Abstract
Identification of a vulnerable atherosclerotic plaque before rupture is an unmet clinical need. Integrating nanomedicine with multimodal imaging has the potential to precisely detect biological processes in atherosclerosis. We synthesized silica-coated iron oxide nanoparticles (SIONs) coated with rhodamine B isothiocyanate and polyethylene glycol and investigated their feasibility in the detection of macrophages in inflamed atherosclerotic plaques of apolipoprotein E-deficient (ApoE−/−) mice via magnetic resonance (MR) and fluorescence reflectance (FR) imaging. In vitro cellular uptake of SIONs was assessed in macrophages using confocal laser scanning microscopy (CLSM). In vivo MR imaging was performed 24 h after SION injection via the tail vein in 26-week-old ApoE−/− mice fed a high-cholesterol diet (HCD). We also performed FR imaging of the extracted aortas from four different mice: two normal-diet-fed C57BL/6 mice injected with saline or 10 mg/kg SIONs and two HCD-fed ApoE−/− mice injected with 5 or 10 mg/kg SIONs. The harvested aortas were cryosectioned and stained with immunohistochemical staining. The CLSM images at 24 h after incubation showed efficient uptake of SIONs by macrophages, with no evidence of cytotoxicity. The in vivo and ex vivo MR and FR images demonstrated SION deposition in the atheroma. Upon immunohistochemical staining of the aorta, CLSM images revealed colocalization of macrophages and SIONs in the atherosclerotic plaque. These results demonstrate that polyethylene glycosylated SIONs could be a highly effective method to identify macrophage activity in atherosclerotic plaques as a multimodal imaging agent.
Introduction
Magnetic nanoparticles (MNPs), which are one of the most widely used nanomaterials, possess great potential for application in clinical diagnostic and therapeutic techniques, which has led to their increasing use in the screening, diagnosis, and treatment of cancer, cardiovascular disease, and neurological disease.1–6 In particular, due to their intrinsic magnetic properties to enhance proton relaxation of specific tissues, the use of MNPs as magnetic resonance (MR) imaging contrast agents is one of the most promising applications of nanomedicine.
Dextran-coated, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs) have been utilized to detect inflammatory plaques in preclinical models, including atheroprone rabbits, due to their excellent biocompatibility and ease of synthesis.7,8 As USPIOs circulate and transiently penetrate the vessel wall, plaque macrophages phagocytose these particles, which results in a strong signal loss on T2-weighted MR imaging. Although USPIOs such as ferumoxtran (Sinerem®, Guerber, Paris, France) have shown promising clinical and preclinical utilities for detecting plaque macrophages, they have several intrinsic weaknesses as contrast agents in molecular imaging, such as a relatively lower relaxivity because of the small iron oxide core. This results in a larger administration dose and difficulty in linking specific ligands to the particles, which would enable the targeting of unique molecules and cells in high-risk plaques.
Silica provides a chemically and mechanically stable scaffold for the iron oxide core, superior biocompatibility with less in vivo toxicity, and an easily functionalized surface for simpler conjugation of various targeting ligands.9–11 In addition, diverse molecules, such as fluorescent dyes or targeting ligands, can simply be integrated into a silica shell.12,13 In this study, we synthesized iron oxide NPs of superparamagnetic particle size and coated the surfaces with organic dye-incorporated silica. This newly synthesized polyethylene glycosylated (PEGylated) silica-coated iron oxide nanoparticle (SION) would serve as a bimodal molecular imaging agent for MR and fluorescence reflectance (FR) imaging.
Since the application of SIONs in the molecular imaging of atherosclerosis is lacking, we investigated the feasibility of SIONs in imaging macrophages within atherosclerotic plaques of apolipoprotein E (ApoE)−/− mice. To accomplish this, we first investigated the efficiency of SION uptake by macrophages and validated the in vivo ability to detect macrophage-rich plaques in ApoE−/− mice via MR and FR imaging.
