, R.; Garcia, R.; Zhu, J.; He, A.; Malone, H.; Martin, ACS Paragon Plus Environment 50 Page 51 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering R.; Behnia, K.; Wang, Z.; Barrett, Y. C.; Garmise, R. J.; Yuan, L.; Zhang, J.; Gandhi, M. D.; Wastall, P.; Li, T.; Du, S.; Salvador, L.; Mohan, R.; Cantor, G. H.; Kick, E.; Lee, J.; Frost, R. J. A. Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils. Cell Metab. 2016, 24 (2), 223–233. https://doi.org/10.1016/j.cmet.2016.07.016. (47) Matoba, T.; Koga, J. ichiro; Nakano, K.; Egashira, K.; Tsutsui, H. Nanoparticle-Mediated Drug Delivery System for Atherosclerotic Cardiovascular Disease. Journal of Cardiology. 2017, pp 206–211. https://doi.org/10.1016/j.jjcc.2017.03.005. (48) Kelley, W. J.; Safari, H.; Lopez-Cazares, G.; Eniola-Adefeso, O. Vascular-Targeted Nanocarriers: Design Considerations and Strategies for Successful Treatment of Atherosclerosis and Other Vascular Diseases. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 2016, pp 909–926. https://doi.org/10.1002/wnan.1414. (49) Jayagopal, A.; Linton, M. F.; Fazio, S.; Haselton, F. R. Insights into Atherosclerosis Using Nanotechnology. Current Atherosclerosis Reports. 2010, pp 209–215. https://doi.org/10.1007/s11883-010-0106-7. (50) Chung, E. J. Targeting and Therapeutic Peptides in Nanomedicine for Atherosclerosis. Exp. Biol. Med. 2016, 241 (9), 891–898. https://doi.org/10.1177/1535370216640940. (51) Caulfield, J. P.; Farquhar, M. G. The Permeability of Glomerular Capillaries to Graded Dextrans: Identification of the Basement Membrane as the Primary Filtration Barrier. J. Cell Biol. 1974, 63 (3), 883–903. https://doi.org/10.1083/jcb.63.3.883. (52) Soo Choi, H.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.; Frangioni, J. V. Renal Clearance of Quantum Dots. Nat. Biotechnol. 2007, 25 (10), 1165– ACS Paragon Plus Environment 51 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 52 of 66 1170. https://doi.org/10.1038/nbt1340. (53) Petros, R. A.; Desimone, J. M. Strategies in the Design of Nanoparticles for Therapeutic Applications. Nature Reviews Drug Discovery. 2010, pp 615–627. https://doi.org/10.1038/nrd2591. (54) Owens, D. E.; Peppas, N. A. Opsonization, Biodistribution, and Pharmacokinetics of Polymeric Nanoparticles. International Journal of Pharmaceutics. 2006, pp 93–102. https://doi.org/10.1016/j.ijpharm.2005.10.010. (55) Gustafson, H. H.; Holt-Casper, D.; Grainger, D. W.; Ghandehari, H. Nanoparticle Uptake: The Phagocyte Problem. Nano Today. 2015, pp 487–510. https://doi.org/10.1016/j.nantod.2015.06.006. (56) Xiao, W.; Lin, J.; Li, M.; Ma, Y.; Chen, Y.; Zhang, C.; Li, D.; Gu, H. Prolonged in Vivo Circulation Time by Zwitterionic Modification of Magnetite Nanoparticles for Blood Pool Contrast Agents. Contrast Media Mol. Imaging 2012, 7 (3), 320–327. https://doi.org/10.1002/cmmi.501. (57) Champion, J. A.; Mitragotri, S. Shape Induced Inhibition of Phagocytosis of Polymer Particles. Pharm. Res. 2009, 26 (1), 244–249. https://doi.org/10.1007/s11095-008-9626-z. (58) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Shape Effects of Filaments versus Spherical Particles in Flow and Drug Delivery. Nat. Nanotechnol. 