Harmonic Ultrasound Imaging of Nanosized Contrast Agents for Multimodal Molecular Diagnoses

The aim of the present work was to demonstrate the possibility of selective detection of nanoparticle contrast agents (NPCAs) on diagnostic echographic images by exploiting the second harmonic component they introduce in the spectra of corresponding ultrasound signals, as a consequence of nonlinear distortion during ultrasound propagation. We employed silica nanospheres (SiNSs) of variable diameter (160 nm, 330 nm, and 660 nm) dispersed in different volume concentrations (range 0.07-0.8%) in agarose gel samples that were automatically scanned through a digital ecograph using narrow-band ultrasound pulses at 6.6 MHz and variable mechanical index (MI range 0.2-0.6). In the first part of the study, the intensity peaks of four different spectral components of the backscattered signal were considered: fundamental (detected in correspondence of the incident ultrasound frequency), subharmonic (detected at half of the fundamental frequency), ultra harmonic (detected at 1.5 times the fundamental frequency), and second harmonic (detected at twice the fundamental frequency). Subsequently, based on the experimental results of the first part of the study and on our recently reported findings, the focus was moved to a detailed comparison between subharmonic and second harmonic trend, which were determined as a function of nanoparticle composition, sample concentration, and MI. The experiments were also repeated on different agarose samples, containing SiNSs covered by an outer shell of smaller magnetic nanoparticles, made of either iron oxide (IO) or FePt-IO nanocrystals. Obtained results show that this new ultrasound-based method for NPCA imaging has a detection sensitivity similar to that of our previously introduced subharmonic-based technique in the presence of 330-nm SiNSs, but performs significantly better in the detection of both the types of "dual mode" NPCAs. The fact that the reported detection method was optimized for identification of 330-nm SiNSs (a sort of "ideal" size for the development of novel tumor-targeting NPCAs) and that the magnetically coated particles are detectable also through magnetic resonance imaging makes the presented second harmonic ultrasound method a valuable solution for the introduction of new protocols for multimodal molecular diagnoses employing only nonionizing radiations.