Methods
Synthesis of fluorescent silica-coated iron oxide nanoparticles (SIONs)
PEGylated SIONs labeled with rhodamine B isothiocyanate (RITC, Sigma-Aldrich, St. Louis, MO, USA) were synthesized following our previous report. 14 Briefly, ferrite (Sigma-Aldrich) was added into polyvinylpyrrolidone (PVP, Sigma-Aldrich) solution. The stabilized ferrite NPs were washed with 10% acetone, followed by centrifugation. Then, the supernatant was removed, and the NPs were resuspended in ethanol. Prior to silica coating, 3-aminopropyltriethoxysilane (APS, Gelest, Morrisville, PA, USA) and RITC were reacted under nitrogen, and the resultant solution was mixed with tetraethoxysilane (TEOS, Gelest). This solution was added to the purified NPs and polymerized by adding ammonia. Next, the resulting NPs were centrifuged and resuspended in basic ethanol containing ASP and polyethylene glycol (PEG) (molecular weight of 460–590 Da; Gelest Inc., Tullytown, PA, USA), followed by additional stirring to obtain SIONs.
Characterization of SIONs
The morphology and size of SIONs were confirmed by transmission electron microscopy (TEM, H-7600, Hitachi Ltd., Japan). Fe concentrations of SIONs were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES, Optima 4300 DV, Perkin-Elmer, Waltham, MA, USA), and T2 relaxation measurements in aqueous solution containing SIONs were performed on a 4.7-T animal MRI system (Biospec 47/40, Bruker, Germany).
In vitro uptake of SIONs in murine macrophages and viability test
RAW 264.7 cells were maintained in culture dishes with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were cultured in a humidified atmosphere of 5% CO2 at 37°C. To examine the capacity of RAW 264.7 cells to phagocytose SIONs, cells were incubated with SIONs at concentrations lower than 100 µg Fe/ml for 24 h under standard culture conditions. The SION-loaded macrophages were washed three times with Dulbecco’s phosphate-buffered saline (DPBS), and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The cellular uptake of SIONs was observed by confocal laser scanning microscopy (CLSM; Zeiss LSM 510, Carl Zeiss, Germany). To assess the potential cytotoxic effect of SIONs, cell viability was assessed in RAW 264.7 cells using an MTT assay.
In vitro MR contrast measurements of RAW 264.7 cells labeled with SIONs
To elucidate the feasibility of SIONs as MR imaging agents, T2*-weighted MR images were verified at different macrophage concentrations in agarose gel phantoms. RAW 264.7 cells were incubated with SIONs (50 µg Fe/ml) for 24 h, and then the cells were prepared at seven different concentrations (0.2, 0.5, 0.8, 1, 2, 8, and 10 × 106 cells/ml) in 1% agarose phantoms. T2*-weighted MR images with a gradient-echo (FLASH) pulse sequence were measured with the following parameters: field of view (FOV) = 3 × 3 cm2, matrix = 128 × 128, slice thickness = 1 mm, repetition time (TR) = 130 ms, number of slices = 5, and number of scans = 4.
MR and FR imaging of the ApoE−/− mouse model of atherosclerosis
All animal experiment procedures were provided in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use Committee (IACUC) in the School of Medicine of The Catholic University of Korea. ApoE−/− mice were fed a 1.2% cholesterol diet from 8 to 26 weeks of age. SIONs (10 mg Fe/kg) were administered via tail vein injection in 26-week-old mice. In vivo T2*-weighted MR imaging was conducted before and 24 h after SION injection. For ex vivo FR imaging, ApoE−/− mice were sacrificed, and their aortas were extracted and fixed in 4% paraformaldehyde solution. C57BL/6 mice fed a normal diet were also injected with SIONs via the tail vein, and their aortas were extracted in a manner similar to that in ApoE−/− mice. We imaged the extracted aortas of C57BL/6 mice with saline injection, C57BL/6 mice with 10 mg/kg SION injection, and two ApoE−/− mice with 5 and 10 mg/kg SION injection using a fluorescence imaging system (MaestroTM; CRi, MA, USA).
Histological evaluation using fluorescence microscopy in the aorta
To evaluate the distribution of accumulated SIONs within the aorta after undergoing in vivo MR imaging, the aortas were extracted and mounted in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA), which were then frozen in liquid nitrogen. Each frozen aorta was cut into a 5 μm thickness using a cryostat (Microm HM 525, Thermo Scientific, MA, USA), and immunohistochemical staining for macrophages was performed. The primary antibody was a mouse monoclonal antibody against CD68 (ED-1, Abcam Inc., MA, USA), and the secondary antibody was a goat anti-rat Alexa Fluor 488-conjugated antibody (Abcam Inc., MA, USA). The aortic sections were stained with DAPI (blue color; Sigma-Aldrich Co., MO, USA) to identify cell nuclei and a mouse monoclonal antibody targeting CD68 (green color) to identify atherosclerosis-associated macrophages. These stained aortic sections were imaged by CLSM.