2007, 2 (4), 249–255. https://doi.org/10.1038/nnano.2007.70. (59) Anselmo, A. C.; Mitragotri, S. Impact of Particle Elasticity on Particle-Based Drug Delivery Systems. Advanced Drug Delivery Reviews. 2017, pp 51–67. https://doi.org/10.1016/j.addr.2016.01.007. (60) Moyer, T. J.; Kassam, H. A.; Bahnson, E. S. M.; Morgan, C. E.; Tantakitti, F.; Chew, T. L.; ACS Paragon Plus Environment 52 Page 53 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Kibbe, M. R.; Stupp, S. I. Shape-Dependent Targeting of Injured Blood Vessels by Peptide Amphiphile Supramolecular Nanostructures. Small 2015, 11 (23), 2750–2755. https://doi.org/10.1002/smll.201403429. (61) Anselmo, A. C.; Zhang, M.; Kumar, S.; Vogus, D. R.; Menegatti, S.; Helgeson, M. E.; Mitragotri, S. Elasticity of Nanoparticles Influences Their Blood Circulation, Phagocytosis, Endocytosis, and Targeting. ACS Nano 2015, 9 (3), 3169–3177. https://doi.org/10.1021/acsnano.5b00147. (62) Zhang, L.; Cao, Z.; Li, Y.; Ella-Menye, J. R.; Bai, T.; Jiang, S. Softer Zwitterionic Nanogels for Longer Circulation and Lower Splenic Accumulation. ACS Nano 2012, 6 (8), 6681– 6686. https://doi.org/10.1021/nn301159a. (63) Banquy, X.; Suarez, F.; Argaw, A.; Rabanel, J. M.; Grutter, P.; Bouchard, J. F.; Hildgen, P.; Giasson, S. Effect of Mechanical Properties of Hydrogel Nanoparticles on Macrophage Cell Uptake. Soft Matter 2009, 5 (20), 3984–3991. https://doi.org/10.1039/b821583a. (64) Kim, Y.; Lobatto, M. E.; Kawahara, T.; Lee Chung, B.; Mieszawska, A. J.; Sanchez-Gaytan, B. L.; Fay, F.; Senders, M. L.; Calcagno, C.; Becraft, J.; Tun Saung, M.; Gordon, R. E.; Stroes, E. S. G.; Ma, M.; Farokhzad, O. C.; Fayad, Z. A.; Mulder, W. J. M.; Langer, R. Probing Nanoparticle Translocation across the Permeable Endothelium in Experimental Atherosclerosis. Proc. Natl. Acad. Sci. 2014, 111 (3), 1078–1083. https://doi.org/10.1073/pnas.1322725111. (65) Lobatto, M. E.; Calcagno, C.; Millon, A.; Senders, M. L.; Fay, F.; Robson, P. M.; Ramachandran, S.; Binderup, T.; Paridaans, M. P. M.; Sensarn, S.; Rogalla, S.; Gordon, R. E.; Cardoso, L.; Storm, G.; Metselaar, J. M.; Contag, C. H.; Stroes, E. S. G.; Fayad, Z. A.; Mulder, W. J. M. Atherosclerotic Plaque Targeting Mechanism of Long-Circulating ACS Paragon Plus Environment 53 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 54 of 66 Nanoparticles Established by Multimodal Imaging. ACS Nano 2015, 9 (2), 1837–1847. https://doi.org/10.1021/nn506750r. (66) Peters, E. B. Endothelial Progenitor Cells for the Vascularization of Engineered Tissues. Tissue Eng. Part B Rev. 2017. https://doi.org/10.1089/ten.teb.2017.0127. (67) Korin, N.; Kanapathipillai, M.; Matthews, B. D.; Crescente, M.; Brill, A.; Mammoto, T.; Ghosh, K.; Jurek, S.; Bencherif, S. A.; Bhatta, D.; Coskun, A. U.; Feldman, C. L.; Wagner, D. D.; Ingber, D. E. Shear-Activated Nanotherapeutics for Drug Targeting to Obstructed Blood Vessels. Science (80-. ). 2012, 337 (6095), 738–742. https://doi.org/10.1126/science.1217815. (68) Sharma, G.; She, Z.