Nanocarrier Drug Release and Blood-Brain Barrier Penetration at Post-Stroke Microthrombi Monitored by Real-Time F&oumlrster Resonance Energy Transfer-Based Detection System (FedEcs)

Nanotechnology holds great promise for improving the delivery of therapeutics to the brain. However, current approaches often operate at the organ or tissue level and are limited by the lack of tools to dynamically monitor cargo delivery in vivo. We have developed highly fluorescent lipid nanodroplets (LNDs) that enable tracking of nanocarrier behaviour at the subcellular level while also carrying a Forster resonance energy transfer (FRET)-based drug delivery detection system (FedEcs) capable of monitoring cargo release in vivo. Using two-photon microscopy, we demonstrate that circulating LNDs in naive mouse brain vasculature exhibit 3D real-time FRET changes, showing size-dependent stability over two hours in blood circulation. Further, in a novel nano-stroke model, dynamic intravital two-photon imaging revealed that LNDs accumulated within cerebral post-ischemic microthrombi, where they released their cargo significantly faster than in normal blood circulation. Furthermore, the blood-brain barrier (BBB) became permeable at the microclot sites thereby allowing accumulated FedEcs-LNDs to cross the BBB and deliver their cargo to the brain parenchyma. This microthrombi-associated translocation was confirmed at the ultrastructural level via volume correlative light-electron microscopy. Consequently, our FedEcs represents a novel tool to quantitatively study the biodistribution and cargo release of nanocarriers at high resolution in real time. By enabling us to resolve passive targeting mechanisms post-stroke, - specifically, accumulation, degradation and extravasation via post-stroke microthrombi - this system could significantly enhance the translational validation of nanocarriers for future treatments of brain diseases.