Ultrasound Molecular Imaging of Blood Vessel Walls and Vulnerable Plaques via CXCR4-Targeted Nanoscale GVs

Abstract

Purpose: C-X-C chemokine receptor 4 (CXCR4) mediates the inflammatory response of atherosclerotic vulnerable plaques (ASVP) and is a potential biomarker of atherosclerotic vulnerable plaques. The purpose of this study was to use the imaging ability of a new type of ultrasound contrast agent, nanoscale biosynthetic gas vesicles (GVs), on the vascular wall and to combine the specific ligand of CXCR4 to construct a targeted molecular probe to achieve early identification of atherosclerotic vulnerable plaques and guide clinical treatment decisions.

Materials and methods: Compared three contrast agents: GVs, the micro-contrast agent SonoVue, and polyethylene glycol (PEG)-modified GVs in the carotid artery. The expression of CXCR4 in atherosclerotic plaques was demonstrated using flow cytometry and immunofluorescence experiments. Cell adhesion and in vivo ultrasound imaging experiments demonstrated their ability to target the nanoscale biosynthetic gas vesicles. The safety of GVs, PEG-GVs, and CXCR4-GVs was tested the CCk8 test, H&E staining, and serum detection.

Results: Strong CXCR4 expression was observed in plaques, whereas little expression was observed in normal vessels. GVs can produce stable contrast signals on the carotid artery walls of rats, whereas PEG-GVs can produce more lasting contrast signals on the carotid artery wall of rats. CXCR4-GVs exhibited excellent binding capability to ox-LDL-induced RAW264.7 cells. Animal experiments showed that compared with Con-GVs, CXCR4-GVs injected plaque imaging signal was stronger and more durable. In vitro scanning of vulnerable plaques in rats injected with fluorescent vesicles demonstrated that CXCR4-GVs oozed through the neovasculars within vulnerable plaques and aggregated in vulnerable plaques. Through the CCK8 test, H&E staining, and serum detection, the safety of CXCR4-GVs was confirmed.

Conclusion: CXCR4-GVs were constructed as targeted molecular probes, which can be proven to have good targeting properties to vulnerable atherosclerotic plaques.

Keywords: CXCR4; nanoscale biosynthetic gas vesicles; ultrasound molecular imaging; vulnerable plaque.

Graphical abstract

Figure 1

Preparation and characterization of GVs, PEG-GVs, and CXCR4-GVs. (A) TEM images of isolated GVs (left and right). The scale bars are 100 nm (left), 50 nm (right), respectively; (B) infra-red spectrogram of GVs, Mal-PEG-GVs, Con-GVs and CXCR4-GVs; (C) Zeta potential of GVs, PEG-GVs, Mal-PEG-GVs and CXCR4-GVs; (D) Size distribution of GVs, PEG-GVs, Mal-PEG-GVs and CXCR4-GVs; Data of (C and D) represent the mean ± SD from 3 independent experiments.

Figure 2

CXCR4 expression in foam cells and plaque tissues. (A) Flow cytometry analysis for the foam cell surface expression level of CXCR4 protein on RAW264.7 cells, MOVAS cells and HUVEC cells. Scale bars are 50 nm; (B) Immunofluorescence Staining in normal vessels and plaque tissues; (C) Quantification analysis of mean fluorescence intensity from (B). *p < 0.05.

Figure 3

In vitro binding of CXCR4-GVs to RAW264.7-derived foam cells. (A) Representative fluorescent microscope images of RAW264.7-derived foam cells incubated with FITC-labeled Con-GVs, FITC-labeled CXCR4-GVs and free targeting peptide + FITC-labeled CXCR4-GVs. Green represents FITC-labeled GVs and blue represents cell nuclei stained with DAPI. Scale bar: 50 µm. (B) Quantification of fluorescence intensity from (A). The data in (B) represent the mean ± SD of three independent experiments. **p < 0.01, ^nsno significance.

Figure 4

In vitro ultrasound contrast imaging of GVs, con-GVs, and CXCR4-GVs. (A) Ultrasound contrast images of GVs, Con-GVs, and CXCR4-GVs at different concentrations (OD₅₀₀ = 0.5–2.0). (B) Quantification of the mean ultrasound signal intensities from (A). Data represent the mean ± SD of three independent experiments.

Figure 5

Vascular wall imaging capabilities of GVs. (A) Contrast-enhanced images of SonVue, GVs, and PEG-GVs after intravenous injection into the normal carotid artery. (B) Contrast-enhanced images of SonVue, GVs, and PEG-GVs after intravenous injection into the plaque carotid artery.

Figure 6

In vivo ultrasound molecular imaging of plaques. (A) Non-linear contrast images of Con-GVs and CXCR4-GVs (OD₅₀₀ = 3.5) at different time points after intravenous injection. (B) Time-intensity curves of Con-GVs and CXCR4-GVs after intravenous injection. (C) Contrast signal intensities of tumors treated with Con-GVs and CXCR4-GVs at 1, 3, 5, and 10 minutes. Data (B and C) represent the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001.

Figure 7

Histological analysis. (A) Representative fluorescence images of plaque sections from rats injected with FITC-labeled Con-GVs or FITC-labeled CXCR4-GVs. Green indicates GVs, red indicates anti-CD31 antibodies, and blue indicates cell nuclei stained with DAPI. Scale bar: 50 µm. (B) Mean fluorescence intensity of plaque sections in three random view fields. Data represent the mean ± SD of three independent experiments. ***p < 0.001.

Figure 8

Biosafety analysis. (A–D) Liver and kidney function indices of rats treated with the same volume of PBS, PEG-GV, and CXCR4-GVs. The units of ALT, AST, and ALP were U/L, and those of BUN and CREA were μmol/L. (E) Representative H&E sections of the main organs (heart, liver, spleen, lung, and kidney) from mice treated with PBS, GVs, PEG-GVs, or CXCR4-GVs for seven days. Scale bars: 100 µm. Data represent the mean ± SD of three independent experiments.