If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Department of Cardiology, The Eighth Affiliated Hospital of Sun Yat-Sen University, Shenzhen, 518033, Guangdong, ChinaGuangdong Innovative Engineering and Technology Research Center for Assisted Circulation, Shenzhen, ChinaNHC Key Laboratory of Assisted Circulation, Sun Yat-sen University, Guangzhou, China
Department of Cardiology, The Eighth Affiliated Hospital of Sun Yat-Sen University, Shenzhen, 518033, Guangdong, ChinaGuangdong Innovative Engineering and Technology Research Center for Assisted Circulation, Shenzhen, China
CD40-targeting magnetic nanoparticles for MRI/optical dual-modality molecular imaging aim to detect the vulnerable plaques.
•
CD40-Cy5.5-SPIONs exhibits promising potentials for noninvasive detection of vulnerable atherosclerotic plaque.
•
CD40-targeting magnetic nanoparticles can better access the risk of atherosclerotic diseases.
Abstract
Background and aims
Acute coronary syndrome caused by vulnerable plaque rupture or erosion is a leading cause of death worldwide. CD40 has been reported to be highly expressed in atherosclerotic plaques and closely related to plaque stability. Therefore, CD40 is expected to be a potential target for the molecular imaging of vulnerable plaques in atherosclerosis. We aimed to design a CD40-targeted magnetic resonance imaging (MRI)/optical multimodal molecular imaging probe and explore its ability to detect and target vulnerable atherosclerotic plaques.
Methods
CD40-Cy5.5 superparamagnetic iron oxide nanoparticles (CD40-Cy5.5-SPIONs), which comprise a CD40-targeting multimodal imaging contrast agent, were constructed by conjugating CD40 antibody and Cy5.5-N-hydroxysuccinimide ester with SPIONs. During this in vitro study, we observed the binding ability of CD40-Cy5.5-SPIONs with RAW 264.7 cells and mouse aortic vascular smooth muscle cells (MOVAS) after different treatments, using confocal fluorescence microscopy and Prussian blue staining. An in vivo study involving ApoE−/− mice fed a high-fat diet for 24–28 weeks was performed.
24 h after intravenous injection of CD40-Cy5.5-SPIONs, fluorescence imaging and MRI were performed.
Results
CD40-Cy5.5-SPIONs bind specifically to tumor necrosis factor (TNF)-α-treated macrophages and smooth muscle cells. Fluorescence imaging results showed that, compared with the control group and the atherosclerosis group injected with non-specific bovine serum albumin (BSA)-Cy5.5-SPIONs, the atherosclerotic group injected with CD40-Cy5.5-SPIONs had a stronger fluorescence signal. T2-weighted images showed that the carotid arteries of atherosclerotic mice injected with CD40-Cy5.5-SPIONs had a significant substantial T2 contrast enhancement effect.
Conclusions
CD40-Cy5.5-SPIONs could potentially serve as an effective MRI/optical probe for vulnerable atherosclerotic plaques during non-invasive detection.
Although impressive progress has been made in the pathogenesis, diagnosis, and treatment of atherosclerosis, atherosclerotic diseases, especially coronary heart disease and cerebral vascular disease, remain the leading causes of morbidity and mortality worldwide [
The worldwide environment of cardiovascular disease: prevalence, diagnosis, therapy, and policy issues: a report from the American College of Cardiology.
