The behaviour of polymeric entrapped thiol-coated GNRs in culture medium under biological conditions was analysed. The in vitro cytotoxicity was studied by a Colony Forming Efficiency assay on immortalized mouse fibroblasts (Balb/3T3) obtaining a dose-effect relationship in which an Inhibitory Concentration at 50% (IC50) was 20.3µM. The suitability of the new nanomaterial as optoacoustic contrast agent was investigated in phantom studies using a hardware platform suitable for retrieving clinically relevant data. Spherical alginate phantoms containing GNR-2-PNP at different concentrations were synthesized and the optoacoustic signal amplitudes were measured as a function of concentration. Signals could be obtained with satisfying SNR down to concentrations of 11 µM corresponding to subtoxic concentration in our in vitro model. The nanomaterial showed to be a suitable and promising contrast agent for different optoacoustic imaging modalities including multispectral approaches.JRC.I.4-Nanobioscience