The lack of a well defined nanosystem that retains its physicochemical properties and can be tracked in complex biological environments is one reason why the study of NP transport across biological barriers is currently so difficult. As a result, surprisingly little is known about the fate of sub-micron particles once they deposit in the airways of the lung. The aim of this study was to design and manufacture a novel nanoparticle (NP) core that would be physically stable, i.e., not aggregate in biological fluids, and act as a tracking system to investigate NP distribution in the lung. Accordingly, covalent fluorescent labeling (to allow particle tracking) of 40% hydrolyzed poly(vinyl alcohol) was undertaken by inducing dissociation of the carboxylic acid group (ArCOO-) of 5 (6)-carboxyfluorescein (CF) which then reacted with the hydroxyl group of poly(vinyl alcohol) (PVA) to produce a covalently linked PVA-CF ester. Polymer purification was followed by NP manufacture and characterization in biological media. In contrast to commercial latex particles which aggregated in both cell culture medium and Hank's balanced salt solution (HBSS), the PVA nanoparticles retained their original size (ca. 220 nm), maintained a neutral surface charge in cell culture medium for 24 h and were not acutely toxic to respiratory cells in vitro.