]. According to the World Health Organization, 17.9 million deaths in 2016 were the consequence of myocardial infarction and stroke, which accounted for 31% of global deaths [
Most acute coronary syndromes are caused by the rupture or erosion of unstable and vulnerable plaques. Vulnerable plaques are characterized by large lipid core, thin fibrous cap, and massive infiltration of inflammatory cells [
]. When a vulnerable plaque ruptures, the necrotic core is exposed, thus stimulating subsequent thrombosis, leading to acute cardiovascular events. Currently, the identification of unstable and vulnerable plaques remains a challenge. Therefore, it is imperative to develop and improve noninvasive imaging techniques to sensitively and accurately detect unstable and vulnerable plaques [
In addition to anatomical imaging, molecular imaging, which focuses on biological processes, has shown great potential for assessing vulnerable plaques [
]. Molecular imaging uses targeted contrast agents/particles or radiotracers to visualize and characterize biological processes at the body, organ, cell, and subcellular levels [
]. Each imaging method has advantages and limitations. Therefore, multimodal imaging that combines multiple imaging methods is being actively sought. MRI has high spatial and temporal resolution. Studies have reported that MRI can distinguish the main components of atherosclerotic plaques, thereby stratifying the risks according to their composition [
]. However, MRI has many limitations when attempting to detect plaques because of its limited contrast sensitivity. Multimodal imaging aims to combine multiple imaging modes to complement each other [
]. The combination of MRI with high spatial resolution and high-sensitivity optical imaging is expected to become a high-resolution and high-sensitivity imaging technology that can achieve high-precision imaging of biological targets [
]. The CD40–CD40L pathway is one of the main pro-inflammatory pathways involved in the development of atherosclerosis, and its activation leads to the up-regulation of a large number of inflammation-related and atherosclerosis-related genes [
]. CD40 is abundantly expressed in endothelial cells, vascular smooth muscle cells (SMCs) and macrophages in atherosclerotic plaques, most notably on the shoulders of plaques [
]. After stimulation by oxidized low-density lipoprotein and tumor necrosis factor (TNF)-α, the expression levels of CD40 in endothelial cells, macrophages, and SMCs were found to be increased [
Oxidized low-density lipoprotein augments and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors limit CD40 and CD40L expression in human vascular cells.
]. It has been reported that blocking the interaction between CD40 and TNF receptor-related factor 6 with small molecule inhibitors delayed the occurrence of atherosclerosis and made the existing atherosclerotic plaques more stable [
]. Therefore, CD40 is a potential target for molecular imaging to detect atherosclerosis, especially vulnerable plaques.
Superparamagnetic iron oxide nanoparticles (SPIONs) have been used as MRI contrast agents because of their superparamagnetism, biocompatibility and high stability [
Fluorescent, superparamagnetic nanospheres for drug storage, targeting, and imaging: a multifunctional nanocarrier system for cancer diagnosis and treatment.
]. In this study, we aimed to construct SPIONs-based multimodal molecular imaging probes targeting CD40 and binding to Cy5.5 to detect and identify vulnerable plaques in vivo using MRI/fluorescence imaging. The cytotoxicity and targeting ability of the nanoprobes were evaluated using RAW 264.7 cells and SMCs, and in vivo MRI and fluorescence imaging was performed to confirm their ability to identify vulnerable plaques.
2. Materials and methods
2.1 Chemical reagents
The CD40 monoclonal antibody(1C10) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Meso-2,3-dimercaptosuccinic acid (DMSA)@Fe3O4 was obtained from Nanjing NanoEast Biotech Co., Ltd. (Nanjing, China). Cy5.5-NHS was purchased from Xi'an Ruixibio Co.,Ltd.(Xi'an, China).N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2-(N-morpholino)-ethanesulfonic acid was purchased from Biofroxx (Einhausen, Germany). Bovine serum albumin (BSA) was purchased from Shanghai YEASEN Biotechnology Co., Ltd. (Shanghai, China).
2.2 Synthesis of CD40-Cy5.5-SPIONs and BSA-Cy5.5-SPIONs
First, we conjugated the antibody to [email protected] 0.3 mL of 0.15 M 2-(N-morpholino)-ethanesulfonic acid buffer (pH 5.5) was added to the 3 mL of [email protected] (4 mg Fe/mL) solution and mixed by vortexing for 1 min 1 mL CD40 antibody (1 mg/mL) was added and the mixture shaken at 37 °C for 0.5 h then 1 mg ethylcarbodiimide hydrochloride (10 mg/mL) was added and the mixture shaken for another 2.5 h for activation. The obtained sample solution was ultrafiltered four times and followed with pure water in a 30 KD ultrafiltration tube, then resuspended in a volume of 10 mL with a concentration of 1 mg Fe/mL. Next, the binding reaction between the Cy5.5 fluorescent dye and CD40-SPIONs was performed. We added 15 μL Cy5.5-NHS solution and shook it at 37 °C for 1 h, and then a 30 KD ultrafiltration tube was used to remove the unbound fluorescent molecules. The final suspension comprised 10 mL (1 mg Fe/mL) with 0.02 M 0.09% NaN3 preservation solution. BSA-Cy5.5-SPIONs were synthesized in a similar manner, however, BSA was used instead of the CD40 antibody.