-G.; Valenta, D. T.; Stallcup, W. B.; Smith, J. W. Targeting of Macrophage Foam Cells in Atherosclerotic Plaque Using Oligonucleotide-Functionalized Nanoparticles. Nano Life 2010, 1 (3–4), 207–214. https://doi.org/10.1142/S1793984410000183. (69) Bhowmick, T.; Berk, E.; Cui, X.; Muzykantov, V. R.; Muro, S. Effect of Flow on Endothelial Endocytosis of Nanocarriers Targeted to ICAM-1. J. Control. Release 2012, 157 (3), 485–492. https://doi.org/10.1016/j.jconrel.2011.09.067. (70) Zhang, L.; Tian, X. Y.; Chan, C. K. W.; Bai, Q.; Cheng, C. K.; Chen, F. M.; Cheung, M. S. H.; Yin, B.; Yang, H.; Yung, W.-Y.; Chen, Z.; Ding, F.; Leung, K. C.-F.; Zhang, C.; Huang, Y.; Lau, J. Y. W.; Choi, C. H. J. Promoting the Delivery of Nanoparticles to Atherosclerotic Plaques by DNA Coating. ACS Appl. Mater. Interfaces 2019, 11 (15), 13888–13904. https://doi.org/10.1021/acsami.8b17928. (71) Strony, J.; Beaudoin, A.; Brands, D.; Adelman, B. Analysis of Shear Stress and Hemodynamic Factors in a Model of Coronary Artery Stenosis and Thrombosis. Am. J. ACS Paragon Plus Environment 54 Page 55 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Physiol. Circ. Physiol. 1993, 265 (5), H1787–H1796. https://doi.org/10.1152/ajpheart.1993.265.5.h1787. (72) Siegel, J. M.; Markou, C. P.; Ku, D. N.; Hanson, S. R. A Scaling Law for Wall Shear Rate Through an Arterial Stenosis. J. Biomech. Eng. 1994, 116 (4), 446. https://doi.org/10.1115/1.2895795. (73) Calin, M.; Stan, D.; Schlesinger, M.; Simion, V.; Deleanu, M.; Constantinescu, C. A.; Gan, A. M.; Pirvulescu, M. M.; Butoi, E.; Manduteanu, I.; Bota, M.; Enachescu, M.; Borsig, L.; Bendas, G.; Simionescu, M. VCAM-1 Directed Target-Sensitive Liposomes Carrying CCR2 Antagonists Bind to Activated Endothelium and Reduce Adhesion and Transmigration of Monocytes. Eur. J. Pharm. Biopharm. 2015, 89, 18–29. https://doi.org/10.1016/j.ejpb.2014.11.016. (74) Iverson, N. M.; Plourde, N. M.; Sparks, S. M.; Wang, J.; Patel, E. N.; Shah, P. S.; Lewis, D. R.; Zablocki, K. R.; Nackman, G. B.; Uhrich, K. E.; Moghe, P. V. Dual Use of Amphiphilic Macromolecules as Cholesterol Efflux Triggers and Inhibitors of Macrophage Athero-Inflammation. Biomaterials 2011, 32 (32), 8319–8327. https://doi.org/10.1016/j.biomaterials.2011.07.039. (75) Chung, E. J.; Mlinar, L. B.; Nord, K.; Sugimoto, M. J.; Wonder, E.; Alenghat, F. J.; Fang, Y.; Tirrell, M. Monocyte-Targeting Supramolecular Micellar Assemblies: A Molecular Diagnostic Tool for Atherosclerosis. Adv. Healthc. Mater. 2015, 4 (3), 367–376. https://doi.org/10.1002/adhm.201400336. (76) Wei, X.; Ying, M.; Dehaini, D.; Su, Y.; Kroll, A. V.; Zhou, J.; Gao, W.; Fang, R. H.; Chien, S.; Zhang, L. Nanoparticle Functionalization with Platelet Membrane Enables Multifactored Biological Targeting and Detection of Atherosclerosis. ACS Nano 2018, 12 ACS Paragon Plus Environment 55 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 56 of 66 (1), 109–116. https://doi.org/10.1021/acsnano.7b07720. (77) Peters, D.; Kastantin, M.; Kotamraju, V. R.; Karmali, P. P.; Gujraty, K.; Tirrell, M.; Ruoslahti, E. Targeting