Amphiphilic core-shell nanoparticles have drawn considerable interest in biomedical applications. The precise control over their physicochemical parameters and the ability to attach various ligands within specific domains suggest shell cross-linked (SCK) nanoparticles may be used as multi-/polyvalent scaffolds for drug delivery. In this study, the biodistribution of four SCKs, differing in size, core composition, and surface PEGylation, was evaluated. To facilitate in-vivo tracking of the SCKs, the positron-emitting radionuclide copper-64 was used. By using biodistribution and microPET imaging approaches, we found that small diameter (18 nm) SCKs possessing a polystyrene core showed the most favorable biological behavior in terms of prolonged blood retention and low liver accumulation. The data demonstrated that both core composition, which influenced the SCK flexibility and shape adaptability, and hydrodynamic diameter of the nanoparticle play important roles in the respective biodistributions. Surface modification with poly(ethylene glycol) (PEG) had no noticeable effects on SCK behavior.