2.3 Characterization of CD40-SPIONs
The morphology and size of the CD40-Cy5.5-SPIONs were observed using transmission electron microscope (FEI Tecnai G2 F20, USA). A 5–10 μL sample was obtained and dropped on copper net. Filter paper was used to absorb excess water from the edge and the sample dried for observation. The magnetic properties of the CD40-Cy5.5-SPIONs were measured using vibrating sample magnetometer (VSM7404, LakeShore, USA). In vitro MRI of CD40-Cy5.5-SPIONs at different concentrations (0–40 μg Fe/mL) was performed using a MesoMR23-060H-I imager (Suzhou Neumag Company, Suzhou, China). The parameters for measuring the transverse relaxation time T2 were as follows: Carr-Purcell-Meiboom-Gill (CPMG) sequence, SFO1(MHz) = 21.3 MHz P1 = 5.8 us, P2 = 9.6 us, SW = 200 KHz, RG1 = 20 db, DRG1 = 1, TW = 5000 ms, TE = 1 ms, NECH = 18000, NS = 4. The parameters of T2-weighted images were as follows: SFO1 (MHz) = 21.3 MHz，RFA90° = 2.6, RFA180° = 3.7，TR = 1600 ms, TE = 50 ms, slice width (mm) = 9.3, Slices = 1, Average = 4，Read Size = 256，Phase Size = 192. The relaxivity value (r2) was calculated from the slope of the linear plots of the r2 relaxation rate (1/T2) compared to the iron concentration.
2.4 Western blotting
The cell lysis buffer for Western blotting and immunoprecipitation (Beyotime, Shanghai, China) was used to isolate the proteins from the HASMCs and the aorta. Proteins were separated by SDS-PAGE and transferred to a 0.45μm PVDF membrane (Millipore, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: CD40 (1:1000, #ab13545, Abcam, UK) and GAPDH (1:2000, #2118S, Cell Signaling Technology). After incubation with anti-rabbit IgG, HRP-linked antibody (1:3000, Proteintech, USA) for 1h, the protein bands were treated with Chemiluminescence reagent (Beyotime, Shanghai, China) and detected using the ChemiDoc™ Touch Imaging System (Bio-Rad, USA).
2.5 Cytotoxicity studies
The viability of RAW 264.7 cells (CL-0190, Procell, Wuhan, China) and HASMCs (#C490, FineTest, Wuhan, China) was assessed using Cell Counting kit-8 (Dojindo, Kumamoto, Japan). RAW 264.7 cells (2 × 104 cells/well)/HASMCs (1 × 104 cells/well) were seeded in 96-well plates and incubated with CD40-Cy5.5-SPIONs at different concentrations (0, 5, 10, 25, 50, 100, and 200 μg Fe/mL) for 24 h. The cells were washed with PBS three times; then, 100 μL fresh medium and 10 μL CCK-8 reagents were added and incubated for 1 h. Absorbance at 450 nm was measured using a microplate reader (Thermo Varioskan LUX, USA).
Calcein/propidium iodide (PI) cell cytotoxicity assay (Beyotime, Shanghai, China) was performed to evaluate the apoptotic effect of CD40-Cy5.5-SPIONs on HASMCs. After HASMCs were pretreated with different concentrations of CD40-Cy5.5-SPIONs, live and dead cells were stained according to the manufacturer's instructions and finally observed by confocal fluorescence microscopy.
Additionally, the Annexin V-FITC/PI Apoptosis Detection Kit (#556547, BD, USA) was used to determine the apoptosis rate of HASMCs. Cells (3 × 105) were seeded in six-well plates and incubated with CD40-Cy5.5-SPIONs (0, 10, and 30 μg Fe/mL) for 24h. The cells were detached by 0.25% trypsin and centrifugated at 1000 rpm for 5 min. After washing twice with PBS, the cells were suspended in 400 μL 1 × binding buffer. Then, 5 μL of Annexin V-FITC was added and cells incubated in the dark for 15 min. Next, 5 μL of PI, and 200 μL of 1 × binding buffer were added before flow cytometry (BD LSRFortessa, USA) to detect cell samples. The results were analyzed using FlowJo 10.4. Apoptotic cells included annexin V+/PI- (early apoptosis) and annexin V+/PI+ (late apoptosis) cells.
