Cardiovascular disease is responsible for the deaths of more than 17 million people worldwide and this rate is expected to grow to over 23 million by the year 2030 [1
]. More strikingly, it is estimated that there is a death every 40 s attributable to cardiovascular disease [2
]. Atherosclerosis is the root cause of the majority of cardiovascular clinical manifestations, which have been significantly curbed due to breakthroughs in drug therapy. However, advanced tools, increasing interest in nanotechnology, and further understanding of the pathology of atherosclerosis elucidated by various animal studies have recently placed researchers in prime position to tackle the complex manifestations of atherosclerosis on a more specific and molecular level.
Atherosclerosis has typically been viewed as a dietary and lipid accumulation disorder. While lipids certainly play a role in lesion formation, they cannot account for all the concerns of atherosclerosis. It is now known that atherosclerosis begins with endothelial damage that can arise as early as adolescence. Lipids such as apolipoprotein B have special affinity for the basal membrane revealed in areas of damaged endothelium [3
]. These regions are well conserved in humans and various animal models and typically occur in curved or branched arteries, which experience disturbed or oscillatory flow dynamics and low shear stress [4
Endothelial damage permits the accumulation and retention of lipids within the subendothelial space where they can be oxidized by oxidative stress-induced molecules and enzymatic products [5
]. Oxidized lipids act as a danger signal to the endothelial cells lining the vessel and the cells begin to increase expression of the inflammatory cell recruitment receptors vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P-selectin, and E-selectin through inflammatory signaling pathways, such as nuclear factor-κB (NF-κB) [6
]. These receptors are used as binding moieties by circulating immune cells such as monocytes, which express conjugate ligands such as very late antigen 4 (VLA-4) for VCAM-1 and lymphocyte function-associated antigen 1 (LFA-1) for ICAM-1 [8
]. The expression of these inflammatory cell recruitment receptors and the production of chemoattractant chemokines lead to increased infiltration of circulating monocytes, which will also express cytokines, thus perpetuating a positive feedback loop.
Upon entering the plaque, monocytes differentiate into macrophages and become activated under the influences of macrophage colony stimulating factor (M-CSF) and tumor necrosis factor-α (TNF-α), both of which are upregulated in plaque cells [10
]. These activated macrophages ingest large amounts of lipids via upregulation of scavenger receptors [11
], eventually becoming foam cells [12
]. These foam cells are prone to apoptosis, releasing damaging cytokines and enzymes that exacerbate the immune response, recruiting more inflammatory cells that amplify the process of plaque formation.
Defective efferocytosis of lipid-laden apoptotic cells also aggravates the situation. Eventually, the formation of a necrotic core, often characterized by the accumulation of cholesterol crystals, marks the transition to a vulnerable plaque that is prone to rupture. In addition to this molecular based degradation, physical destabilization and degradation of the fibrous cap is a consequence of pro-inflammatory signaling. The fibrous cap is composed mostly of collagen secreted by vascular smooth muscle cells migrating into the plaque from the medial layer of the vessel [13
]. The upregulation of matrix metalloproteinases (MMP-3 and -9) leads to the degradation of the collagenous cap [10
]. These cumulative events are all a result of chronic and non-resolving inflammation [14
] that can be countered by anti-inflammatory cytokines, such as interleukin-10 (IL-10) [15
]. IL-10 and other anti-inflammatory cytokines help to influence the ratio of activated pro-inflammatory “M1” macrophages to pro-healing “M2” macrophages via the janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway [16
]. In the event that the thinned fibrous cap ruptures, damaging lipids, enzymes, cytokines, calcium, and dead cell fragments are released into the blood, stimulating the formation of a thrombus that can quickly occlude the artery, leading to acute clinical events [17
Interest in the potential to apply nanotechnology to cardiovascular disease has been high for some years [19
], allowing for developments using nanoparticle research specifically for atherosclerosis. Nanoparticles (NPs) are uniquely suited to combat atherosclerosis given their ability to encapsulate various therapeutics such as nucleic acids, drugs, proteins, and even cells. Encapsulation serves two purposes in the field of nanomedicine: firstly, it protects the in vivo environment from harmful drugs or off-target effects by ensuring that drug release is controlled via material properties and/or targeted to the affected area via surface properties. Secondly, encapsulation protects labile cargo from degradation and/or other unwanted modifications. In addition, the high surface area to volume ratio of nanoparticles makes them ideal for surface functionalization for the purpose of targeting plaque components and/or evading the body’s immune system and clearance. The two most popular moieties incorporated onto nanoparticles are targeting ligands (antibodies, peptides, aptamers, or small molecules) specific for plaque components and PEGylation, which confers stealth and stability in vivo.
