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Gas vesicles (GVs) are gas-filled protein nanostructures that can regulate the buoyancy of microorganisms such as cyanobacteria and archaea. Recent studies have shown that GVs have the potential to be used as ultrasound molecular imaging probes in disease diagnosis and treatment. However, the mechanism of the inflation and deflation of GVs remains unclear, which hampers the preservation of GVs and gas replacement. In the present study, the environmental pH value was found to be an important factor in regulating the inflation and deflation of GVs. It can not only regulate the inflation and deflation of GVs in vivo to make Microcystis sp. cells present distinct levitation state, but also regulate the inflation and deflation of purified GVs in vitro , and the regulation process is reversible. Our results may provide a technical support for the large-scale production and preservation of biosynthetic ultrasound molecular imaging probes, especially for gas replacement to meet different diagnostic and therapeutic needs, and would facilitate the application of biosynthetic ultrasound molecular imaging probes.

Gas vesicles are a unique class of gas-filled protein nanostructures which are commonly found in cyanobacteria and Halobacterium . The gas vesicles may scatter sound waves and generate harmonic signals, which enabled them to have the potential to become a novel ultrasound contrast agent. However, the current hypertonic cracking method for isolating gas vesicles contains tedious operational procedures and is of low yield, thus not suitable for large-scale application. To overcome these technical challenges, we developed a rapid and efficient method for isolating gas vesicles from Microcystis. The new H 2 O 2 -based method increased the yield by three times and shortened the operation time from 24 hours to 7 hours. The H 2 O 2 method is not only suitable for isolation of gas vesicles from laboratory-cultured Microcystis , but also suitable for colonial Microcystis covered with gelatinous sheath . The gas vesicles isolated by H 2 O 2 method showed good performance in ultrasound contrast imaging. In conclusion, this new method shows great potential for large-scale application due to its high efficiency and wide adaptability, and provides technical support for developing gas vesicles into a biosynthetic ultrasonic contrast agent.

Objective: To investigate the effect and safety of Escherichia coli ( E.coli ) expressing gas vesicle (GVs) for enhancing the efficacy of tumor ablation by high intensity focused ultrasound (HIFU) in tumor-bearing mice., Objective: Thirty-two female BALB/c mice were used to establish mouse models bearing 4T1 tumor, which were randomized into GVs group [ E.coli BL21 (AI)-PET28a-Arg1] and control group (PBS), and the efficacy of HIFU ablation was evaluated by examining coagulative necrotic volume and pathology of the tumors. Another 104 BALB/c mice were also randomly divided into GVs group and control group, and body weight changes of the mice were recorded on days 1, 4 and 15 after intravenous injection of E.coli containing GVs or PBS. White blood cells, red blood cells, hemoglobin and platelet counts and liver and renal function parameters of the mice were detected, and serum levels of TNF-α and IL-1β were examined using ELISA. The pathological changes in the liver and spleen were evaluated using HE staining to assess the safety of the treatments., Objective: HIFU ablation resulted in a significantly greater volume of coagulative necrosis and severer tissue damage in GVs group than in the control group ( P < 0.001). In the 104 BALB/c mice without tumor cell inoculation, intravenous injection of E.coli expressing GVs, as compared with PBS, did not significantly affect body weight or cause changes in white blood cell, red blood cell and platelet counts or hemoglobin level ( P 1=0.59, P 2=0.27, P 3=0.76, P 4=0.81). The liver and kidney function parameters ( P 1=0.12, P 2=0.46, P 3=0.62, P 4=0.86) and serum levels of TNF-α and IL-1β ( P 1=0.48, P 2=0.56) were all comparable between GVs group and control group. No obvious pathological changes were detected in the liver and spleen tissues in either GVs group or the control group., Objective: E.coli expressing GVs is safe for enhancing the ablation effect of HIFU in tumor-bearing mice.