Nanomedicine relies on the effective internalization of nanoparticles combined with polymeric nanocarriers into living cells. Thus, exploration of internalization is essential for improving the efficacy of nanoparticle-based strategies in clinical practice. Here, we investigated the physicochemical internalization of gold nanoparticles (AuNPs) conjugated with hydrophobic polyampholytes into cell-sized liposomes at a low but nonfrozen temperature. The hydrophobic polyampholytes localized in the disordered phase of the membrane, and internalization of AuNPs was enhanced in the presence of hydrophobic polyampholytes together with incubation at -3 degrees C as compared to 25 degrees C. These results contribute toward a mechanistic understanding for developing a model nanomaterials-driven delivery system based on hydrophobic polyampholytes and low temperature.