Acoustic protein nanostructures for contrast ultrasound

Abstract

Gas vesicles (GVs) are nanosized protein-encased gas particles produced in species of Bacteria and Archea. Similar to microbubbles, GVs can generate harmonic signals when exposed to ultrasound, making it an alternative agent for contrast imaging. In addition, their protein shell allows for continuous gas exchange with the environment resulting in a physically stable long-lasting structure. A further advantage of gas vesicles is their size (40-500 nm), which has the potential for extravasation out of leaky vasculatures of tumors. This can potentially allow for targeting molecular biomarkers outside of the vasculature. Using the Vevo®2100 system (FUJIFILM VisualSonics, Toronto, Canada) operating at 18MHz, we imaged mouse tumor models to optimize and improve signal enhancement. GVs were isolated and purified from Halobacterium sp. NRC-1 and quantified using the optical density at 500nm (OD500). Mouse tumor models were generated using Lewis Lung Carcinoma cells injected into SHO mice and imaged after 14 days. A total of 300 µL of GVs at a concentration of 40 OD500 was injected intravenously at an infusion rate of 500 µL/min. Non-linear contrast enhancement in tumors was clearly visible after infusion of GVs, with wash-in curves showing high SNR. Unfortunately, in contrast to the liver where a prolong enhancement was observed, a rapid loss of contrast was seen in tumors post-injection. Inhibition of the RES is a well-known method to improve circulation of nanosized particles. The saturation of liver Kupffer cells using a double injection showed better signal retention (40-70%) increase following the second administration of GVs, in comparison to the first. Additionally, the disruption of Kupffer cell function using either GdCl3 or Intralipid also resulted in prolonged signal retention. To examine a more biocompatible method of blocking GVs detection by the RES, the surface of GVs were modified with the addition of PEG chains of varying lengths. A 5 kDa PEG length showed 75% signal retention during the washout phase of contrast enhancement.