Many of the applications of NPs in atherosclerosis have focused on imaging (Figure 1
). Ultrasound imaging using contrast-enhancing agents such as gas-filled particles or microbubbles has been ongoing for decades [20
]. Though not quite using nanoparticles, ultrasound imaging has nevertheless inspired numerous nano-scale imaging and treatment options, and has been used for atherosclerosis [21
]. However, there have also been interesting less conventional uses of nanoparticles in modelling, sensing, and elucidating the biology of plaque progression in atherosclerotic mice and hyperlipidemic rabbits. NPs may even participate in the treatment of atherosclerosis, acting by virtue of their material-based natural interactions with plaque components rather than by the delivery of therapeutics. Nanoparticles vary in their molecular makeup, with inorganic components being more useful for studies involving physical phenomena (imaging, photodynamic therapy, etc.) and organic materials being typically chosen for their ability to interact with cargo (protection, encapsulation, controlled release, etc.) and for their increased biocompatibility and biodegradability. However, NPs made of both materials can be targeted to the plaque by fine tuning their size, shape, surface properties, and ligand coating. Exploring these various phenomena can add exciting perspective to the numerous reviews discussing experimental nanoparticle interventions for atherosclerosis, which have focused on imaging or drug delivery [23
]. Thus, this review article illustrates how nanoparticles are advancing the research of atherosclerosis using animal models through the probing of fundamental biological interactions, delivering therapeutics, and reporting back to researchers via imaging or sensing modalities as a function of the materials chosen.
5. Conclusions and Perspectives for Future Work
Many types of nanoparticles have made their way into all areas of research into atherosclerosis including its pathology, sensing, therapeutic delivery strategies, imaging, and development of more physiologically relevant models. From a bioengineering perspective, the convergence of advancements in both atherosclerosis pathology and nanoparticle materials research provides a unique nano-sized window through which we can see the future of diagnosis and treatment. In fact, the gamut of advanced nanoparticle formulations available today is so wide that researchers seem to be able to therapeutically intervene in atherosclerosis at every stage of disease progression (Figure 7
). With the advances in materials that allow for fine tuning of NP properties, tracking, reporting, and sensing, it will not be long before nanoparticles become standard tools to uncover the underlying events in atherosclerosis progression. At the same time, researchers have found and continue to find new and interesting therapeutics to encapsulate within NPs for delivery, each with its own unique set of design criteria and constraints. Furthermore, NPs can be evaluated on smaller and smaller microfluidic models as we approach true disease-on-a-chip models.
The delivery of biotherapeutics seems uniquely suited to addressing the underlying inflammation at the heart of atherosclerosis. The aforementioned strategies involving the delivery of RNAi, which led to an increased deposition of collagen and stable fibrous cap, were promising. Furthermore, the delivery of biotherapeutics has the potential to extend into the exciting areas of gene editing, whole genome, and personalized medicine strategies in the context of atherosclerosis. However, there are still relatively few studies and more work needs to be done to corroborate these findings in more and larger animal models.
The translational potential of NP-mediated strategies remains unknown as regulatory agencies must adapt novel methods of evaluation for these complex technologies. What seems necessary for new strategies to succeed is interdisciplinary cooperation between research fields. It is no longer enough to design and synthesize novel NPs and evaluate their efficacy/safety in one cell type in a petri dish. They must be tested in vivo or in as close to an in vivo environment as possible on microchips or in silico. The importance of screening NP materials for toxicity during experiments in vitro and in vivo should not be overlooked as well. Targeting the endothelium in a safe and effective way is the ultimate goal for NP-mediated therapies [133
], but it may induce added damage, which can accelerate atherosclerosis lesion formation [144
There is no shortage of constraints in using nanotechnology for any purpose in biology. However, researchers continue to identify novel targets for imaging, diagnosing, and treating atherosclerosis using nanoparticles. With the recent combination of therapeutics delivered directly and specifically to the vulnerable plaque, a targeted NP-based theranostic treatment based on NP-elucidated pathologies is becoming more possible.