Design a dual-response two-photon fluorescent probe for simultaneous imaging of mitochondrial viscosity and peroxynitrite in a thrombosis model

Venous thromboembolism is a sudden cardiovascular disease that can lead to death, and its pathologic development is closely related to vascular viscosity and inflammation. However, direct evidence from in vivo is really scarce. The key limitation is that the combined probes cannot detect multiple markers simultaneously, which may lead to unreliable results. Therefore, to develop a single probe that can simultaneously monitor the variations of viscosity in the vascular microenvironment as well as inflammation level during venous thrombosis. A dual-responsive two-photon fluorescent probe, Cou-ONOO, was designed and synthesized. Cou-ONOO provides a visualization tool for monitoring the viscosity of the vascular as well as the inflammatory marker ONOO¯ during thromboembolism via dual-channel simultaneous imaging. As a single probe that can recognize dual targets, Cou-ONOO effectively avoids the problems from unreliable results caused by complex synthesis and differences in intracellular localization, diffusion, and metabolism of different dyes as using combinatorial probes. Using Cou-ONOO, simultaneous imaging the variations of viscosity and ONOO¯at the cellular and tissue levels was successfully performed. In addition, Cou-ONOO also successfully visualized and tracked the viscosity of the vascular microenvironment and ONOO¯ during venous embolism in mice. Experimental results show that both viscosity and inflammation are abnormally overexpressed in the microenvironment at the thrombus site during venous thrombosis. An intuitive visualization tool to elucidate the variations of viscosity as well as inflammation level in the vascular microenvironment during thrombosis was provided, which will facilitate a better clinical understanding of the pathological process of thrombosis.