Biosynthetic Gas Vesicles from Halobacteria NRC-1: A Potential Ultrasound Contrast Agent for Tumor Imaging

Abstract

Ultrasound contrast agents are valuable for diagnostic imaging and drug delivery. Generally, chemically synthesized microbubbles (MBs) are micro-sized particles. Particle size is a limiting factor for the diagnosis and treatment of many extravascular diseases. Recently, gas vesicles (GVs) from some marine bacteria and archaea have been reported as novel nanoscale contrast agents, showing great potential for biomedical applications. However, most of the GVs reported in the literature show poor contrast imaging capabilities due to their small size, especially for the in vivo condition. In this study, we isolated the rugby-ball-shaped GVs from Halobacteria NRC-1 and characterized their contrast imaging properties in vitro and in vivo. Our results showed that GVs could produce stable and strong ultrasound contrast signals in murine liver tumors using clinical diagnostic ultrasound equipment at the optimized parameters. Interestingly, we found these GVs, after systemic administration, were able to perfuse the ischemic region of a tumor where conventional lipid MBs failed, producing a 6.84-fold stronger contrast signal intensity than MBs. Immunohistochemistry staining assays revealed that the nanoscale GVs, in contrast to the microscale MBs, could penetrate through blood vessels. Thus, our study proved these biosynthesized GVs from Halobacterium NRC-1 are useful for future molecular imaging and image-guided drug delivery.

Modification of PEG reduces the immunogenicity of biosynthetic gas vesicles

“…Compared to conventional ultrasonic contrast agents, nanoscale GVs extracted from Halobacterium NRC-1 can perfuse tumor ischemic regions and generate contrast signals due to their small particle size ( Wei et al, 2022 ). However, similar to conventional nanoparticles, most of GVs after tail vein injection were usually uptake by liver macrophages, resulting in a weak signal in the tumor ( Kreuter, 1996 ; Le Floc’h et al, 2018 ).…”

“…Hydrophobic GvpA forms the spindle-shaped skeleton, and the hydrophilic GvpC is arranged in the outer structure of the protein shells ( Distribution, 2012 ; Hill and Salmond, 2020 ; Volkner et al, 2020 ). Our previous studies demonstrated GVs from Halobacterium NRC-1 bacteria have about 200 nm particle size and exhibited excellent contrast imaging performance by using of clinical diagnostic ultrasound equipment at the optimized parameters ( Wei et al, 2022 ). However, owing to the shells composed of proteins, GVs are easily removed by macrophages of the reticuloendothelial system (RES) and probably bring with some immunogenicity or side effects, limiting their use in the future clinical practice ( Ling et al, 2020 ; Yan et al, 2020 ).…”

Ultrasound Molecular Imaging of Epithelial Mesenchymal Transition for Evaluating Tumor Metastatic Potential via Targeted Biosynthetic Gas Vesicles

“…Preparation and Characterization of E-cad-GVs and N-cad-GVs: GVs were isolated and purified from Halobacterium Salinarum NRC-1 as previously described. [24] Briefly, the bacteria were cultured for 2 weeks in a shaker at 37 °C at 220 rpm, then transferred into a separatory funnel to allow the bacteria with GVs to float to the top of the medium. The floated bacteria were collected and lysed by the hypotonic shock method.…”

“…Lipid MBs were fabricated according to the previous study. [ 24 ] The MBs used in this work were ≈1‐5 µm in particle size. The ultrasound contrast images were obtained after 60 s by using an ultrasound diagnostic system (Resona 7, Mindray, China).…”

“…Recently, biosynthetic gas vesicles (GVs) with ≈200 nm particle size from Halobacterium NRC-1 (Figure S1) have been demonstrated excellent ultrasound contrast imaging performance, [22][23][24] providing an ideal alternative to microbubbles to design the targeted nanobubbles for imaging of markers on tumor cells. In this study, we designed E-cad-and N-cadtargeted nanobubbles through conjugating the anti-E-cad or anti-N-cad antibodies to biosynthetic GVs and evaluated their molecular imaging performance for monitoring the status or balance of E-cad and N-cad of tumor cells during tumor progression (Figure 1).…”