Real‐Time In Vivo Detection of Nanocarrier Number and Velocity in the Cerebrovasculature Using Hot Band Absorption

ABSTRACT Nanocarriers play a critical role in the transport and targeted delivery of therapeutic agents. However, real‐time in vivo observation of individual nanocarriers in the circulatory system is challenging due to the need for high spatiotemporal resolution and sensitivity. Here, DNA‐stabilized silver nanoclusters (DNA640), which exhibit anti‐Stokes fluorescence upon hot‐band absorption (HBA), are loaded into cationic mesoporous silica nanoparticles (CMSN) and coated with liposomes. These constructs (DNA640‐CMSN‐liposome) exhibit improved resistance to chloride ions (Cl − ) compared to bare DNA640, ensuring that they are traceable in the cerebrovasculature. For imaging of the cerebrovasculature, adeno‐associated viral vectors (AAV) encoding for albumin‐mNeonGreen (Alb‐mNG) expression are used to secrete the label into the bloodstream allowing for visualization of the vasculature, exhibiting negligible spectral crosstalk with DNA640. Combining two‐photon fluorescence imaging and HBA‐based fluorescence correlation spectroscopy enables real‐time observation of the flow velocity and particle number of DNA640‐CMSN‐liposome within individual capillaries, demonstrating potential for pharmacokinetic and hemodynamic studies.