Photophysics of Cy3-encapsulated calcium phosphate nanoparticles

Progress toward clinical application of biodegradable fluorescent calcium phosphate (CP) nanoparticles as a bioimaging agent requires detailed knowledge of chromophore interaction with CP. As readouts of this cargo-matrix interaction, we determined the principle photophysical properties of Cy3 encapsulated in CP nanparticles (CPNPs) using steady-state and time-resolved fluorescence spectroscopy. Fluorescence correlation spectroscopy (FCS)-determined diffusion coefficients and associated hydrodynamic radii confirmed the presence of highly monodisperse CPNPs with radii ranging from 7 to 10 nm. Single CP nanoparticles were 20 times brighter than free dye molecules because of a CP-induced 5-fold increase in quantum efficiency and encapsulation of four dye molecules per particle. Solvatochromic shifts resulting from hydrogen bonding between free dye and solvent or restricted intramolecular mobility by solvent viscosity were absent when Cy3 was encapsulated in CP. Encapsulation-mediated increases in radiative decay rates and decreases in nonradiative decay rates resulting in longer fluorescence lifetimes of Cy3 were attributed to solvent and CP-related local refractive indices and restricted flexibility of dye by rigid CP. Enhanced brightness of CPNPs enabled imaging of single nanoparticles under epifluorescence using both standard and total internal reflection fluorescence (TIRF) modes with camera exposure times on the order of tens of milliseconds. These enhanced photophysical properties together with excellent biocompatibility make CPNPs ideal for bioimaging applications ranging from single-molecule tracking to in vivo tumor detection and offer the possibility of timed codelivery of drugs to control cell function.