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In order to investigate the impact of the particle interface characteristics on the biological fate, PGAS nanoparticles are covalently and noncovalently coated with <jats:italic>N<\/jats:italic>\u2010(2\u2010hydroxypropyl) methacrylamide (HPMA) copolymers. HPMA copolymer\u2010modified PGAS nanoparticles have similar particle sizes, but less negative zeta\u2010potentials. Nanoparticles are double labeled with the fluorescent dyes DiR (noncovalently) and DYOMICS\u2010676 (covalently bound to HPMA copolymer), and their biodistribution is investigated noninvasively by multispectral optical imaging. Both covalent and noncovalent coatings cause changes in the pharmacokinetics and biodistribution in healthy and tumor\u2010bearing mice. In addition to the intended tumor accumulation, high signals of both fluorescent dyes are also observed in other organs, including liver, ovaries, adrenal glands, and bone. 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