AbstractHere, hybrid polymer‐lipid nanoparticles are designed as colloidal carriers for Rapamycin, to improve the aqueous drug stability and to support the drug repositioning for cancer treatment, that is, against rhabdomyosarcoma (RMS). With this aim, Rapamycin – loaded hybrid nanoparticles are produced by using as nanoparticle core a graft copolymer obtained from the functionalization of the α,β‐poly(N‐2‐hydroxyethyl)‐DL‐aspartamide (PHEA) with Rhodamine B (RhB), Polylactic acid (PLA), the PHEA‐g‐RhB‐g‐PLA, and different phospholipids, that is, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) coated, pegylated and Mannose/PEG, for the surface coating. The drug loading of these samples allows for controlled release, and improves drug stability at pH 5.5 and 7.4 compared to the free drug. Chemical‐physical characterization confirms the nanostructure size below 200 nm, ideal for systemic administration, and easy re‐dispersibility in aqueous media. Moreover, biological characterization to test the potential use as antitumor agent shows induction of cytotoxicity in human rhabdomyosarcoma (RD) and macrophage (RAW) cell lines in a time‐ and concentration – dependent manner, and stimulated autophagy, comparable to the free drug. The uptake study following the fluorescence of the copolymer reveals that the hybrid nanoparticles are internalized by both tested cell lines, with a significantly higher amounts of internalized particles in the case of surface mannosylated and/or pegylated systems.