n-Butanol Enables OMVs as Heat-Responsive Nanometer Ultrasound Contrast Agents for Tumor Imaging

Extracellular vesicles, such as Escherichia coli outer membrane vesicles (OMVs), have been widely explored as carriers for contrast agents, yet their application in ultrasound imaging remains limited. In this study, heat-responsive nB-OMVs are developed by loading n-butanol into OMVs (nB-OMVs). Upon heating, the decreased solubility of n-butanol triggered phase separation within the vesicles, resulting in enhanced ultrasound signals, including fundamental, harmonic, and subharmonic components. Experimental results demonstrated that nB-OMVs retained the native morphology of OMVs, along with their tumor-targeting accumulation and tumor growth inhibition capabilities. nB-OMVs injection induces red blood cell extravasation into tumors, under near-infrared (NIR) irradiation, these extravasated RBCs acted as photothermal mediators, generating a localized thermal environment that activated intratumoral nB-OMVs and further amplified the ultrasound signal. In B-mode and contrast-enhanced ultrasound (CEUS) imaging, tumor grayscale intensities increased by 4.6- and 8.5-fold, respectively. This work provides a promising strategy for ultrasound-based tumor imaging using extracellular vesicles.