Enhanced Bioluminescence Imaging Of Inflammation Based On Dual-color Nanobubbles

Inflammation is an immunological response involved in various inflammatory disorders ranging from neurodegenerative diseases to cancers.In the inflammatory process,the heme-containing enzyme myeloperoxidase(MPO),which is abundantly expressed in neutrophils,monocytes,and macrophages,plays a key role.Luminol has been reported to detect MPO activity in an inflamed area through a light-emitting reaction.However,this method is limited by low tissue penetration and poor spatial resolution.Ultrasound imaging is unique and is characterized with low cost,real time,and high spatial resolution.With the use of ultrasound contrast agents,the ultrasound imaging can delicately delineate the microvasculature in deep tissues.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),enabling MPO-dependent inflammation imaging in deep tissues.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.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.We demonstrated a luminescing NB employing an energy transfer relay that integrates BRET and FRET to red-shift luminol-emitted blue light to red light,enabling MPO-dependent inflammation imaging in deep tissues.The luminescing NBs caused a 24-fold increase in detectable luminescence emission over luminol alone in mice challenged by lipopolysaccharide.In addition,the NBs can also be utilized as an ultrasound contrast agent.Compared with commercially available microbubbles,the luminescing NBs show comparable ultrasound contrast-enhancing capability but smaller size and higher concentration.In an animal model of breast cancer,the luminescing NBs combined with luminol clearly show the MPO activity in the tumor,whereas the contrast-enhanced ultrasound imaging delineates the anatomical structure and vasculature development of the inflamed region indicated by bioluminescence imaging.In addition,multi-injection of luminol and the NBs did not cause any observable toxicity.In conclusion,this methodology provides a noninvasive and highly sensitive means for accurate visualization of inflammatory diseases.