Preclinical in vitro studies of drug candidates for anticancer therapy are generally conducted on well-established 2D cell models. Unfortunately, these models are unable to mimic the properties of in vivo tumors. However, in vitro 3D models (spheroids) have been proven to be superior in reflecting the tumor microenvironment. Diethyldithiocarbamate (DDC⁻) is the active metabolite of Disulfiram, an approved drug for alcoholism and repurposed for cancer treatment. DDC⁻ binds copper in a molar ratio of 2:1 resulting in a water-insoluble Cu(DDC)₂ complex exhibiting anticancer activities. Delivery of the Cu(DDC)₂ complex using nanoparticulate carriers provides decisive advantages for a parental application. In this study, an injectable liposomal Cu(DDC)₂ formulation was developed and the toxicity was compared with a 2D neuroblastoma and a 3D neuroblastoma cell model. Our results indicate that Cu(DDC)₂ liposomes complied with the size requirements of nanoparticles for intravenous injection and demonstrated high drug to lipid ratios as well as colloidal stability upon storage. Furthermore, an efficient cytotoxic effect on neuroblastoma 2D cell cultures and a very promising and even more pronounced effect on 3D cell cultures in terms of neuroblastoma monoculture and neuroblastoma co-culture with primary cell lines was proven, highly encouraging the use of Cu(DDC)₂ liposomes for anticancer therapy.