Simultaneous direct and indirect assessments of singlet oxygen generation during vascular‐targeted photodynamic therapy with thrombin molecular beacon

Abstract Vascular‐targeted photodynamic therapy (V‐PDT) offers a precise therapeutic approach for treating diseases associated with abnormal vasculature. The therapeutic efficacy of type II V‐PDT mainly relies on the vascular response to singlet oxygen ( 1 O 2 ), which could convert prothrombin into thrombin and then result in vasocontraction and subsequent tissue ischemia. In this study, the photosensitizer pyropheophorbide‐a (Pyro) was conjugated with the fluorescent molecule 5‐carboxy‐X‐rhodamine (Rox) through a thrombin‐cleavable peptide, forming a thrombin‐activated molecular beacon Pyro‐thrombin‐cleavable peptide‐Rox (PPR). Furthermore, a novel multimodal imaging system was developed for simultaneously imaging near‐infrared (NIR) 1 O 2 luminescence at around 1270 nm and Rox fluorescence, which could be used to directly and indirectly assess the 1 O 2 generation during V‐PDT for an in vivo model, respectively. It was found that the vasoconstrictions are positively correlated with both 1 O 2 luminescence intensity and Rox fluorescence intensity, respectively. For this, the PPR could serve as a therapeutic PS and as an indirect indicator for 1 O 2 generation during V‐PDT, which has the advantage of higher sensitivity compared to the direct measurement of 1 O 2 luminescence.