Review

Metal Nanoparticles in Atherosclerosis: Applications and Potential Toxicity

Fatemeh Imanparas 1,2, Mahmood Doosti 2, Mohammad Ali Faramarzi 1*

1Dpartment of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 1417614411, Iran.

2Department of Medical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran 1417614411, Iran.

* Corresponding author. E-mail: faramarz@tums.ac.ir  

Received: Aug. 15, 2015; Accepted: Sept. 26, 2015; Published: Sept. 30, 2015.

Citation: Fatemeh Imanparast, Mahmood Doosti and Mohammad Ali Faramarzi. Metal Nanoparticles in Atherosclerosis: Applications and Potential Toxicity. Nano Biomed. Eng. 2015, 7(3), 111-127.

DOI: 10.5101/nbe.v7i3.p111-127.

Abstract

As a chronic inflammatory disease, atherosclerosis is responsible for thousands of deaths worldwide each year, and it imposes massive economic costs on individuals and on society. Because of its high importance, the discovery of sensitive and accurate strategies for imaging, targeted drug delivery, and therapeutic monitoring of this condition is essential. In recent years, continuous research has achieved remarkable successes in the use of nanotechnology in the molecular imaging and treatment of atherosclerosis. Among various nanoparticles — such as metallic, polymeric, and lipid — metallic nanoparticles are being considered due to their unique properties for use in treatment and imaging. It should be taken into consideration that some of the metal nanoparticles themselves can cause adverse biological effects, and these effects should be considered important risk factors in the pathological pathways leading up to atherosclerosis. This review provides a description of the applications and potential toxicity of metal nanoparticles in atherosclerosis.

Keywords: Atherosclerosis; Metals; Imaging; Drug delivery; Nanoparticles; Toxicity

Abbreviations

NPs, Nanoparticles; CVD, Cardiovascular disease; CHD, Coronary heart disease; ox-LDL, Oxidized low-density lipoprotein; VSMCs, Vascular smooth muscle cells; NO, Nitric oxide; eNOS, Endothelial NO synthase; ROSs, Reactive oxygen species; O2.-, Superoxide; VCAM-1, Vascular cell adhesion molecule-1; ICAM-1, Intercellular adhesion molecule-1; M-CSF, Monocyte colony-stimulating factor; SRs, Scavenger receptors; MMPs, Matrix- degrading metalloproteinases; PDGF, Platelet-derived growth factor; FGF-2, Fibroblast growth factor-2; TGF-β, Transforming growth factor-β; MRI, Magnetic particle imaging; US, Ultrasound; PET, Positron emission tomography; CT, Computed tomography; SPECT, Single photon emission computed tomography; USPIO, Ultra-small particles of iron oxide; SPIONs, Superparamagnetic iron oxide nanoparticles; AHA, American Heart Association; Au, Gold; Au- HDL, Gold high-density lipoprotein nanoparticles; PPTT, Plasmonic photothermal therapy; NIR, Near- infrared; IVUS/IVPA, Intravascular ultrasound and photoacoustic; AgNPs, Silver NPs; AGE, Advanced glycation end; MPs, Microparticles; TiO2, Titanium dioxide; Fe2O3, Iron oxide; Y2O3, Yttrium oxide; ZnO, Zinc oxide; HAECs, Human aortic endothelial cells; Zn, Zinc; HUVEC, Human umbilical vein endothelial cells.

Introduction

In recent years, nanoparticles (NPs) have been used in the diagnosis, treatment, and monitoring of human diseases such as cancer and cardiovascular disorders due to their limited retention to binding sites and mild side-effects [1−3]. Among various types of NPs, metal NPs exhibit unique diagnostic and therapeutic properties and have been extensively applied for medical purposes [4, 5]. Atherosclerosis, one of the most prevalent chronic vascular inflammatory diseases, arises by endothelial dysfunction, imbalance in lipid metabolism, and maladaptive immune response [6, 7]. Metal NPs have many applications for imaging and targeted drug delivery to each step in the pathogenesis of atherosclerosis. Therefore, they can be of use in preventing the pathophysiological consequences of atherosclerosis, such as thrombotic strokes [8−12]. Although metal NPs have various applications, they may have limitations and may be toxic to humans [13−17]. This review provides a description of the benefits and risks of metal NPs in the treatment or induction of atherosclerosis. It also describes metal NP-based strategies regarding site- selective delivery of diagnostic probes to therapeutic targets of atherosclerosis disease. The targeted use of metal NPs and their toxicity are dependent on disease mechanisms. Therefore, before a description of the benefits and risks of metal NPs is given, a summary of the cellular and molecular mechanisms of atherosclerosis is useful to clarify the role of metal nanoparticles in atherosclerosis.