2.6 In vitro fluorescence detection and prussian blue staining
RAW 264.7 cells and MOVAS (CRL-2797, ATCC) were stimulated with TNF-α (100 ng/mL) for 24 h and then incubated with nanoparticles (20 μg Fe/mL) for 8 h. In order to verify the ability of CD40-Cy5.5-SPIONs to bind cells by targeting CD40, excessive CD40 antibody was added to TNF-α+anti-CD40+ CD40-Cy5.5-SPIONs group 1h before the addition of nanoparticles to block the competitive binding site. The cells were washed with PBS three times and fixed with 4% paraformaldehyde for 20 min. Finally, 4’,6-diamidino-2-phenylindole (DAPI) was added and cells incubated for 5 min before washing with PBS three times. The detection of fluorescence was performed by a confocal laser scanning microscope (Olympus, Japan).
Prussian blue staining is a common method used to detect iron oxide nanoparticles. After fixing with 4% paraformaldehyde for 20 min, the cells were stained with Perls staining solution for 20 min, and counterstained with nuclear fast red solution, and examined by lightmicroscopy.
2.7 Animal models of atherosclerosis
All animal experiments were performed according to a protocol approved by the Animal Ethical and Welfare Committee of the Eighth Affiliated Hospital, Sun Yat-sen University (Shenzhen, China; 2019-115-02). The 8-week-old male C57BL/6J and ApoE−/− mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). After 1 week of acclimatization, ApoE−/− mice were fed a high-fat diet (HFD) (catalog# D12108C, Research Diets) for approximately 24–28 weeks to establish the atherosclerosis model; C57 mice were provided a regular diet as a control group.
2.8 In vivo fluorescence/MRI imaging
To confirm the in vivo targeting ability of the CD40-Cy5.5-SPIONs, fluorescence imaging and MRI were performed.
Fluorescence imaging was performed using a PerkinElmer IVIS Lumina II instrument. Before fluorescence imaging, the mouse neck was depilated and fasted. In vivo imaging was performed 24 h after injection of 200 μL of CD40-Cy5.5-SPIONs or BSA-Cy5.5-SPIONs. The imaging parameters were as follows: binning, 4; F/stop 2; exposure time, 1s; excitation filter, 640; and emission filter, Cy5.5. Subsequently, the mice in the HFD group were euthanized to harvest the entire aorta for ex vivo imaging. Quantitative analyses of fluorescence imaging results were performed using Living Image Software (Caliper Life Sciences).
MRI was performed using a 9.4 T small animal MRI system (BioSpec94/30 USR, Bruker, USA). PD-T2WI was performed before and 24 h after administration of CD40-NPs respectively using the following parameters: repetition time [TR]/echo time [TE] = 3058/13 ms, field of view = 25 mm × 25 mm, matrix = 256 × 256 and slice thickness = 0.5 mm. To calculate the T2-weighted signal change, the following formula was used: %SIchanges = (SIpost - SIpre)/SIpre × 100%, where SIpre and SIpost are the signal intensities of carotid atherosclerotic plaque 24 h before and after the injection of nanoparticles, respectively.
2.9 Pathological analysis
To evaluate the extent of atherosclerotic lesions, en face oil Red O (ORO) staining, HE staining, and Masson staining were performed. The entire aorta was removed after the mice were euthanized, and the adipose tissue around the blood vessels was separated. The blood vessels were immersed in 60% isopropanol for 3 s, then dipped in oil Red O staining solution for 60 min, and immersed in 60% isopropanol for differentiation until the plaques were orange or bright red and the other vessel parts were almost colorless.
To assess the accumulation of CD40-Cy5.5-SPIONs in atherosclerotic plaques, paraffin sections of the aorta were evaluated Prussian blue staining.
2.10 Statistical analysis
Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software Inc., United States). The data were presented as mean ± standard deviation. Student'st-tests were used for comparisons between the two groups. Statistical significance was set at p < 0.05. The analysis among multiple-groups was conducted using one-way analysis of variance test. (significant differences: p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***).
3. Results
3.1 The expression of CD40 in macrophages, smooth muscle cells and atherosclerosis plaque
The expression level of CD40 in TNF-α-treated RAW 264.7 cells and HASMCs were verified using Western blotting. Compared to the control group, the expression of CD40 was significantly enhanced in the TNF-α group (Fig. 1A and B). After 24–28 weeks of the high-fat diet, the mice underwent imaging. Before imaging, to confirm the establishment of an animal model of vulnerable atherosclerotic plaques, we performed aortic oil Red staining.
Fig. 1The expression of CD40 in macrophages, smooth muscle cells and atherosclerosis plaque.