Atherosclerosis by Using Modular, Multifunctional Micelles. Proc. Natl. Acad. Sci. 2009, 106 (24), 9815–9819. https://doi.org/10.1073/pnas.0903369106. (78) O’Brien, K. D.; Allen, M. D.; McDonald, T. O.; Chait, A.; Harlan, J. M.; Fishbein, D.; McCarty, J.; Ferguson, M.; Hudkins, K.; Benjamin, C. D.; Lobb, R.; Alpers, C. E. Vascular Cell Adhesion Molecule-1 Is Expressed in Human Coronary Atherosclerotic Plaques: Implications for the Mode of Progression of Advanced Coronary Atherosclerosis. J. Clin. Invest. 1993, 92 (2), 945–951. https://doi.org/10.1172/JCI116670. (79) Mlinar, L. B.; Chung, E. J.; Wonder, E. A.; Tirrell, M. Active Targeting of Early and MidStage Atherosclerotic Plaques Using Self-Assembled Peptide Amphiphile Micelles. Biomaterials 2014, 35 (30), 8678–8686. https://doi.org/10.1016/j.biomaterials.2014.06.054. (80) Calderon, A. J.; Muzykantov, V.; Muro, S.; Eckmann, D. M. Flow Dynamics, Binding and Detachment of Spherical Carriers Targeted to ICAM-1 on Endothelial Cells. Biorheology 2009, 46 (4), 323–341. https://doi.org/10.3233/BIR-2009-0544. (81) Han, J.; Zern, B. J.; Shuvaev, V. V.; Davies, P. F.; Muro, S.; Muzykantov, V. Acute and Chronic Shear Stress Differently Regulate Endothelial Internalization of Nanocarriers Targeted to Platelet-Endothelial Cell Adhesion Molecule-1. ACS Nano 2012, 6 (10), 8824– 8836. https://doi.org/10.1021/nn302687n. (82) Papademetriou, I.; Tsinas, Z.; Hsu, J.; Muro, S. Combination-Targeting to Multiple Endothelial Cell Adhesion Molecules Modulates Binding, Endocytosis, and in Vivo Biodistribution of Drug Nanocarriers and Their Therapeutic Cargoes. J. Control. Release ACS Paragon Plus Environment 56 Page 57 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering 2014, 188, 87–98. https://doi.org/10.1016/j.jconrel.2014.06.008. (83) Fischer, J. W. Role of Hyaluronan in Atherosclerosis: Current Knowledge and Open Questions. Matrix Biology. 2018. https://doi.org/10.1016/j.matbio.2018.03.003. (84) Beldman, T. J.; Senders, M. L.; Alaarg, A.; Pérez-Medina, C.; Tang, J.; Zhao, Y.; Fay, F.; Deichmöller, J.; Born, B.; Desclos, E.; Van Der Wel, N. N.; Hoebe, R. A.; Kohen, F.; Kartvelishvily, E.; Neeman, M.; Reiner, T.; Calcagno, C.; Fayad, Z. A.; De Winther, M. P. J.; Lutgens, E.; Mulder, W. J. M.; Kluza, E. Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis. ACS Nano 2017, 11 (6), 5785–5799. https://doi.org/10.1021/acsnano.7b01385. (85) Sanchez-Gaytan, B. L.; Fay, F.; Lobatto, M. E.; Tang, J.; Ouimet, M.; Kim, Y.; Van Der Staay, S. E. M.; Van Rijs, S. M.; Priem, B.; Zhang, L.; Fisher, E. A.; Moore, K. J.; Langer, R.; Fayad, Z. A.; Mulder, W. J. M. HDL-Mimetic PLGA Nanoparticle To Target Atherosclerosis Plaque Macrophages. Bioconjug. Chem. 2015, 26 (3), 443–451. https://doi.org/10.1021/bc500517k. (86) Marrache, S.; Dhar, S. Biodegradable Synthetic High-Density Lipoprotein Nanoparticles for Atherosclerosis. Proc. Natl. Acad. Sci. 2013, 110 (23), 9445–9450. https://doi.org/10.1073/pnas.1301929110. (87) Zhao, Y.; Black, A. S.; Bonnet, D. J.; Maryanoff, B. E.; Curtiss, L. K.; Leman, L. J.; Ghadiri, M. R. In