In this study, we report, for the first time, the synthesis of two original nanosystems, based on gold Au(III) and copper Cu(II): simple gold–copper nanoparticles (Cu0AuNPs) and enriched monopicolinate cyclam (L1)–Cu(II)–Au(III)-complex (L1@Cu2+AuNPs). The two nanomaterials differ substantially by the chelation or not of the Cu(II) ions during the NPs synthesis process. The two hybrid nanoparticles (Cu0AuNPs; L1@Cu2+AuNPs) were deeply studied from the chemical and physical point of view, using many different analytical techniques such as Raman and UV–vis spectroscopy, electron transmission microscopy, and dynamic light scattering. Both nanosystems show morphological and good chemical stability at pH 4 values and in physiological conditions during 98 h. Undifferentiated and neural differentiated murine embryonic stem cells were used as a model system for in vitro experiments to reveal the effects of NPs on these cells. The comparative study between Cu0AuNPs and L1@Cu2+AuNPs highlights that copper chelated in its +2 oxidation state in the NPs is more functional for biological application.