Results
Synthesis and characterization of SIONs
Monodispersed SIONs incorporating RITC were synthesized via a modified procedure of our previous work, in which this NP coating process with silica and PEG was conducted to provide highly biocompatible properties of the SIONs, resulting in a long blood half-life of approximately 3 h. 15 According to this procedure, approximately 10 nm diameter core iron oxide was first prepared, and sequential modification was performed by a RITC-incorporated silica and PEG coating. The final SIONs were obtained with an average size of 90 ± 7 nm (Figure 1(a)). MR T2 contrast behavior of SIONs was examined using a 4.7-T MRI system, and then the T2 relaxivity (r2) of SIONs revealed approximately 150 s−1mM−1, indicating a linear correlation with SION concentrations (Figure 1(b)).
Characterization of SIONs. Representative TEM images exhibited well-dispersed SIONs with an average size of 90 ± 7 nm (scale bar: 100 nm) (a). The relaxation rate R2 (=1/T2) was linearly dependent on the SION concentration (b). The T2 relaxivity (r2) calculated from the fitted line was 152.2 ± 6.7 s−1mM−1.
Cellular uptake and in vitro magnetic properties of SIONs in murine macrophages
RITC-containing SIONs were effectively taken up by macrophages following 24 h of incubation, and there was a concentration-dependent increase in the intracellular accumulation of SIONs (Figure 2(a)). After macrophages were incubated with 50 µg Fe/ml of SIONs for 24 h, nearly all cells were loaded with the SIONs. The intracellular Fe content in the SION-treated macrophages at 50 μg Fe/ml was 28 ± 6 pg Fe/cell, which was comparable to the content reported by others with a similar cell type and particle size. No sign of cytotoxicity was observed up to 100 µg Fe/ml of SIONs within the following 24 h of incubation (Figure 2(b)).
In vitro cellular uptake and MR phantom imaging of SIONs in mouse macrophages (RAW 264.7 cells). CLSM images after a 24-h incubation of RITC-containing SIONs at different SION concentrations are shown (400× magnification) (a). Cytotoxicity test of the SIONs in RAW 264.7 cells (b). In vitro MR phantom imaging of SION-loaded macrophages (c). T2*-weighted 4.7 T MR images of agarose gel suspension after a 24-h incubation of SION-treated macrophages (50 µg Fe/ml) depending on cell densities between 2 × 105 and 1 × 107 cells/ml.
The T2*-weighted MR images of the agarose phantom were measured by treating 100 µg Fe/ml of SIONs at different concentrations of macrophages. Representative MR images of SION-loaded macrophages with cell densities between 2 × 105 and 1 × 107 cells/ml are shown in Figure 2(c), in which signal intensity (SI) attenuation was observed depending on the cell density. This SI loss in the T2*-weighted MR images of the in vitro phantom demonstrated that SIONs could be used for in vivo imaging of monocyte/macrophage infiltrates in inflammatory sites.
In vivo MR images of the ApoE−/− mouse model
In vivo T2*-weighted MR images were taken before and after SION (10 mg/kg) injections in the ApoE−/− mouse model. In the MR images taken before SION injection (Figure 3(a)), the ascending and descending aortas showed no signal changes in their respective aortic walls. By comparison, the MR images taken after SION injection demonstrated strong focal signal loss (arrowheads) in the aortic wall (Figure 3(b)). This signal loss was a result of SION accumulation mainly in macrophages infiltrating the atherosclerotic plaque, indicating that in vivo MR imaging following biocompatible SION injection provided information on SION-deposited macrophage infiltration in the atherosclerotic plaque.
In vivo T2 MR images of ApoE−/− mice taken before (a) and after (b) SION injection (10 mg Fe/kg) via the tail vein (scale bar: 5 mm).
Ex vivo FR images of the aortas
To clarify SION deposition in the atherosclerotic plaque, ex vivo FR images were obtained from the extracted aortas after MR imaging (Figure 4). C57BL/6 mice injected with saline showed no fluorescence signal, whereas C57BL/6 mice injected with 10 mg/kg SIONs exhibited minimally visible blue-colored fluorescence. ApoE−/− mice treated with 5 mg/kg SIONs presented signal enhancement at the aortic root; however, a stronger red fluorescence signal was noted in ApoE−/− mice injected with 10 mg/kg SIONs. These FR images confirmed the results seen in the in vivo MR images.