Pathogenesis of Atherosclerosis

Cardiovascular disease (CVD) is the most common cause of death in modern industrialized countries and an increasingly frequent cause of death in developing countries [8, 18]. The main types of CVD are coronary heart disease (CHD) and stroke that atherosclerosis is the pathology that causes them, it is a multifactorial disease and involves several cell types [19, 20]. This section summarizes the cellular and molecular mechanisms of atherosclerosis. In summary, the pathologic steps that cause atherosclerosis include endothelial dysfunction initiated by the risk factors, inflammation, foam cell formation (up-take of the oxidized low-density lipoprotein (ox-LDL) by macrophages), migration and proliferation of vascular smooth muscle cells (VSMCs), plaque rupture, and thrombus formation [21-23]. Because metal NPs can be used at each step of the pathogenesis of atherosclerosis, and because their specificity for each step is dependent on targeting by specific molecules involved in every step, the above steps are described briefly below. Atherosclerosis is initiated through damage of the endothelium by risk factors such as hypertension, diabetes, elevated plasma homocysteine concentrations, smoking, and high levels of LDL [24−28]. The effects of these risk factors on endothelial cells manifest as molecular changes. These molecular changes are primarily due to a decrease in normal nitric oxide (NO) production by endothelial NO synthase (eNOS), which is due to an increase in reactive oxygen species (ROSs) production by an increase in the expression and activation of the superoxide (O .-)- producing enzymes, especially NADH2  oxidase. Increased O2.- decreases NO production by eNOS, and this enzyme produces O .- rather than nitric oxide (Fig. 1) [29]. NO modulates many processes involved in the normal activity of endothelial cells, and ROSs modulates many processes involved in atherosclerosis, such as inflammation, apoptosis, VSMC proliferation, angiogenesis, and matrix turnover. The final result of these molecular changes is endothelial dysfunction [30, 31]. Endothelial dysfunction increases endothelial expression of selectins and cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) [32]. Platelets adhere to dysfunctional endothelial cells and release inflammatory factors such as proteases and vasoactive substances, which leads to recruitment and adhesion of neutrophils and monocytes to this area [33, 34]. First, leukocytes roll along the endothelial surface of the vessel wall by selectins — P-selectin, for example — and then adhere tightly to the endothelium by VCAM-1 and ICAM-1 [32]. Tight junctions between endothelial cells are destroyed due to endothelial injury; as a result, the endothelium permeability increases, and leukocytes can migrate through the endothelium into the intima. In the intima, monocytes are influenced by growth factors such as monocyte colony-stimulating factor (M-CSF) and cytokines such as TNF-α and IFN-γ, and they then differentiate to macrophages. Due to increased permeability of the endothelium, LDL infiltrates into the blood vessel wall intima and is there modified to oxidized LDL by ROSs [35]. During the maturation process of macrophages, expression of various scavenger receptors (SRs) — including SRs-A and CD36 — that can bind and internalize modified LDL increase. Due to increased SRs-A and CD36 expression on macrophages and ox-LDL in the intima, macrophages internalize ox-LDL, resulting in the accumulation of ox-LDL in the macrophages and the formation of foam cells (Fig. 2). Foam cells release growth factors and cytokines — which are involved in lesion progression — and matrix-degrading metalloproteinases (MMPs) — which stimulate matrix degradation [36−38]. VSMCs are stimulated by growth factors and cytokines — such as platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), and transforming growth factor-β (TGF-β) — and migrate from the media into the intima. As a result of the formation of a fibrous cap, fatty streaks develop to advanced atherosclerotic plaques (Fig. 3). MMPs permit the migration of VSMCs from the media into the intima; as a result, the plaques develop into more complex lesions. Macrophages under the influence of inflammatory cytokines such as IFN-α also undergo apoptosis, which can result in necrotic core formation [23, 37, 39]. Plaque rupture can occur by thinning of the fibrous cap due to VSMCs, macrophage apoptosis, and inflammation occurring within the plaque. Plaque rupture can result in activation of proteases such as serine proteases and MMPs, leading to degradation of extracellular proteins, such as collagen, and thinning of the cap. Research also suggests that microvessels form more frequently in unstable plaques than in stable plaques; in other words, microvessel rupture actually causes the instability in unstable plaque [37, 40−42]. Plaque rupture exposes highly thrombogenic plaque constituents to the bloodstream, leading to platelet aggregation and thrombus formation. A thrombus can completely occlude the blood vessel, resulting in heart attack or stroke. Some important biomarkers that are used as targets for NPs in imaging and drug delivery for atherosclerosis are presented in Table 1 [10, 12, 43-52].

