" Drug-initiated" nitroxide-mediated synthesis of two well-defined, heterotelechelic polymer prodrugs ( Mn = 1960-5260 g·mol-1, Đ = 1.31-1.37) was performed by using the newly developed nitroxide exchange reaction. These polymers comprised, at the chain end, gemcitabine (Gem) as anticancer drug and either cyanine 7.5 (Cy7.5) as a near-infrared (NIR) dye suitable for in vivo imaging or biotin (Biot) for cancer cell targeting. These materials were co-nanoprecipitated into fluorescently labeled polymer prodrug nanoparticles of average diameter in the 100-180 nm range with narrow particle size distribution and variable surface amounts of biotin. Nanoparticles containing 15 wt % biotinylated polymer showed superior uptake and the highest cytotoxicity in vitro on A549 human lung cancer cells. In vivo, on A549 tumor bearing mice, biotinylated nanoparticles showed significantly higher efficacy than free Gem and maintained the same anticancer activity than nontargeted nanoparticles without inducing prohibitive body weight loss. Biotinylated polymer prodrug nanoparticles did not result in an improved anticancer activity or significant increase in tumor accumulation, which may be the result of a nonoptimal biotin surface display and/or insufficient affinity toward the target. They however displayed delayed liver accumulation compared to nonbiotinylated counterparts, suggesting the premise of a stealth property likely due to the hydrophilic tetraethylene glycol-Biot positioned at the nanoparticle surface. This work showed for the first time the applicability of this simple construction method to in vivo imaging and cancer cell targeting and might stimulate the design of new functional materials for biomedical applications.