Imaging Carotid Plaque Burden in Living Mice via Hybrid Semiconducting Polymer Nanoparticles-Based Near-Infrared-II Fluorescence and Magnetic Resonance Imaging

Abstract

The majority of atherothrombotic events (e.g., cerebral or myocardial infarction) often occur as a result of plaque rupture or erosion in the carotid, and thereby it is urgent to assess plaque vulnerability and predict adverse cerebrovascular events. However, the monitoring evolution from stable plaque into life-threatening high-risk plaque in the slender carotid artery is a great challenge, due to not enough spatial resolution for imaging the carotid artery based on most of reported fluorescent probes. Herein, copolymerizing with the small molecules of acceptor-donor-acceptor-donor-acceptor (A-D-A'-D-A) and the electron-donating units (D'), the screened second near-infrared (NIR-II) nanoprobe presents high quantum yield and good stability, so that it enables to image slender carotid vessel with enough spatial resolution. Encouragingly, NIR-II nanoprobe can effectively target to intraplaque macrophage, meanwhile distinguishing vulnerable plaque in carotid atherosclerosis in living mice. Moreover, the NIR-II nanoprobe can dynamically monitor the fresh bleeding spots in carotid plaque, indicating the increased risk of plaque instability. Besides, magnetic resonance imaging is integrated with NIR-II fluorescence imaging to provide contrast for subtle structure (e.g., narrow lumen and lipid pool), via incorporating ultrasmall superparamagnetic iron oxide into the NIR-II nanoprobe. Thus, such hybrid NIR-II/magnetic resonance imaging multimodal nanoprobe provides an effective tool for assessing carotid plaque burden, selecting high-risk plaque, and imaging intraplaque hemorrhage, which is promising for reducing cerebral/ myocardial infarction-associated morbidity and mortality.

Fig. 1.

Hybrid SPNs for noninvasive time-lapse imaging of carotid atherosclerosis progression in C57BL/6 male mice via NIR-II fluorescence and MRI, which is promising for assessing and monitoring the high-risk pathological changes within carotid atherosclerotic plaque in living body.

Fig. 2.

Molecular engineering and synthesis of semiconducting polymers and characteristics of NIR-II fluorescent or NIR-II/MRI hybrid nanoparticles. (A) The synthetic routes for NIR-1, NIR-2, or NIR-3. (B) The preparation of SPNs(NIR-1) and SPNs(NIR-1)@ SPIO and diagram of NIR-II or MR imaging. (C) The density functional theory molecular orbital plots of NIR-1. (D) Absorption spectra of NIR-1, NIR-2, and NIR-3 in toluene. (E) Emission spectra of NIR-1, NIR-2, and NIR-3 in toluene. (F) Absorption spectra of SPNs(NIR-1), SPNs(NIR-2), and SPNs(NIR-3) in water (10 μg/ml). Inset indicated aqueous solutions of SPNs(NIR-1), SPNs(NIR-2), and SPNs(NIR-3). (G) The emission spectra of SPNs(NIR-1), SPNs(NIR-2), and SPNs(NIR-3) in water. (H) Representative TEM image of SPNs(NIR-1). (I) NIR-II images of SPNs(NIR-1), SPNs(NIR-2), SPNs(NIR-3), and ICG (10 μg/ml) under excitation of 808 nm (1 W/cm²). (J) The NIR-II fluorescence intensity from (I). (K) Fluorescence intensity of SPNs(NIR-1) under 808-nm laser irradiation (1 W/cm²) for different concentrations (10 to 100 μg/ml). (L) The quantized NIR-II fluorescence intensity of SPNs(NIR-1), SPNs(NIR-2), SPNs(NIR-3), and ICG (10 μg/ml) under 808-nm laser irradiation (1 W/cm²) for different times. (M) Representative TEM image of SPNs(NIR-1)@SPIO. (N) The r₁ (1/T₁) and r₂ (1/T₂) water proton spin relaxivities of SPNs(NIR-1)@SPIO. (O) T₂WI images of SPNs(NIR-1)@SPIO at different Fe concentrations (mM) using a 7.0-T MRI scanner. (P) NIR-II fluorescence imaging of SPNs(NIR-1)@SPIO.