Vivo Efficacy of HDL-like Nanolipid Particles Containing Multivalent Peptide Mimetics of Apolipoprotein A-I. J. Lipid Res. 2014, 55 (10), 2053–2063. https://doi.org/10.1194/jlr.m049262. (88) Mansukhani, N. A.; Peters, E. B.; So, M. M.; Albaghdadi, M. S.; Wang, Z.; Karver, M. R.; Clemons, T. D.; Laux, J. P.; Tsihlis, N. D.; Stupp, S. I.; Kibbe, M. R. Peptide Amphiphile ACS Paragon Plus Environment 57 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 58 of 66 Supramolecular Nanostructures as a Targeted Therapy for Atherosclerosis. Macromol. Biosci. 2019. https://doi.org/10.1002/mabi.201900066. (89) Massberg, S.; Brand, K.; Grüner, S.; Page, S.; Müller, E.; Müller, I.; Bergmeier, W.; Richter, T.; Lorenz, M.; Konrad, I.; Nieswandt, B.; Gawaz, M. A Critical Role of Platelet Adhesion in the Initiation of Atherosclerotic Lesion Formation. J. Exp. Med. 2002, 196 (7), 887–896. https://doi.org/10.1084/jem.20012044. (90) Gawaz, M.; Stellos, K.; Langer, H. F. Platelets Modulate Atherogenesis and Progression of Atherosclerotic Plaques via Interaction with Progenitor and Dendritic Cells. Journal of Thrombosis and Haemostasis. 2008, pp 235–242. https://doi.org/10.1111/j.15387836.2007.02867.x. (91) Song, Y.; Huang, Z.; Liu, X.; Pang, Z.; Chen, J.; Yang, H.; Zhang, N.; Cao, Z.; Liu, M.; Cao, J.; Li, C.; Yang, X.; Gong, H.; Qian, J.; Ge, J. Platelet Membrane-Coated NanoparticleMediated Targeting Delivery of Rapamycin Blocks Atherosclerotic Plaque Development and Stabilizes Plaque in Apolipoprotein E-Deficient (ApoE −/− ) Mice. Nanomedicine Nanotechnology, Biol. Med. 2019, 15 (1), 13–24. https://doi.org/10.1016/j.nano.2018.08.002. (92) Vijayan, V.; Uthaman, S.; Park, I.-K. Cell Membrane-Camouflaged Nanoparticles: A Promising Biomimetic Strategy for Cancer Theragnostics. Polymers (Basel). 2018, 10 (9), 983. https://doi.org/10.3390/polym10090983. (93) Hahn, C.; Schwartz, M. A. The Role of Cellular Adaptation to Mechanical Forces in Atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 2008, pp 2101–2107. https://doi.org/10.1161/ATVBAHA.108.165951. (94) Orr, A. W.; Sanders, J. M.; Bevard, M.; Coleman, E.; Sarembock, I. J.; Schwartz, M. A. ACS Paragon Plus Environment 58 Page 59 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering The Subendothelial Extracellular Matrix Modulates NF-ΚB Activation by Flow. J. Cell Biol. 2005, 169 (1), 191–202. https://doi.org/10.1083/jcb.200410073. (95) Sindhwani, S.; Syed, A. M.; Ngai, J.; Kingston, B. R.; Maiorino, L.; Rothschild, J.; MacMillan, P.; Zhang, Y.; Rajesh, N. U.; Hoang, T.; Wu, J. L. Y.; Wilhelm, S.; Zilman, A.; Gadde, S.; Sulaiman, A.; Ouyang, B.; Lin, Z.; Wang, L.; Egeblad, M.; Chan, W. C. W. The Entry of Nanoparticles into Solid Tumours. Nat. Mater. 2020, 19 (5), 566–575. https://doi.org/10.1038/s41563-019-0566-2. (96) Flores, A. M.; Ye, J.; Jarr, K. U.; Hosseini-Nassab, N.; Smith, B. R.; Leeper, N. J. Nanoparticle Therapy for Vascular Diseases. Arterioscler. Thromb. Vasc. Biol. 2019. https://doi.org/10.1161/ATVBAHA.118.311569. (97) Maruf, A.; Wang, Y.; Yin, T.; Huang, J.; Wang, N.; Durkan, C.; Tan, Y.; Wu, W.; Wang, G. Atherosclerosis Treatment with Stimuli-Responsive Nanoagents: Recent Advances and Future Perspectives. Advanced Healthcare Materials. 