Purpose: To overcome the nonspecific toxicity and immunogenicity of immunotoxins, PE38KDEL was used as a modle protein toxin to prepare PE38KDEL-loaded poly(lactic-co-glycolic acid)(PLGA) targeted nanoparticles, which was covalently conjugated with a targeted ligand, Fab fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2) to construct PE38KDEL-Nanoparticles-HER (PE-NPs-HER).Method: small size (~100 nm) NPs with desirable drug loading to link with rhuMAbHER2 were fabricated with double emulsion method.The characterization of targeted NPs, such as particle size, size distribution and morphology were evaluated by dynamic light-scattering detector and TEM, respectively.Micro BCA assay was used to determine the drug encapsulation efficiency and the amount of drug released from PE-NPs.The presence of Fab on nanoparticles was confirmed by Flow cytometry.Result:The significantly higher cytotoxicity was observed while PE-NPs-HER binded to HER2 in breast cancer cells in vitro comparing with PE-NPs that lack anti-HER2 Fab.In vivo therapy studies with HER2-overexpressing tumor xenograft model showed the superiority of PE-NPs-HER over all other treatments, including immunotoxins PE-HER constructed by chemically coupling PE38KDEL to rhuMAbHER2 and other controls.More importantly, the PE-NPs-HER was well tolerated in mice with a higher LD50 [LD50 of 6.86 ± 0.47 mg/kg vs.2.21 ± 0.32 mg/kg for PE-NP-HER vs PE-HER (mean ± SD); n =3)].Notably, PE-NPs-HER was less susceptible to inactivation by anti-PE38KDEL neutralizing antibodies in vitro compared with PE-HER.Conclusion:The bioconjugates PE-NPs-HER may represent a potentially useful strategy for a immunotoxins therapeutic application.