ROS-sensitive Dyes in Lipid Nanoparticles for in vivo Imaging

Reactive oxygen species (ROS) actively participate in many metabolic processes in liver cells, including cell proliferation and apoptosis. Intracellular ROS not only contribute in signaling, but also oxidize lipids, proteins, and DNA. As a result, ROS are involved in the development of many diseases. Lifetime visualization of ROS using sensors that are sensitive to the presence of free radicals is an urgent but difficult task due to short lifetimes of radicals and varied concentrations in different cell compartments. We synthesized lipid nanoparticles (LNP) with average diameters $100 \pm 10$ nm and two different ROS sensitive dyes (L-012 and hydrocyanine 5) for effective detection of ROS in liver cells. We demonstrated that L-012 sodium salt leaks from the nanoparticles, while hydrocy5 labeled LNP are stable and shows an increase of fluorescence in more than 5 times after reaction with hydrogen peroxide. We also validated animal model with increased ROS production by scanning ion-conductance microscopy. Both lipopolysaccharide (LPS) and carbon tetrachloride (CCl4) treatment caused intensive ROS burst in 24 h after injection. Using fluorescent tomography, we analyzed LNP biodistribution in Balb/c mice and observed its preferable accumulation in liver. Thus, incorporation of ROS-sensitive dyes into LNP can improve the stability of the sensors, minimize degradation during blood circulation, and increase its accumulation in the target organ. Creating delivery systems for lifetime ROS imaging can be a useful tool for studying changes in ROS in liver diseases.