2019. https://doi.org/10.1002/adhm.201900036. (98) Chen, P.-Y.; Qin, L.; Li, G.; Wang, Z.; Dahlman, J. E.; Malagon-Lopez, J.; Gujja, S.; Cilfone, N. A.; Kauffman, K. J.; Sun, L.; Sun, H.; Zhang, X.; Aryal, B.; Canfran-Duque, A.; Liu, R.; Kusters, P.; Sehgal, A.; Jiao, Y.; Anderson, D. G.; Gulcher, J.; FernandezHernando, C.; Lutgens, E.; Schwartz, M. A.; Pober, J. S.; Chittenden, T. W.; Tellides, G.; Simons, M. Endothelial TGF-β Signalling Drives Vascular Inflammation and Atherosclerosis. Nat. Metab. 2019. https://doi.org/10.1038/s42255-019-0102-3. (99) Wencong, S.; Cheng-Lin, Z.; Lingshan, G.; Lei, H.; Yao-Yu, G.; Dan, Q.; Lei, Z.; Nana, J.; Fung, C. T.; Li, W.; Yu, T. X.; Jiang-Yun, L.; Yu, H. Endothelial TFEB (Transcription Factor EB) Restrains IKK (IκB Kinase)-P65 Pathway to Attenuate Vascular Inflammation ACS Paragon Plus Environment 59 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 60 of 66 in Diabetic Db/Db Mice. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (4), 719–730. https://doi.org/10.1161/ATVBAHA.119.312316. (100) Qu, D.; Wang, L.; Huo, M.; Song, W.; Lau, C.-W.; Xu, J.; Xu, A.; Yao, X.; Chiu, J.-J.; Tian, X. Y.; Huang, Y. Focal TLR4 Activation Mediates Disturbed Flow-Induced Endothelial Inflammation. Cardiovasc. Res. 2019, 116 (1), 226–236. https://doi.org/10.1093/cvr/cvz046. (101) Yi, S.; Zhang, X.; Sangji, M. H.; Liu, Y.; Allen, S. D.; Xiao, B.; Bobbala, S.; Braverman, C. L.; Cai, L.; Hecker, P. I.; DeBerge, M.; Thorp, E. B.; Temel, R. E.; Stupp, S. I.; Scott, E. A. Surface Engineered Polymersomes for Enhanced Modulation of Dendritic Cells During Cardiovascular Immunotherapy. Adv. Funct. Mater. 2019. https://doi.org/10.1002/adfm.201904399. (102) Nakashiro, S.; Matoba, T.; Umezu, R.; Koga, J. I.; Tokutome, M.; Katsuki, S.; Nakano, K.; Sunagawa, K.; Egashira, K. Pioglitazone-Incorporated Nanoparticles Prevent Plaque Destabilization and Rupture by Regulating Monocyte/Macrophage Differentiation in ApoE -/-Mice. Arterioscler. Thromb. Vasc. Biol. 2016. https://doi.org/10.1161/ATVBAHA.115.307057. (103) Lee, S. D.; Tontonoz, P. Liver X Receptors at the Intersection of Lipid Metabolism and Atherogenesis. Atherosclerosis 2015. https://doi.org/10.1016/j.atherosclerosis.2015.06.042. (104) Leren, T. P. Sorting an LDL Receptor with Bound PCSK9 to Intracellular Degradation. Atherosclerosis. 2014. https://doi.org/10.1016/j.atherosclerosis.2014.08.038. (105) Bittencourt, M. S.; Cerci, R. J. Statin Effects on Atherosclerotic Plaques: Regression or Healing? BMC Medicine. 2015. https://doi.org/10.1186/s12916-015-0499-9. ACS Paragon Plus Environment 60 Page 61 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering (106) Wong, J.; Quinn, C. M.; Brown, A. J. Statins Inhibit Synthesis of an Oxysterol Ligand for the Liver X Receptor in Human Macrophages with Consequences for Cholesterol Flux. Arterioscler. Thromb. Vasc. Biol. 2004. https://doi.org/10.1161/01.ATV.0000148707.93054.7d. (107) Qiu, G.; Hill, J. S. Atorvastatin Inhibits ABCA1 Expression and Cholesterol Efflux in THP1 Macrophages by an LXR-Dependent Pathway. J. Cardiovasc. Pharmacol. 