A computational methodology based on Metropolis Monte Carlo (MC) and the weighted histogram analysis method (WHAM) has been developed to calculate the absolute binding free energy between functionalized nanocarriers (NC) and endothelial cell (EC) surfaces. The calculated NC binding free energy landscapes yield binding affinities that agree quantitatively when directly compared against analogous measurements of specific antibodycoated NCs (100 nm in diameter) to intracellular adhesion molecule-1 (ICAM-1) expressing EC surface in in vitro cell-culture experiments. The effect of antibody surface coverage (σ s ) of NC on binding simulations reveals a threshold σ s value below which the NC binding affinities reduce drastically and drop lower than that of single anti-ICAM-1 molecule to ICAM-1. The model suggests that the dominant effect of changing σ s around the threshold is through a change in multivalent interactions; however, the loss in translational and rotational entropies are also important. Consideration of shear flow and glycocalyx does not alter the computed threshold of antibody surface coverage. The computed trend describing the effect of σ s on NC binding agrees remarkably well with experimental results of in vivo targeting of the anti-ICAM-1 coated NCs to pulmonary endothelium in mice. Model results are further validated through close agreement between computed NC rupture-force distribution and measured values in atomic force microscopy (AFM) experiments. The three-way quantitative agreement with AFM, in vitro (cell-culture), and in vivo experiments establishes the mechanical, thermodynamic, and physiological consistency of our model. Hence, our computational protocol represents a quantitative and predictive approach for model-driven design and optimization of functionalized nanocarriers in targeted vascular drug delivery.absolute binding free energy | Monte Carlo | targeted drug delivery | multivalent interactions | antibody surface coverage T argeted delivery of functionalized nanocarriers (i.e., NCs coated with specific targeting ligands) to endothelium remains an important design challenge in pharmacological and biomedical sciences. The use of functionalized NCs offers a wide range of targeting options through tunable design parameters (size, shape, type, method of functionalization, etc.). This necessitates a multiparameter optimization for achieving efficacious targeting in drug delivery applications (1) including vascular-targeting in oncology (2-4).Rational design of functionalized NCs faces many challenges owing to the complexities of molecular and geometric parameters surrounding receptor-ligand interactions and NCs (5-9), lack of accurate characterization of hydrodynamic, physico-chemical barriers for NC uptake/arrest (10-14), and uncertainty in targeting environment in vivo (15)(16)(17).Among the factors impacting the design of NCs and therapeutic agents are: (i) binding affinity (18) Recently the binding affinity of functionalized NCs to ICAM-1 expressing EC surface has been studied experim...