The expression of CD40/GAPDH in (A) RAW 264.7 cells and (B) HASMCs upon TNF-α treatment was detected by (C) en face oil Red O staining and (D) Western blotting. (E) HE staining, and (F) Masson staining of the aorta from the control group and HFD group. (scale bars = 100 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
A large number of lipid droplets formed in the aortas of HFD mice (Fig. 1C). H&E and Masson staining further confirmed the formation of vulnerable atherosclerotic plaques, such as large lipid cores, thin fiber caps, and cholesterol crystals (Fig. 1E and F). Additionally, the expression of CD40 in the aorta of HFD mice was significantly higher than that of the control group, indicating that CD40 has the potential to become a target molecule for vulnerable atherosclerosis (Fig. 1D).
3.2 The characterization of CD40-Cy5.5-SPIONs
The transmission electron microscopy image showed that CD40-Cy5.5-SPIONs (Fig. 2A) were spherical and had good dispersibility, with an average particle size of 9.18 nm (±1.66 nm) (Fig. 2B). The magnetization curve exhibited an S-shaped curve, indicating that CD40-Cy5.5-SPIONs were superparamagnetic, and its saturation magnetization as high as 205.22 emu/g (Fig. 2C). To determine the negative MRI contrast agent performance of the CD40-Cy5.5-SPIONs, in vitro MRI was performed. The T2-weighted image of the nanoparticles became darker (the weaker red color displayed in the pseudo-color image) as the iron concentration increased (Fig. 2E). Additionally, the r2 value was 45.752 mM−1s−1 based on the linear relationship between the iron concentration and 1/T2, which indicated that it can enhance MRI contrast and is suitable for use as a T2-weighted image contrast agent for MRI (Fig. 2D).
(A) TEM image of CD40-Cy5.5-SPIONs. (B) Size distribution histogram of CD40-Cy5.5-SPIONs. (C) Magnetic hysteresis loops of CD40-Cy5.5-SPIONs. (D) T2 relaxation rate (1/T2, S-1) of CD40-Cy5.5-SPIONs. (E) MRI T2-weighted images of CD40-Cy5.5-SPIONs at different concentrations (0, 2.5, 5, 10, 20, and 40 μg Fe/mL).
The cytotoxicity of CD40-Cy5.5-SPIONs was assessed using CCK8 assay, Calcein/PI cell cytotoxicity assay, and flow cytometry. The cell viability of RAW 264.7 cells and HASMCs incubated with CD40-Cy5.5-SPIONs with the concentration range of 0–200 μg Fe/mL for 24h showed no significant difference (Fig. 3A and B). The results of Calcein/PI cell viability assay showed that CD40-Cy5.5-SPIONs with concentrations of 10, 30 and 50 μg Fe/mL did not cause significant HASMCs death (Fig. 3C). Additionally, consistent with the aforementioned experimental results, the flow cytometry results revealed the low cytotoxicity of the CD40-Cy5.5-SPIONs (apoptosis rate: control group 5.19 ± 0.14% versus 10 μg Fe/mL group 5.64 ± 0.33% versus 30 μg Fe/mL group 5.58 ± 0.39%, p > 0.05) (Fig. 3D). In conclusion, these results indicate that nanoparticles have good biocompatibility and safety.
The CCK8 assay results of RAW 264.7 cells (A) and HASMCs (B), (C) calcein/PI cell cytotoxicity assay in HASMCs, and (D) flow cytometry results for evaluating the cytotoxicity of CD40-Cy5.5-SPIONs (scale bars = 100 μm).
3.4 The specific targeting ability of CD40-Cy5.5-SPIONs in vitro
Confocal imaging results showed that TNF-α-treated RAW 264.7 cells co-incubated with CD40-Cy5.5- SPIONs exhibited remarkably higher fluorescence signals compared to those of nontreated RAW 264.7 cells (Fig. 4A). Only weak fluorescence signals were observed in TNF-α-treated RAW 264.7 cells co-incubated with BSA-Cy5.5- SPIONs. When we added excess CD40 antibody before adding CD40-Cy5.5-SPIONs, the binding of CD40-Cy5.5-SPIONs to RAW 264.7 cells was significantly reduced. Similarly, it was observed that MOVAS pretreated with TNF-α bound a large amount of CD40-Cy5.5- SPIONs, while when excess CD40 antibody was added in advance, the CD40-Cy5.5-SPIONs bound by MOVAS were considerably reduced (Fig. 4A). Furthermore, Prussian blue staining also showed a consistent trend (Fig. 4B). These results indicated that CD40-Cy5.5- SPIONs have an excellent ability to target and bind to TNF-α--treated RAW 264.7 cells and MOVAS through CD40.