Fig. 1 The production of NO in endothelial cells and control of vascular tone. (a) A normal endothelial cell. Normal levels of NO are produced from L-arginine by eNOS in the presence of BH4 as its cofactor. NO modulates many processes involved in the normal activity of endothelial cells, such as regulation of vascular tone by conversion of GTP to cGMP as an activator of protein kinase G. An important action of this enzyme is vascular smooth muscle relaxation, which leads to vasodilation. (b) A dysfunctional endothelial cell and eNOS uncoupling. Cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes mellitus, and−chronic smoking increase expression and activation of O2·- producing enzymes (especially NADH2 oxidase) that increase ROSs in the vascular wall. O2·- reacts avidly with vascular NO to form ONOO-. The cofactor BH4 is highly sensitive to oxidation by ONOO-. Diminished levels of BH4 promote O2·- production by eNOS; this is referred to as eNOS uncoupling.

Fig. 2 Foam cell formation. In normal circumstances, tight junctions between endothelial cells prevent the entry of monocytes into the sub-endothelial space (intima). Initial damage of the endothelium by cardiovascular risk factors (1) results in an inflammatory response. As a result, ROSs production increases (2). ROSs cause breakdown of tight junctions (3) and expression of P-selectin and cellular adhesion molecules (CAMs) (4). High LDLs enter into the sub-endothelial space (5) and oxide by ROSs (6). P-selectin and CAMs recruit circulating monocytes (7−8), monocytes enter into the artery wall from the bloodstream (9), and there the monocytes differentiate into macrophages via pro-inflammatory factors such as M-CSF (10). Pro-inflammatory factors act on macrophages, leading to increased expression of downstream scavenger receptors (SRs) on their surface (11). Macrophages then ingest oxidized LDL by SRs (12) and convert to foam cells (13).

Fig. 3 Formation of the plaque. VSMCs are stimulated by MMPs and growth factors — such as transforming growth factor-β (TGF-β) — and they then migrate from the media into the intima. VSMCs and extracellular matrix fibers such as collagens and fibronectin form a fibrous cap and develop foam cells to advanced atherosclerotic plaques.

Table 1: Some of the important biomarkers of atherosclerosis for targeting imaging and drug delivery

Metal Nanoparticles and Atherosclerosis

Metal nanoparticles have many applications in industry and in medicine [53−55]. In recent years, many efforts have been made to develop early detection methods for molecular and cellular abnormalities leading to atherosclerosis and to develop treatments for preventing fatal consequences such as heart attacks and strokes [56−59]. From the expression of the molecular mechanisms leading to atherosclerosis described in the second section, it can be inferred that several key steps — including endothelial dysfunction, inflammation, foam cell formation, VSMC migration and proliferation, plaque rupture, and thrombus formation — are involved in this disease, and that each of these steps can be targeted by imaging techniques for diagnosis and treatment. Conventional imaging techniques in the diagnosis of atherosclerosis include magnetic particle imaging (MRI), ultrasound (US), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT) [60−64]. Although these methods are only capable of detecting cellular and molecular changes in advanced stages of atherosclerosis, scientists are currently looking at ways to detect atherosclerosis as early as possible [65, 66]. NPs can be used in the prevention, control, diagnosis, treatment, and screening of atherosclerosis. Targeted NPs are considered efficient tools for localizing and staging of atherosclerosis and for identifying it as quickly as possible in patient check-ups. Various materials — such as liposomes for ultrasound imaging, polyamidoamine (PAMAM) and diaminobutane (DAB) dendrimers for MIR, gold NPs for optical coherent tomography, quantum dots for fluorescence tomography, and iron microparticles or dextran coated ultra-small particles of iron oxide (USPIO) for MIR — are used to enhance image quality and sensitivity to the various stages of atherosclerosis [67]. Metal NPs possess unique chemical and physical properties and have various applications in medicine according to their size and type. Due to the resonant oscillations of their free electrons in the presence of light, metal NPs have unique photothermal and optical properties. They also have the ability to radiate light, which can be used in imaging, and rapid conversion into heat, which is used in the treatment of artherosclerosis [68]. Although the use of metal NPs in the diagnosis and treatment of diseases such as atherosclerosis is being considered, it should be noted that NPs of certain types and particle sizes can disrupt the body’s homeostasis and can cause diseases such as atherosclerosis [14, 69]. In the following sections, metal NP applications in the monitoring, diagnosis, and treatment of atherosclerotic disease will be discussed. The adverse effects of some metal NPs, which may lead to atherosclerosis, will also be discussed [69].

