Bioengineered gas vesicle nanostructure increases the ultrasound backscattered signal amplitude in presence of tumoral enzyme Cathepsin B

Gas vesicles (GVs), nanostructure gas-filled protein-shelled compartments, act as ultrasound contrast agents (UCAs). Recent works report bioengineered GvpC, one of the shell proteins, potentializes interactions with specific molecules in tumor microenvironment. An important target of investigation is Cathepsin B (CTSB), an enzyme produced by several tumors. CTSB acts in the tumoral microenvironment to increase tumor growth and invasion. This work objective relied on bioengineering the GVs to promote the GvpC-CTSB interaction with a subsequent increase of ultrasound (US) signal backscattered. GvpC was engineered to contain a known CTSB cleavage sequence re-added in striped GV (ΔGV). Western Blot (WB) test was used to compare the GVwt (wild type) and GV* (GV engendered) before and after 24 hours reaction with CTSB. US images for PBS (negative control), GVtw, ΔGV, GV* and CTSB+GV*, were obtained in 21MHz (5% total power) and regions-of-interest (ROIs) over the final B-mode image were determined. The WB test for GVwt resulted in mean (±SD) GvpC concentrations of 25169 (±8574) and 32873 (±9113) and GV* was 32487 (±6867) and 12763 (±2846), at t0 and t24, respectively. The US image gray level intensities for the ROIs related to the samples of PBS, GVwt, ΔGV, GV* and CTSB+GV* were 55.8 (±17.6), 47.4 (±16.6), 46.1 (±22.8) and 150.2 (±24), respectively. These results reveal an increase in the US signal backscattered from GV* after CTSV reaction. This increase is likely related to a direct GvpC degradation by CTSB, without affecting the GV* structure. In conclusion, high frequency US B-mode images showed the GV* with the potential to act as an UCA sensitive to the CTSB presence in tumoral microenvironment.