FR and CLSM images in atherosclerotic plaques. SION deposition in atheroma as detected by FR imaging in extracted aortas of C57BL/6 and ApoE−/− mice (scale bar: 5 mm) (a). Representative CLSM images of CD68+ macrophages in the aortic root (scale bar: 200 µm) (b) and intimal lesions of the aortic root (scale bar: 20 µm) (c) treated with SIONs. Images of ApoE−/− mouse aortas after immunohistochemical staining for SIONs (red), nuclei (stained with DAPI; blue), and macrophages (stained with CD68; green).
Colocalization of SIONs and macrophages within atherosclerotic plaques
Atherosclerotic lesions in the aorta were assessed using DAPI (blue color) staining to identify nuclei and mouse monoclonal antibody targeting CD68 (green color) staining to identify macrophages. Based on fluorescence images of the aortic root section, macrophage areas in aortic root plaques (indicated by green fluorescence) overlapped with SION-derived fluorescence signals (red fluorescence). In particular, CD68+ intimal macrophages within plaque lesions were clearly colocalized with SIONs, presenting yellow signals.
Discussion
High-risk atherosclerotic plaques are characterized by their specific cellular and biological compositions. In this setting, macrophages are mainly involved in plaque disruption and thrombus formation, which are responsible for both fatal and nonfatal cardiovascular disease. Approaches to imaging macrophages have been extensively investigated to identify vulnerable atherosclerotic plaques in vivo. In this study, we investigated the cellular targeting and multimodal imaging capabilities of magnetic NPs in an experimental mouse atherosclerosis model and observed clear cellular uptake of SIONs in macrophages in vitro and in vivo. The SIONs allowed imaging of cellular inflammation in a mouse model of atherosclerosis via in vivo MR imaging and ex vivo multimodal imaging systems. The SIONs allowed for the successful imaging of macrophages embedded in atherosclerotic plaques.
Traditional cardiovascular imaging has focused on anatomy, but current molecular imaging is being expanded to interrogate pathological perspectives of initiation and progression of atherosclerotic plaques. These approaches are based on identifying stage-specific molecular markers or inflammatory cells, which can be detected by various contrast agents. USPIO particles, which are representatively used as T2 MR contrast agents, are taken up by macrophages and can induce a significant decrease in in vivo MR imaging signals in inflammatory lesions of atherosclerotic plaques, as has been reported in human studies. 16 A clear correlation of MR imaging signals with macrophage densities was introduced in a murine atherosclerosis model by using Gd3+-loaded micelles as a contrast agent. 17 Additionally, increased 18 F-fluorodeoxyglucose ( 18 F-FDG) uptake in human atherosclerotic plaques was detected by positron emission tomography depending on the number of macrophages. 18 Imaging atherosclerosis with SPIO particles, however, is less understood than the other contrast agents mentioned above. We demonstrated the possibilities for this type of particle, such as novel atherosclerosis-targeted imaging NPs and a noninvasive imaging system to assess inflammation in atherosclerosis.
SIONs utilize two mechanisms to improve their use in biomedical applications: a core-shell structure and polymeric coating. The size, shape, and surface modification of nanoparticles can be adjusted by physical and chemical modifications to not only allow for an increase in magnetic properties but also improve its impact on the in vivo behavior of NPs.6,19–21 In its simplest form, magnetic NPs consisting of an inorganic core and biocompatible outer coating layer stabilize the particle under a physiological environment. Numerous strategies have been attempted to enhance coat NPs, forming “core-shell structures”. Some NPs, for example, gold-coated NPs, have proven useful in molecular imaging but have limitations with respect to long-term biocompatibility. Silica coating of NPs theoretically allows for better biocompatibility and prevents the potential degradation of the inner core and encapsulated molecules, including alternative diagnostic agents or drugs.12,13 In addition, the polymeric coating is largely used to (i) prevent magnetic NPs from aggregating under physiological conditions; (ii) act as a barrier to protect the magnetic core from the aqueous solvent; and (iii) allow for sites for chemical modification with targeting agents, such as peptides or antibodies.22,23 As a representative example, PEG has been widely studied and proposed as an efficient strategy for an NP coating for biomedical applications.