Fig. 4Fluorescence imaging and Prussian blue staining of CD40-targeting specificity of CD40-Cy5.5-SPIONs in vitro.
(A) Confocal microscopy images of RAW 264.7 cells and MOVAS incubated with nanoparticles. CD40 antibody was added to the TNF-α + anti-CD40 + CD40-Cy5.5-SPIONs group prior to the introduction of CD40-Cy5.5-SPIONs (scale bars = 50 μm). (B) Prussian blue staining of MOVAS treated in the same way (scale bars = 200 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
At 24 h after the injection of CD40-Cy5.5-SPIONs or BSA-Cy5.5-SPIONs, the mice were subjected to fluorescence imaging. The fluorescence signal of the neck of the HFD group injected with CD40-Cy5.5-SPIONs was significantly higher than that of the control group and the HFD + BSA-Cy5.5-SPIONs group, indicating that CD40-Cy5.5-SPIONs target atherosclerotic plaques for binding (Fig. 5A). To further accurately compare the fluorescence signal intensity of the aorta, in vitro fluorescence imaging of the separated aorta was also performed. The in vitro fluorescence images showed that the fluorescence signal of the aorta in the HFD + CD40-Cy5.5-SPIONs group was significantly higher than that in the HFD + BSA-Cy5.5-SPIONs group (Fig. 5B). In addition, the fluorescence signal of aorta was significantly stronger than that of cervical lymph nodes (Fig. 5B).
Fig. 5In vivo fluorescence images and T2-weighted MR images of atherosclerotic mice after intravenous injection of nanopaticles.
(A) In vivo fluorescence images of Control + CD40-Cy5.5-SPIONs group, HFD + BSA-Cy5.5-SPIONs group, and HFD + CD40-Cy5.5-SPIONs group, respectively. (B) In vitro fluorescence images of the aorta and the lymph node. (C and D) In vivo T2-weighted MR images of HFD fed mice before and 24h after injection CD40-Cy5.5-SPIONs and BSA-Cy5.5-SPIONs, respectively. (E) Prussian blue staining and immunohistochemical of the HFD fed mice aorta. Iron is indicated by arrows. (scale bars = 200 μm) (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
T2-weighted MRI images were acquired before and 24 h after intravenous injection. The signal of the carotid artery wall of atherosclerotic mice was unevenly enhanced, and the arterial wall was thickened (Fig. 5D). At 24 h after intravenous administration of CD40-Cy5.5-SPIONs, the carotid wall of the mice showed an enhanced negative signal (darkening), whereas the carotid artery signal of the mice in the BSA-Cy5.5-SPIONs group showed no significant change.
Prussian blue staining consistently confirmed that more CD40-Cy5.5-SPIONs were deposited in atherosclerotic plaques than BSA-Cy5.5-SPIONs (Fig. 5E). The results of immunohistochemistry showed that CD40-Cy5.5-SPIONs preferentially concentrated in the regions of CD40 expression in atherosclerotic plaques. The macrophage marker CD68 and smooth muscle cell marker α-SMA were significantly expressed in the high expression region of CD40 (Fig. 5E). These data indicate that CD40-Cy5.5-SPIONs also exhibit outstanding targeting in vivo.
4. Discussion
Myocardial infarction and ischemic stroke caused by the rupture of inflammation-driven vulnerable atherosclerotic plaques cause significant mortality in developed countries [
]. The assessment and prediction of cardiovascular risks are key issues in contemporary cardiovascular medicine.
The coronary artery calcification score, coronary computed tomographic angiography, carotid intima-media thickness, and ankle-brachial index have been used to assist with arteriosclerotic cardiovascular disease risk assessments [
2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.
]. However, these techniques mainly rely on anatomical imaging to quantify the plaque burden rather than an evaluation of the biological characteristics of unstable or vulnerable plaques [
]. Therefore, with the development of personalized medicine, the role of molecular imaging based on non-invasive imaging technology for identifying vulnerable plaques has gained strong interest. In the present study, we designed CD40-Cy5.5-SPIONs to detect vulnerable atherosclerotic plaques by targeting CD40.
