In this investigation, we have designed and synthesized an amphiphilic co-polymer with hyper-branched poly(amine-ester) and polylactide (HPAE-co-PLA) to generate nanoparticles (NPs). These have been used to encapsulate a highly active hydrophobic anti-tumor agent, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT). Encapsulation in NPs was done in an effort to increase the anti-tumor activity of this agent by facilitating its delivery to tumor cells. We have also examined and optimized the formulation parameters of the NPs that alter their drug-loading capacity and their physical, chemical and biological properties. The resulting NPs exhibited high Bp4eT-loading capacity and substantial stability in aqueous solution. In vitro drug release studies demonstrated a controlled drug release profile with increased release at acidic pH. Anti-tumor proliferation assays showed that both free drug and drug-encapsulated NPs markedly inhibited tumor cell proliferation in a time- and concentration-dependent manner. Direct microscopic observation revealed that the fluorescent NPs were taken up by cells and localized, in part, in organelles consistent with lysosomes. These results demonstrate a feasible application of the amphiphilic hyper-branched co-polymer, HPAE-co-PLA, as nanocarriers for intracellular delivery of potent anti-tumor agents.