Combined therapy with gemcitabine and tyrosine-kinase inhibitors (i.e., sunitinib) has already demonstrated important benefits in pancreatic cancer treatment. Further therapeutic advantage could be achieved by their co-loading in a single nanoscale system, which enables (i) the co-existence of drugs with different mechanisms of action and pharmacokinetic profiles and (ii) the fine tuning of their release rate overcoming the rapid clearance often observed with free drugs. In this context, the already validated squalenoylation approach has been applied to the design of a multidrug nanoparticle (NP) made by co-self-assembly of the squalene-based prodrugs of gemcitabine (SQGem) and sunitinib (SQSun). We hypothesized that co-delivering of SQGem and SQSun in a single nanoparticle was capable to increase their cytotoxicity on MIA PaCa-2 pancreatic cancer cells compared to the monodrug NPs. Nevertheless, multidrug NPs (i.e., SQGem/SQSun NPs) were as efficient as the physical mixture of the individual monodrug NPs (SQGem NPs + SQSun NPs) thus suggesting that the cytotoxicity raised from the exposure of the cells simultaneously to the two bioconjugates rather than to their original loading into a single or two different nanoparticles. To be noted that the lack of differences in static 2D cultures does not exclude a different behavior in dynamic conditions in vivo. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.