Bioluminescence Imaging of Inflammation in Vivo Based on Bioluminescence and Fluorescence Resonance Energy Transfer Using Nanobubble Ultrasound Contrast Agent

Inflammation is an immunological response involved in various inflammatory disorders ranging from neurodegenerative diseases to cancers. Luminol has been reported to detect myeloperoxidase (MPO) activity in an inflamed area through a light-emitting reaction. However, this method is limited by low tissue penetration and poor spatial resolution. Here, we fabricated a nanobubble (NB) doped with two tandem lipophilic dyes, red-shifting luminol-emitted blue light to near-infrared region through a process integrating bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET). This BRET–FRET process caused a 24-fold increase in detectable luminescence emission over luminol alone in an inflammation model induced by lipopolysaccharide. In addition, the echogenicity of the BRET–FRET NBs also enables perfused tissue microvasculature to be delineated by contrast-enhanced ultrasound imaging with high spatial resolution. Compared with commercially available ultrasound contrast agent, the BRET–FRET NBs exhibited comparable contrast-enhancing capability but much smaller size and higher concentration. This bioluminescence/ultrasound dual-modal contrast agent was then successfully applied for imaging of an animal model of breast cancer. Furthermore, biosafety experiments revealed that multi-injection of luminol and NBs did not induce any observable abnormality. By integrating the advantages of bioluminescence imaging and ultrasound imaging, this BRET–FRET system may have the potential to address a critical need of inflammation imaging.