Targeting Nanostrategies for Imaging of Atherosclerosis

Abstract

Despite the progress in cardiovascular research, atherosclerosis still represents the main cause of death worldwide. Clinically, the diagnosis of Atherosclerotic Cardiovascular Disease (ASCVD) relies on imaging methodologies including X-ray angiography and computed tomography (CT), which however still fails in the identification of patients at high risk of plaque rupture, the main cause of severe clinical events as stroke and heart attack. Magnetic resonance imaging, which is characterized by very high spatial resolution, could provide a better characterization of atherosclerotic plaque (AP) anatomy and composition, aiding in the identification of "vulnerable" plaques. In this context, hydrogel matrices, which have been demonstrated able to boost relaxometric properties of Gd-based contrast agents (CAs) by the effect of Hydrodenticity, represent a valuable tool towards the precision imaging of ASCVD improving the performance of this class of CAs while reducing systemic toxicity. In particular, hydrogel nanoparticles encapsulating Gd-DTPA can further contribute to providing CA-specific accumulation in the AP by nanoparticle surface decoration triggering an active targeting of the AP with the overall effect of allowing an earlier and more accurate diagnosis. In this work, we tested crosslinked Hyaluronic Acid Nanoparticles (cHANPs) in the complex environment of human atherosclerotic plaque. In addition, the surface of cHANPs was decorated with the antibody anti-CD36 (Ab36-cHANPs) for the active targeting of AP-associated macrophages. Results demonstrate that the Hydrodenticity of cHANPs and Ab36-cHANPs is preserved in this complex system and, preliminarily, that interaction of these probes with the AP is present.

Figure 1

Characterization of Ab-ST-cHANPs and Ab-cHANPs. (a) Size distribution of cHANPs, Ab36-ST-cHANPs, and Ab36-cHANPs by dynamic light scattering (DLS). Transmission electron microscopy (TEM) images of (b) cHANPs, (c) Ab36-ST-cHANPs, and (d) Ab36-cHANPs. (e) Relaxometric properties of cHANPs, Ab36-ST-cHANPs, and Ab36-cHANPs by Minispec Benchtop Relaxometer.

Figure 2

Scanning electron microscopy (SEM) of transversal sections of a human AP (a) tunica intima with detached basal lamina (red arrow head). (b) Interrupted or damaged endothelial layer (black arrow head). (c) Fibrinous reticulum in the area with absent endothelial lining. (d) Cells in the endothelial layer showing pseudopodia (white arrow head) and microvilli.

Figure 3

Transmission electron microscopy (TEM) of a cross section of a human AP showing (a) lipid droplets of different sizes, surrounded by calcium deposits (red arrows) and dispersed in the fibrinous reticulum. (b) Interspersed cholesterol crystals (C and black lines). (c) Damaged tunica intima (yellow arrow heads) showing the absence of endothelial cells, as observed in SEM imaging.

Figure 4

Ex vivo MRI of six APs. (a) Bidimensional MR image of APs injected with (1) free Gd-DTPA at 12 μM, (2) cHANPs loaded with 12 μM of Gd-DTPA, and (3) Ab36-cHANPs loaded with 10.66 μM of Gd-DTPA. (b) Normalized T1 in the selected ROI over time.

Figure 5

Transmission electron microscopy (TEM) of a section of a human AP injected with cHANPs and Ab36-cHANPs. Cellular localization of (a) cHANPs (black arrow head—mitochondria) and (b) Ab36-cHANPs (red arrow head—cell nucleus).