The emerging demand for programmable functionalization of existing base nanocarriers necessitates development of an efficient approach for cargo loading that avoids nanoparticle redesign for each individual application. Herein, we demonstrate in vivo a postformulation strategy for lipidic nanocarrier functionalization with the use of a linker peptide, which rapidly and stably integrates cargos into lipidic membranes of nanocarriers after simple mixing through a self-assembling process. We exemplified this strategy by generating a VCAM-1-targeted perfluorocarbon nanoparticle for in vivo targeting in atherosclerosis (ApoE-deficient) and breast cancer (STAT-1-deficient) models. In the atherosclerotic model, a 4.1-fold augmentation in binding to affected aortas was observed for targeted vs. nontargeted nanoparticles (P< 0.0298). Likewise, in the breast cancer model, a 4.9-fold increase in the nanoparticle signal from tumor vasculature was observed for targeted vs. nontargeted nanoparticles (P< 0.0216). In each case, the nanoparticle was registered with fluorine (19 F) magnetic resonance spectroscopy of the nanoparticle perfluorocarbon core, yielding a quantitative estimate of the number of tissue-bound nanoparticles. Because other common nanocarriers with lipid coatings (eg, liposomes, micelles, etc.) can employ this strategy, this peptide linker postformulation approach is applicable to more than half of the available nanosystems currently in clinical trials or clinical uses.—Pan, H., Myerson, JW, Hu, L., Marsh, JN, Hou K., Scott, MJ, Allen, JS, Hu, G., San Roman, S., Lanza, GM, Schreiber, RD, Schlesinger, PH, Wickline, SA Programmable nanoparticle functionalization for in vivo targeting.