Fig. 3.

NIR-II fluorescence imaging and MRI of carotid atherosclerosis plaque. (A) Schematic diagram for preparation of mice mode with atherosclerotic plaque of carotid artery (AS mice) and NIR-II fluorescence/MRI imaging. (B) The H&E-stained images of RCA and LCA. Green curve indicated plaque in lumen. (C) NIR-II fluorescence imaging of cervical region of AS mice post intravenously injected with SPNs(NIR-1). (D) The fluorescence intensity profiles along the red dashed line in RCA and LCA (C). (E and F) Representative T₂WI images of cervical region of 2 slices of AS mice pre- and postinjection (24 h) of SPNs(NIR-1)@SPIO. Inset pictures enlarged the LCA (green or orange circles), white arrows indicated plaque, and blue circles indicated muscle. (G) The T₂WI plaque-to-muscle ratio in (E) and (F). (H) Representative confocal images of Raw264.7 cells incubated with Cy5.5-SPNs(NIR-1) for various times. (I) The relative fluorescence intensity of Cy5.5-SPNs(NIR-1) in (H). (J) Representative confocal images of C166 and RAW264.7 cells pretreated with LPS or not and incubated with Cy5.5-SPNs(NIR-1), respectively. (K) The relative fluorescence intensity (%) of Cy5.5-SPNs(NIR-1) in (J).

Fig. 4.

Distinguishing unstable plaque in vivo via NIR-II imaging. (A) Schematic diagram for NIR-II fluorescence imaging of carotid plaque in AS mice and AS mice + AIP, using healthy mice as control, after intravenous injection of SPNs(NIR-1) (n = 3). (B) Representative biopsy of LCAs seperated from healthy mice, AS mice, and AS mice + AIP, as well as corresponding H&E-stained images of LCAs and lungs. Green curve indicated plaque, black curve indicated a soft lipid pool, and red curve indicated lung interstitial inflammation. (C) The body temperature changes in various groups. (D) The neutrophile granulocyte (%) and WBC count for various groups. (E and F) NIR-II fluorescence images of healthy mice, AS mice, and AS mice + AIP at different times post intravenous injection of SPNs(NIR-1), and NIR-II fluorescence images of ex vivo carotid arteries for various groups. (G) The fluorescence intensity of LCAs at different time points for various groups in (E). (H) The fluorescence intensity of LCAs and RCAs for various groups in (F). (I) The TG, TC, HDL-C, and low-density lipoprotein control (LDL-C) levels for various groups. (J) Representative fluorescence confocal images of ex vivo carotid arteries, stained by DAPI (nucleus), F4/80 (macrophage), and α-SMA (smooth muscle actin). Blue, green, and red curves indicated lumens. (K) The relative fluorescence intensity of F4/80⁺ cells in (J). (L) Heat map for fluorescence intensity of LCA, F4/80⁺ cells, neutrophile, WBC, body temperature, lung wet/dry (W/D) ratio, and plaque area.

Fig. 5.

Imaging IPH in vivo. (A) Schematic diagram for NIR-II fluorescence imaging of atherosclerosis mice (named AS mice) and atherosclerosis mice combined with small IPH (named AS + small IPH) and large IPH (named AS + large IPH), after intravenous injection of SPNs(NIR-1) (n = 3). (B) Representative biopsy of LCA from AS mice, AS mice + small IPH, and AS mice + large IPH, as well as corresponding H&E-stained images of LCAs. The marked area by green curve indicated plaque, and black curve indicated blood micro-injection. (C and D) NIR-II fluorescence images of AS mice, AS mice + small IPH, and AS mice + large IPH at different times postinjection of SPNs(NIR-1), and NIR-II fluorescence images of ex vivo carotid arteries. Green curve indicated LCAs; orange curve indicated RCAs. (E and F) The fluorescence intensity of LCAs and RCAs at different time points for various groups in (C). (G) The fluorescence intensity of LCAs and RCAs for various groups in (D).