50-nm gas-filled protein nanostructures to enable the access of lymphatic cells by ultrasound technologies

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need of microbubbles, which cannot transverse many biological barriers due to their large size. Here we introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles that we referred to as _50nm GVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to our knowledge, the smallest stable, free-floating bubbles made to date. _50nm GVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected _50nm GVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen-presenting cells adjacent to lymphocytes. We anticipate that _50nm GVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas-filled nanomaterials.