Optimization of ultrasound contrast agent for high frequency ultrasound molecular imaging using subharmonic oscillation

Ultrasound molecular imaging visualizes disease biomarkers using targeted ultrasound contrast agents (t-UCA). Different lipids result in UCA with different acoustic behavior. In this study, subharmonic (SH) response of MicroMarker UCA and four homemade UCA with different lipid coatings were compared in vitro with a high frequency ultrasound system (Vevo 2100 with MS250 probe). UCA with different size distribution were made with DSPC (UCA A and C) or DPPC (UCA B and D) as main lipid by sonication or Vialmix. We transmitted long bursts (20-cycle, 30 MHz, 10% power, MS250 probe) with pulse inversion sequence for optimized SH imaging. The effect of the UCA concentration on SH imaging was tested using two concentrations: 8.0×10⁶ and 4.0×10⁵ microbubbles per milliliter. For A, B and D type UCA, attenuation effect is dominant at high concentration. MicroMarker and C type UCA had the highest and most spatially homogeneous distribution of SH response throughout the entire UCA area at high concentration. SH amplitude of MicroMarker dropped about 11 dB when UCA concentration was reduced by a factor of 20. For this low concentration, SH amplitude of B type UCA was 10 dB higher than that for MicroMarker and was homogeneous throughout the UCA area. We showed that for 30 MHz transmit frequency, at low concentration of UCA, DPPC microbubbles have higher SH response than DSPC ones and at high concentration, smaller bubbles (MicroMarker and C) produced the most homogeneous SH responses. Our results suggest that for molecular imaging applications where UCA concentration is low, our B-type and MicroMarker UCA are suitable choices for SH imaging.