A Bioengineered Cathepsin B-sensitive Gas Vesicle Nanosystem That Responds With Increased Gray-level Intensity of Ultrasound Biomicroscopic Images

Abstract

ObjectivesThis work aimed to promote the interaction of a modified Gas Vesicle (GV) with Cathepsin B (CTSB) protease and analyzed their backscattered signal by Ultrasound (US). MethodsWe modified the sequence of the gene coding for GvpC to contain a CTSB cleavage and expressed the protein in an Escherichia coli recombinant system. The protein was purified and added to GVs preparations in which the original GvpC was removed (ΔGV), constituting the modified GV (GV*). Western Blot (WB) test was used to compare GVs with GvpC and engineer GvpC at starting (T0) and after 24 hours (T24) reacting with CTSB. A 21 MHz US B-mode and nonlinear contrast-mode (5% total power) imaged US phantoms having samples of GVwt, ΔGV (stripped GV), GV*, and CTSB+GV*. Also, a 21MHz US B-mode imaged US phantoms having a tumor cell line extracellular fraction (TCEF) and the TCEF+GV* sample. A 100% total US power was applied to collapse the GV structure. ResultsIn WB we detected a decrease in engineered GvpC levels 24 h after the incubation of GV* with CTSB, compared to the concentration at T0, suggesting that CTSB cleaved the engineered GvpC. Regions-of-interest over image of phantom cross-sections were determined and the B-mode image mean gray level intensity resulted in a significant (p<0.05) increase comparing CTSB+GV* with PBS (control), GVwt, ΔGV and GV*. Non-linear mode image gray level intensity from CTSB+GV* increased by 11.79, 7.86 and 14.75 dB from samples containing GVwt, ΔGV and GV*, respectively. GV preparations incubated with TCEF and the TCEF+GV* sample showed an increase of 81% in signal compared to TCEF+GVwt. ConclusionsThe increased ultrasound backscattered signal intensity suggests GVs as a potential biosensor for protease activity, possibly aiding the detection of protease-rich tissue regions.