Deep in vivo two-photon imaging of blood vessels with a new dye encapsulated in pluronic nanomicelles

Our purpose is to test if Pluronic® fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.