Iron oxide nanoparticles and atheroscle-rosis

Today, molecular and cellular imaging for non- invasive control of the biological processes at each stage of a disease are being considered, and in the case of atherosclerosis, access to contrast agents is a necessity. Superparamagnetic iron oxide nanoparticles (SPIONs) are considered an efficient contrast agent, and they have many applications, especially in revealing inflammatory diseases and identifying the cell surface markers of various diseases such as cancer and atherosclerosis [11, 51,70−73]. Therefore, the use of SPIONs in molecular imaging of the various stages of atherosclerosis provides early detection and monitoring of treatment effectiveness. Although SPIONs have been used in imaging for many years, efforts are focused on improving their surface and targeting strategies for identifying specific cell-surface molecules (Table 2) [9, 10, 12, 45, 74−89]. This section will offer a glimpse of the structural and functional properties of SPIONs and provide a description of their applications in atherosclerosis, particularly in imaging, surface modification, and targeting to increase their effectiveness. Finally, the negative effects that they may have in the pathogenesis of this disease will be discussed. SPIONs typically consist of two parts: an iron oxide core (Fe3O4 and γ Fe2O3) and a hydrophilic coating. They are available in a wide range of sizes, including oral-SPION at between 300 nm and 3.5 μm, standard SPIO (SSPION) at 60-150 nm, and an ultra-small SPIO (USPION) of approximately 10−40 nm [90]. There are two general biological and chemical methods for the synthesis of metal NPs, but the most common chemical methods for production of SPIONs are co-precipitation (ferrous and ferric co-precipitation in an alkaline solution) and microemulsions (suspending a ferrous salt-surfactant precipitate to an aqueous solution, then adding a base to form a magnetic precipitate) [90, 91]. Surface coating agents — such as citrate, dextran, polycarboxymethyl dextran, PLGA, and silica — are used for colloid stability and biocompatibility of SPIONs [90, 92−93].As discussed above, the size and particular coating of SPIONs effect their pharmacokinetic characteristics, such as phagocytosis capacity, bloodstream clearance, the time required for detection of loss of signal on MRI, toxicity, and the mechanisms of their endocytosis into target cells [94, 95]. Such as the time required for detection of a loss of signal on MRI for citrate-coated SPIONs is much shorter than that of polymer-coated SPIONs [87, 88]. Wagner et al. studied accumulation of citrate-coated SPIONs in atherosclerotic plaques, and the results of this study indicated that the time required for detection of loss of signal on MRI for these SPIONs is 1 h after intravenous injection (compared to 24 to 48 h for polymer-coated SPIONs) with a dose 0.05 of mmol Fe/kg (compared to 0.2−1 mmol Fe/Kg for nanoparticles coated with polymer [88]. Due to their small size, USPIONs are able to escape the phagocytic systems of the liver and the spleen, so they have a long half-life in the bloodstream. This allows for their uptake by macrophages throughout the body, such as the macrophages in the atherosclerotic plaque (Fig. 4) [96, 97]. Due to their appropriate biocompatibility and bioavailability, dextran-coated USPIONs are a suitable contrast agent in MRI of atherosclerotic plaques. Some forms of dextran-coated USPIONs, such as Ferumoxtran-10, are produced commercially and used for imaging of atherosclerotic plaques and for the monitoring of the effects of drugs such as statins [9, 11, 56, 77, 98]. Morris et al. used UPIONs to assess the effects of P38 MAPK inhibitor (SB239063) on atherosclerotic plaque [99]. Appropriate surface modifications of SPIONs may even be helpful in avoiding their adverse effects; vascular endothelial dysfunction is caused by SPIONs through increased oxidative stress, and stimulates the expression of adhesion molecules that the surface modification of SPIONs can reduce these adverse effects [87]. In addition to appropriate coating of the surface, specific functional groups on SPION surfaces for atherosclerotic biomarkers for having minimum signal-to-background ratio, minimal time for detecting signal loss of MRI and limited retention of them at atherosclerotic plaque are required [76]. Therefore, researchers need a detailed understanding of the mechanisms of atherosclerotic plaque formation and the identification of specific cell surface biomarkers such as SR-As on macrophages