Ultrasound characterization of oxygen contrast agents produced during the reaction of hydrogen peroxide with catalase-loaded nanoparticles

We present a novel class of ultrasound (US) contrast agents, which enable de novo production of echogenic O₂ microbubbles (MBs) in vivo in regions with elevated hydrogen peroxide (H₂O₂). The agent is a 200 nm silica shell nanoparticle loaded with catalase (catSHEL) that catalyzes H₂O₂ into water (H₂O) and oxygen (O₂). Importantly, this reactive process produces O₂ MBs visible during US imaging. We have shown that this agent can detect elevated levels of H₂O₂ in kidneys with acute kidney injury (AKI). The purpose of this in vitro study is to further characterize the US properties of these O₂ MBs. When exposed to US energy (mechanical index, MI = 0.07), a marked increase in the 2^nd harmonic signal was recorded after the addition of H₂O₂ when the catSHELs were suspended in 25% plasma + phosphate buffered saline (PBS) versus PBS alone (13.1 vs. 1.7 dB). This harmonic gain abated at higher plasma concentrations (6.0 dB at 100% plasma) and higher (MI = 0.10) and lower (MI = 0.03) US transmit powers (8.8 and 5.7 dB, respectively). The harmonic signal also increased with increasing surfactant concentration. Again, the response was greatest at moderate acoustic power. O₂ MBs produced in PBS alone were transient and relatively large, while those produced at increasing plasma and surfactant concentrations were smaller and longer lasting, suggesting that they are stabilized in plasma or surfactant. Overall, nonlinear US imaging may be suitable for the local visualization of O₂ MBs produced in response to AKI.