The key to molecular imaging is choosing a suitable molecular target and imaging platform. CD40−CD40L signaling has an important role in eliciting plaque inflammation [
The reduced atherosclerotic lesion size, of the CD40−/−ApoE−/− mouse model induced a stable plaque phenotype, that is, a limited abundance of inflammatory cells and thickened fibrous caps. Additionally, inhibition of the interaction between CD40 and its downstream adaptor molecule TNF receptor-related factor 6 almost completely abolished the development of atherosclerosis [
]. During our study, the expression of CD40 was significantly upregulated in TNF-α-treated HASMCs and the aortic tissue of the HFD group. Therefore, highly expressed CD40 has the potential to serve as an imaging target for detecting vulnerable plaques.
Ultrasound imaging is a quick and convenient method, but it relies on operator experience, and the penetration depth is insufficient [
]. Positron emission tomography and single-photon emission computed tomography have relatively limited spatial resolutions. X-ray-based computed tomography has a short scan time and high spatial resolution. However, ionizing radiation exposure has been a problem that cannot be ignored [
]. In contrast, MRI has high spatial and temporal resolution, no radiation, and excellent soft-tissue contrast that can distinguish the main components of atherosclerotic plaques [
]. However, optical imaging has the advantages of high sensitivity and simple operation. Therefore, combining optical imaging and MRI for complementary multimodal imaging is expected to result in highly accurate and sensitive imaging of atherosclerotic plaques [
MRI contrast agents are divided into T1 and T2 MRI contrast agents that produce high-contrast signals on T1-weighted images and low-contrast signals on T2-weighted images, respectively. Typical T1 contrast agents are paramagnetic gadolinium (Gd), iron oxide nanoparticles are T2 contrast agents. Because of the nephrotoxicity caused by Gd-based contrast agents, SPIONs were selected as probe carriers during this research [
]. During in vivo applications, the surface of the SPIONs must be covered with biocompatible polymers to prevent agglomeration and improve stability and biocompatibility. Additionally, it can provide binding functional groups to target ligands or drugs [
]. Mejías et al. confirmed that high-concentration dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles (MNP) have low toxicity to NCTC 1469 cells [
]. Therefore, we chose DMSA-coated magnetic iron oxide particles as the probe carriers.
Molecular imaging includes passive and active targeting strategies. Passive targeting relies on enhanced permeability and retention effects of the vessel. A large number of studies have used non-specific uptake of SPIONs by macrophages in plaques to reflect inflammation in plaques [
The ATHEROMA (Atorvastatin Therapy: effects on Reduction of Macrophage Activity) Study. Evaluation using ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging in carotid disease.
]. Active targeting strategies combine contrast agents with molecular targets on the surface of target cells or tissues to achieve the accumulation of contrast agents at specific sites, thereby improving the specificity and imaging contrast of contrast agents [
]. During this study, the binding of SPIONs with CD40 and Cy5.5 may be useful for identifying the molecular characteristics of plaque and improving the sensitivity and accuracy of early detection of vulnerable plaques.
Biological safety is a prerequisite for its clinical application. Although T2-weighted contrast agents based on iron oxide have been approved by the United-States. Food and Drug Administration for use in clinical trials, studies have reported that the cytotoxicity of SPIONs is affected by surface coating materials and surface modification [
]. Therefore, it is necessary to verify the cytotoxicity of SPIONs modified with CD40 antibody and Cy5.5 fluorescent dye. The CCK8 assay, live/dead cell staining, and flow cytometry confirmed the low toxicity of CD40-Cy5.5-SPIONs, indicating their suitability for in vivo experiments.
In practical applications, SPIONs require sufficient blood circulation time to accumulate in the target area and to be detected by imaging technology; they should be able to pass through the capillaries of organs and tissues to avoid vascular blockage [
]. Transmission electron microscopy images showed that CD40-Cy5.5-SPIONs had a diameter of 9.18 nm (±1.66 nm), uniform particle size, and good dispersion. Additionally, the hysteresis curve results showed that CD40-Cy5.5-SPIONs exhibited superparamagnetism, with a saturation magnetization of 205.22 emu/g. The transverse relaxation rate R2 of CD40-Cy5.5-SPIONs was 45.752 mM−1s−1. T2-weighted images darkened with increasing of iron concentration. These results indicate that CD40-Cy5.5-SPIONs are suitable MRI T2 contrast agents for in vivo imaging.