and VSMCs in plaques [100-101]. Segers et al. reported successful development of SR- A-targeted USPIO and its use in animal models for imaging of atherosclerotic plaques [10]. Investigations have shown that macrophage burden and the presence of plaque calcifying microvesicles are appropriate criteria for plaque characterization. However, the presence of calcifying microvesicles is a much more efficient criteria for determining the American Heart Association (AHA) type and amount of plaque instability [11, 102, 103]. Citrate-coated SPIONs accumulate in macrophages and microvesicles, whereas polymer-coated SPIONs are only able to accumulate in macrophages [88]. Although it is often said that vulnerable plaques have a thin fibrosis cap with a lipid-rich core, they may also have other molecular and histopathological characteristics. The development of efficient diagnostic methods for the identification of biomarkers for each step involved in the pathogenesis of atherosclerosis can be used to determine the appropriate therapeutic strategies for the prevention of thrombosis [76, 104−106]. In 2012, Burden et al. reported successful development of R832-USPION and R826-USPION for targeting of VCAM-1 (as a biomarker of inflammation) and phosphatidylserine (as a biomarker of apoptosis). SPIONs can also be used for targeted drug delivery such as transfection of therapeutic genes to different cells involved in the pathogenesis of this disease [107, 108]. Due to the high importance of endothelial injury at the start of the molecular and cellular changes leading to atherosclerosis, the vascular endothelium is a therapeutic target for genetic modification by therapeutic gene transfection to resistance against damage [109]. Namgung et al. developed hybrid SION-branched polyethylenimine magnetoplexes for therapeutic gene transfection to endothelial cells (Fig. 4) [58]. SPIONs mainly undergo endocytosis into monocytes/macrophages by using non-specific receptors, which is known as passive targeting [10]. In endocytosis of SPIONs into macrophages, cytokines and receptors such as SR-A and MAC-1 are involved [10, 51, 58, 70, 110, 111]. Litovsky et al. reported that the uptake of SPIONs into macrophages in the presence of TNF-α, IL-1β and interferon-γ takes place multiple times [70]. Different cell types involved in the pathophysiology of atherosclerosis express SR-A receptors at a high-level, and these receptors play a role in many processes leading to plaque formation. In lipoprotein endocytosis in particular they play a key role [100, 101], so they probably also have a role in the uptake of SPIONs into macrophages. Raynal et al. reported the relationship between SPIONs endocytosis and SR-A by comparing the role of these receptors in endocytosis of ferumoxide (dextran-coated iron oxide NPs, 120-180 nm) and ferumoxtran10 (dextran- coated iron oxide NPs, 15-30 nm). The results of their experiments showed that ferumoxide endocytosis is inhibited in the presence of specific ligands of SR- A, polyinosinic acid, and fucoidan, but these have no effect on the endocytosis of ferumoxtran10. Consequently, the SR-A receptors may play no role in small-sized SPIONs endocytosis; other receptors are probably involved [111]. As integrin Mac-1 receptors on the surface of monocytes, particularly activated monocytes (macrophages), express at high levels in inflammation and involve in processes such as leukocyte adhesion to endothelial cells [112]. It is likely that these receptors are also involved in the uptake of SPIONs, particularly small SPIONs [10]. Muhlen et al. studied the role of Mac-1 in endocytosis amino-PVA SPIONs and ferumoxtran10 NPs with different coatings and similar sizes (~30 nm) in vitro, results showed endocytosis of both types of nanoparticle was inhibited in the presence of anti- Mac-1 antibodies. Therefore, Mac-1 receptors are involved in the endocytosis of small SPIONs with different coatings [113]. Muhlen et al. surveyed Mac-1’s role in the endocytosis of SPIONs into macrophages in vivo by comparing the uptake of anti- Mac-1 antibody-targeted SPIONs to the uptake of non- targeted SPIONs. However, contrary to expectations, no differences in NPs uptake by macrophages in either targeted or non-targeted SPIONs were observed [51]. This result was inconsistent with the result in vitro, perhaps due to the fact that Muhlen used large particles with a size of approximately 100 nm for the in vivo studies. It is recommended that both in vitro and in vivo studies be done simultaneously with the small SPIONs.