| The clinical use of immunotoxins is severely limited by nonspecific toxicity of its toxin protein.To overcome this limitation,PE38KDEL was used as a model protein toxin to prepare PE38KDEL-Ioaded poly(lactic-co-glycolic acid)(PLGA) nanoparticles(PE-NP) by double-emulsion evaporation method.The PE38KDEL-loaded anti-HER2 targeted nanoparticles(PE-NP-HER)were fabricated by conjugation of Fab' fragments of a humanized anti-HER2 humanized monoclonal antibody(rhHER2-mAb) onto the surface of nanoparticles according to carbodiimide method.The processing parameters of NP preparation were optimized by single factor study and orthogonal design.The pharmaceutical characterization of nanoparticles made with optimized prescription and technic,such as particle size,zeta potential,drug loading,encapsulation efficiency,Fab' conjugation efficiency and drug release were evaluated.Particle size,zeta potential and morphology of PE-NP-HER and PE-NP were observed by dynamic light-scattering detector(DLS) and transmission electron microscope(TEM).The results showed both NPs were quiet round and homogenous with a 100～130nm average size distribution.And it could be speculated that Fab' had presented on the PE-NP surface due to the increase in particle size(106nm→125nm) and the change of zeta potential(-35 mV→12 mV).Micro BCA assay was used to determine the drug content,encapsulation efficiency, drug release in vivo of PE-NP and Fab' conjugation efficiency on the PE-NP-HER.The drug content and encapsulation efficiency of optimized prescription were 7.2±0.3μg/mg and 30.1±1.5%(n=9) respectively,and the accumulative drug release had a burst of 28.3%within 24 h and approached 50%in the first 96 h,the conjugation efficiency was about 19.4μg Fab'/mg NP.The biological activity of the PE38KDEL during the PLGA NP preparation procedure was evaluated by MTT assay.The results indicated that the activity of PE38KDEL was well-protected by protectant in the inner water phase during preparation.Recognition property,cytotoxicity against targeted cell and the antitumor mechanism of PE-NP-HER were studied in vitro experiment.The binding ability of PE-NP-HER(biological activity of Fab') was demonstrated by flow cytometry(FCM).The binding and internalization of PE-NP-HER to BT-474 cells were analyzed by laser scanning confocal microscopy (LSCM).Cytotoxicity against HER2-over expressed breast cancer BT-474 cells was analyzed using cellular cytotoxicity assays.The FCM result showed PE-NP-HER could recognize and bind to the targeted cell,the biological activities of Fab' on the NP were well protected.The binding and internalization of targeted NP were detected on the BT-474 cell membrane only after a 5-minute incubation and in the cytoplasm of cells after a 2 h incubation by LSCM. PE-NP-HER had remarkable in vitro cytotoxicity against HER2-overexpressing breast cancer BT-474 cells(Cell viability of 26±3%).Progressively,in developed HER2-overexpressing tumor xenograft model,administration of PE-NP-HER(0.9 mg/kg) showed a much better therapeutic efficacy in inhibiting tumor growth compared with PE-HER and other controls:the final mean tumor volume was 13±6 mm~3(mean±SD;n=8)(P＜0.01).In addition,PE-NP-HER were well tolerated in mice with a much higher MTD(maximally tolerated dose) over the control immunotoxin PE-HER constructed by chemically coupling PE38KDEL to rhHER2-mAb(2.92 mg/kg vs.0.92 mg/kg).Conclusion:The PE38KDEL-loaded HER2-targeted nanoparticles had dramatically reduced systematically nonspecific toxicity(especially hepatoxicity) of immunotoxin PE-HER by PLGA encapsulation.Comparing with PE-HER,PE-NP-HER showed a more remarkable antitumor efficacy both in vitro and in vivo experiment.Targeted NP provided a selective drug delivery to tumor which was conformed in the HER2-overexpressing tumor xenograft model therapeutic experiment.
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