The targeting ability of CD40-Cy5.5-SPIONs in vitro was evaluated in TNF-α stimulated RAW 264.7 cells and MOVAS. Fluorescence and Prussian blue staining consistently showed that compared with the control group, considerable CD40-Cy5.5-SPIONs accumulated on RAW 264.7 cells and MOVAS in the TNF-α group, and almost no accumulation of BSA-Cy5.5-SPIONs in the TNF-α group. After addition of excess CD40 antibody to block the competitive binding site, the binding of CD40-Cy5.5-SPIONs to RAW 264.7 cells and MOVAS decreased significantly, indicating the ability of CD40-Cy5.5-SPIONs to bind to CD40.
In vivo fluorescence imaging and MRI were performed for ApoE−/− mice fed the HFD for 24–28 weeks. Aortic oil red O staining showed severe plaque formation in the aorta, and HE staining of paraffin sections of the aorta showed that the plaques had a significant lipid necrotic core and thin fiber caps. CD40 was significantly up-regulated in ApoE−/− mice group. The fluorescence image showed that, compared with the control group and atherosclerotic mice injected with the nontargeted probe BSA-Cy5.5-SPIONs, there was a strong fluorescent signal in the neck of atherosclerotic mice administered CD40-Cy5.5-SPIONs. Furthermore, in vitro fluorescence imaging showed that CD40-Cy5.5-SPIONs aggregated more significantly than nontargeted BSA-Cy5.5-SPIONs in the mouse aorta. MRI T2-weighted imaging demonstrated that the carotid artery signal of atherosclerotic mice was considerably decreased (negative signal enhancement) at 24 h after the injection of CD40-Cy5.5-SPIONs. Prussian blue staining consistently confirmed that CD40-Cy5.5-SPIONs accumulated to a greater extent in the diseased aorta. These in vivo experimental results demonstrated the excellent targeting and localization ability of CD40-Cy5.5-SPIONs.
Despite these advantages, our study has some limitations. For example, CD40-Cy5.5-SPIONs have not been used for the dynamic observation of atherosclerotic plaques at different stages, and SPIONs have been reported to achieve targeted therapy by combining drug molecules [
]. Future studies should construct SPIONs containing anti-atherosclerotic drugs to achieve the integration of diagnosis and treatment.
4.1 Conclusion
In conclusion, we have constructed a CD40-targeted MRI/fluorescence multimodal imaging nanoprobe with good biocompatibility. CD40-Cy5.5-SPIONs specifically bind to atherosclerotic plaques and have the potential to be used as probes for detecting atherosclerotic plaques, thus providing more helpful information for the diagnosis of vulnerable plaques, and contributing to the accurate and individual diagnosis of atherosclerosis.
Financial support
This study was supported by the Science and Technology Planning Project of Shenzhen Municipality (No. JCYJ20180306173433984), Shenzhen Key Medical Discipline Construction Fund (No. SZXK002), and Shenzhen Key Clinical Discipline Funds (ZDXKJF-01002).
CRediT authorship contribution statement
Qimin Wu: conducted the experiments, Formal analysis. Wei Pan: conceived and designed of the study, conducted the experiments. Guifu Wu: conceived and designed of the study. Fensheng Wu: Formal analysis. Yousheng Guo: revised the manuscript accordingly, All authors have read and approved the final manuscript. Xinxia Zhang: conceived and designed of the study, revised the manuscript accordingly.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We are grateful for the experimental platform provided by the Central Laboratory of the Eighth Affiliated Hospital of Sun Yat-sen University. We are also grateful to all the participants and sponsors for their support.
References
Laslett L.J.
Alagona Jr., P.
Clark 3rd, B.A.
Drozda Jr., J.P.
Saldivar F.
et al.
The worldwide environment of cardiovascular disease: prevalence, diagnosis, therapy, and policy issues: a report from the American College of Cardiology.
Oxidized low-density lipoprotein augments and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors limit CD40 and CD40L expression in human vascular cells.
Fluorescent, superparamagnetic nanospheres for drug storage, targeting, and imaging: a multifunctional nanocarrier system for cancer diagnosis and treatment.
2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.
The ATHEROMA (Atorvastatin Therapy: effects on Reduction of Macrophage Activity) Study. Evaluation using ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging in carotid disease.
00084-9&id=ga1.jpg){kind=link}
00084-9&id=gr1.jpg){kind=link}
00084-9&id=gr2.jpg){kind=link}
00084-9&id=gr3.jpg){kind=link}
00084-9&id=gr4.jpg){kind=link}
00084-9&id=gr5.jpg){kind=link}