Targeted drug delivery to endothelial cells lining the vasculature can improve treatment of many pathologies. Intercellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein overexpressed in many diseases, is a good determinant for endothelial targeting of drug nanoparticles (NPs). In this study we synthesized surfactant-free, FITC-labeled poly(lactic-co-glycolic) acid (PLGA) NPs coated with anti-ICAM, and used fluorescence microscopy and radiotracing to study their interaction with endothelial cells in culture and in vivo. These NPs were stable in storage conditions and degraded in conditions mimicking intracellular lysosomes. Furthermore, NPs showed specific ICAM-1 binding, which was enhanced in diseased-like conditions, followed by efficient uptake and lysosomal trafficking via the CAM-mediated pathway. Intravenous administration of NPs in mice resulted in organ-specific accumulation, most prominently the lungs. Hence, surfactant-free, FITC-labeled anti-ICAM PLGA NPs enabled the study of NP interactions with biological systems, which along with their fast degradation profile in physiological-like conditions, will guide future therapeutic applications.