SIONs, a new contrast particle of SPIOs with some of the specialized structures mentioned above, extended the use of MR imaging to cardiovascular diseases and had several advantages over currently available macrophage-targeting USPIOs detecting atherosclerosis.8,16,24 SPIOs approximately 150 nm in diameter are more susceptible to phagocytosis than USPIOs approximately 30 nm in diameter, and earlier reports confirmed that phagocytic uptake of iron oxide increased with increasing particle size.25,26 This conception was applied to the currently available SPIO and USPIO, as Metz et al. 27 and Lunov et al. 28 showed that in vitro loading of human monocytes for MR imaging is most effectively obtained with SPIO compared to USPIO. SIONs have an average diameter of 90 nm, which is larger than that of USPIO; therefore, we can assume that SIONs are more efficiently phagocytosed by macrophages based on their size.
SIONs have a higher value of T2 relaxivity than do USPIO-based particles. Ferumoxides, a commercially available SPIO, have 160 s−1mM−1 T2 relaxivity at 0.47 T. The T2 relaxivity of ferumoxtran, an actively investigated USPIO, is 80 s−1mM−1 at 37°C and 0.47 T. Because of its lower T2 relaxivity value, more ferumoxtran is needed to produce the same signal intensity in the T2 MR image. Given the same amount of iron oxide NPs, ferumoxides elicit a higher signal intensity than ferumoxtran. The T2 relaxivity of the SIONs in our current study is 150 s−1mM−1, which is approximately twice that of currently available USPIO particles. This implies that 50% fewer SIONs can create the same signal intensity as that by USPIO particles.
Coating the SIONs with PEG gives these particles several benefits, as mentioned above. PEG is nontoxic, nonimmunogenic and resistant to protein aggregation of magnetic NPs and conveys an extended circulation time. 29 Magnetic NPs coated with charged polymers are prone to opsonization due to strong electrostatic interactions between their surface and plasma proteins. By contrast, neutral polymers contain an abundant number of neutral and hydrophilic groups that offer excellent resistance against opsonization. SPIO was originally investigated as a contrast agent for liver and spleen imaging because of its selective uptake by Kupffer cells and reticuloendothelial systems. On the other hand, USPIO is small enough to evade the reticuloendotherlial systems. The half-life of ferumoxide in rats, which is 0.1 h, is shorter than that of ferumoxtran, 1.4‒3 h. Coating the SIONs with PEG rendered them able to evade uptake by the reticuloendothelial systems, resulting in a longer half-life in the blood of rats (approximately 2 h). Therefore, SIONs are more likely to be phagocytosed by macrophages in the blood and other inflamed tissues, such as atherosclerotic lesions.
Coating NPs with silica and PEG provides multifunctional capacities. Ma et al. 30 described core-shell magnetic NPs composed of iron oxide cores approximately 10 nm in diameter surrounded by a SiO2 shell 10‒15 nm thick. In this study, an organic dye, tris (2,2′-bipyridine) ruthenium, was incorporated with a second silica shell to provide a luminescence signal and prevent quenching by interacting with the magnetic core. In recent years, a wide variety of in vitro and in vivo applications have been demonstrated, suggesting that silica-coated NPs allow peripheral labeling of cancer cells31,32 and mesenchymal stem cells.33,34 In addition, antibody-conjugated silica-coated NPs allow for multitarget monitoring of bacterial species.35,36 Surface modification of magnetic NPs with PEG and folic acid has been used to facilitate their uptake to specific cancer cells for diagnosis and treatment purposes.37–39
Our main study limitation is that the colocalization of SION and macrophages bound by CD68 antibodies in the CLSM images does not necessarily mean that the same macrophages were directly inside pre-established atherosclerotic lesions. We cannot be sure that SIONs truly accumulated at the site of atherosclerotic lesions based on the CLSM images. In addition, the results obtained in the murine atherosclerosis model may not fully reflect the pathogenesis in the human athrogenesis, thereby limiting direct translation into clinical setting. Accordingly, many aspects of clinical applications concerning e.g. clinical dose, iron oxide content, and signal properties should be further refined and examined in order to extent this study toward clinical trials, including multiple comparisons with currently used imaging agents.
Conclusion
Our findings indicate that silica-coated magnetic fluorescent NPs are a superior contrast agent owing to their unique silica shells and polymeric coatings. Our study may provide the foundation for the noninvasive assessment of cellular components of vulnerable plaques in concert with structural atherosclerosis in MR imaging. Assessment of atherogenesis in the early stage of development using this noninvasive imaging modality would allow for early treatment before the disease processes. Further studies are required to monitor the safety and application of SIONs in atherosclerosis in humans.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Research Council of Science & Technology (NST) grant by the Korean government (MSIT) (KSH, CAP-18-02-KRIBB) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1A02049346).