2008. https://doi.org/10.1097/FJC.0b013e318167141f. (108) Tall, A. R.; Yvan-Charvet, L. Cholesterol, Inflammation and Innate Immunity. Nature Reviews Immunology. 2015. https://doi.org/10.1038/nri3793. (109) Guo, Y.; Yuan, W.; Yu, B.; Kuai, R.; Hu, W.; Morin, E. E.; Garcia-Barrio, M. T.; Zhang, J.; Moon, J. J.; Schwendeman, A.; Eugene Chen, Y. Synthetic High-Density LipoproteinMediated Targeted Delivery of Liver X Receptors Agonist Promotes Atherosclerosis Regression. EBioMedicine 2018. https://doi.org/10.1016/j.ebiom.2017.12.021. (110) Virmani, R.; Burke, A. P.; Farb, A.; Kolodgie, F. D. Pathology of the Vulnerable Plaque. Journal of the American College of Cardiology. 2006. https://doi.org/10.1016/j.jacc.2005.10.065. (111) Tabas, I.; Williams, K. J.; Borén, J. Subendothelial Lipoprotein Retention as the Initiating Process in Atherosclerosis: Update and Therapeutic Implications. Circulation. 2007. https://doi.org/10.1161/CIRCULATIONAHA.106.676890. (112) Fredman, G.; Tabas, I. Boosting Inflammation Resolution in Atherosclerosis: The Next Frontier for Therapy. American Journal of Pathology. 2017. https://doi.org/10.1016/j.ajpath.2017.01.018. (113) Bäck, M.; Yurdagul, A.; Tabas, I.; Öörni, K.; Kovanen, P. T. Inflammation and Its ACS Paragon Plus Environment 61 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 62 of 66 Resolution in Atherosclerosis: Mediators and Therapeutic Opportunities. Nature Reviews Cardiology. 2019. https://doi.org/10.1038/s41569-019-0169-2. (114) Fredman, G.; Hellmann, J.; Proto, J. D.; Kuriakose, G.; Colas, R. A.; Dorweiler, B.; Connolly, E. S.; Solomon, R.; Jones, D. M.; Heyer, E. J.; Spite, M.; Tabas, I. An Imbalance between Specialized Pro-Resolving Lipid Mediators and pro-Inflammatory Leukotrienes Promotes Instability of Atherosclerotic Plaques. Nat. Commun. 2016. https://doi.org/10.1038/ncomms12859. (115) Mallat, Z.; Besnard, S.; Duriez, M.; Deleuze, V.; Emmanuel, F.; Bureau, M. F.; Soubrier, F.; Esposito, B.; Duez, H.; Fievet, C.; Staels, B.; Duverger, N.; Scherman, D.; Tedgui, A. Protective Role of Interleukin-10 in Atherosclerosis. Circ. Res. 1999. https://doi.org/10.1161/01.res.85.8.e17. (116) Saxena, A.; Khosraviani, S.; Noel, S.; Mohan, D.; Donner, T.; Hamad, A. R. A. Interleukin10 Paradox: A Potent Immunoregulatory Cytokine That Has Been Difficult to Harness for Immunotherapy. Cytokine 2015. https://doi.org/10.1016/j.cyto.2014.10.031. (117) Bouhlel, M. A.; Derudas, B.; Rigamonti, E.; Dièvart, R.; Brozek, J.; Haulon, S.; Zawadzki, C.; Jude, B.; Torpier, G.; Marx, N.; Staels, B.; Chinetti-Gbaguidi, G. PPARγ Activation Primes Human Monocytes into Alternative M2 Macrophages with Anti-Inflammatory Properties. Cell Metab. 