Erythrocyte incubation as a method for free-dye presence determination in fluorescently labeled nanoparticles

The field of nanotheranostics encompasses the integration of nanosized carriers in cancer imaging, diagnosis, and therapy. The use of nanomedicines for theranostic application typically depends on direct visualization of the nanocarriers. Normally fluorescent probes are attached to nanocarriers for biodistribution measurement through fluorescence imaging. However continued, noninvasive assurance that the fluorescent probe remains bound to the carrier has proven elusive. Mature erythrocytes, also known as red blood cells, are incapable of endocytosis. As a consequence, when incubated with fluorescently labeled particles, they do not show any signal coming from the membrane or the cytoplasm. Yet, these cells readily take up free BODIPY fluorescent dyes into their membranes. Here we show that incubation of nanoparticles with erythrocytes is a rapid and reliable method for the detection of unbound dye present within a nanoparticle sample, as the detection of a fluorescent signal coming from the cells can only be due to unbound dye present in the sample. We test the method on both sulfonate and PEG terminated gold nanoparticles, and we determine the minimum concentration of detectable dye for a specific gold nanoparticle sample.