2007. https://doi.org/10.1016/j.cmet.2007.06.010. (118) Almerighi, C.; Sinistro, A.; Cavazza, A.; Ciaprini, C.; Rocchi, G.; Bergamini, A. 1α,25Dihydroxyvitamin D3 Inhibits CD40L-Induced pro-Inflammatory and Immunomodulatory Activity in Human Monocytes. Cytokine 2009. https://doi.org/10.1016/j.cyto.2008.12.009. (119) Hermansson, A.; Johansson, D. K.; Ketelhuth, D. F. J.; Andersson, J.; Zhou, X.; Hansson, G. K. Immunotherapy with Tolerogenic Apolipoprotein B-100-Loaded Dendritic Cells ACS Paragon Plus Environment 62 Page 63 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering Attenuates Atherosclerosis in Hypercholesterolemic Mice. Circulation 2011. https://doi.org/10.1161/CIRCULATIONAHA.110.973222. (120) Allen, S. D.; Liu, Y. G.; Kim, T.; Bobbala, S.; Yi, S.; Zhang, X.; Choi, J.; Scott, E. A. Celastrol-Loaded PEG-: B -PPS Nanocarriers as an Anti-Inflammatory Treatment for Atherosclerosis. Biomater. Sci. 2019. https://doi.org/10.1039/c8bm01224e. (121) Nurunnabi, M.; Khatun, Z.; Badruddoza, A. Z. M.; McCarthy, J. R.; Lee, Y. K.; Huh, K. M. Biomaterials and Bioengineering Approaches for Mitochondria and Nuclear Targeting Drug Delivery. ACS Biomaterials Science and Engineering. 2019. https://doi.org/10.1021/acsbiomaterials.8b01615. (122) Wang, Z.; Guo, W.; Kuang, X.; Hou, S.; Liu, H. Nanopreparations for Mitochondria Targeting Drug Delivery System: Current Strategies and Future Prospective. Asian Journal of Pharmaceutical Sciences. 2017. https://doi.org/10.1016/j.ajps.2017.05.006. (123) Gomes, E.; Shorter, J. The Molecular Language of Membraneless Organelles. Journal of Biological Chemistry. 2019. https://doi.org/10.1074/jbc.TM118.001192. (124) Schuster, B. S.; Reed, E. H.; Parthasarathy, R.; Jahnke, C. N.; Caldwell, R. M.; Bermudez, J. G.; Ramage, H.; Good, M. C.; Hammer, D. A. Controllable Protein Phase Separation and Modular Recruitment to Form Responsive Membraneless Organelles. Nat. Commun. 2018. https://doi.org/10.1038/s41467-018-05403-1. (125) Bhatia, S. N.; Ingber, D. E. Microfluidic Organs-on-Chips. Nat. Biotechnol. 2014, 32 (8), 760–772. https://doi.org/10.1038/nbt.2989. (126) Nguyen, L. T. H.; Muktabar, A.; Tang, J.; Dravid, V. P.; Thaxton, C. S.; Venkatraman, S.; Ng, K. W. Engineered Nanoparticles for the Detection, Treatment and Prevention of Atherosclerosis: How Close Are We? Drug Discovery Today. 2017. ACS Paragon Plus Environment 63 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 64 of 66 https://doi.org/10.1016/j.drudis.2017.07.006. (127) Niu, N.; Xu, S.; Xu, Y.; Little, P. J.; Jin, Z. G. Targeting Mechanosensitive Transcription Factors in Atherosclerosis. Trends in Pharmacological Sciences. 2019. https://doi.org/10.1016/j.tips.2019.02.004. (128) Libby, P.; Ridker, P. M.; Hansson, G. K. Progress and Challenges in Translating the Biology of Atherosclerosis. Nature 2011, 473 (7347), 317–325. https://doi.org/10.1038/nature10146. ACS Paragon Plus Environment 64 Page 65 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Biomaterials Science & Engineering TABLE OF CONTENTS Examples of nanomaterials used to treat atherosclerosis ACS Paragon Plus Environment 65 ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Examples of nanomaterials used to treat atherosclerosis 338x190mm (96 x 96 DPI) ACS Paragon Plus Environment View publication stats Page 66 of 66