In vitro and in vivo gene delivery mediated by anti-EGF receptor immunoliposomes and immunolipoplexes

Abstract

Conventional liposomes and lipoplexes, non-viral gene delivery systems, are known to effectively reduce the side effects of viral delivery systems. The electrically neutral or negatively charged surfaces of liposomes have been known to have a lower affinity to serum proteins resulting in reduced macrophage recognition as well as lower cell cytotoxicity than positively charged lipoplexes. Meanwhile, cationic lipoplexes can effectively load their payload, negatively charged nucleic acids. In this study, anti-EGFR (epithermal growth factor receptor) immunoliposomes and immunolipoplexes were formulated and their targeting and gene delivery efficiencies were compared in in vitro and in vivo models of cancer expressing EGFR. The plasmid DNA encoding reporter genes (luciferase) was encapsulated in the neutral liposomes by the freeze-thaw method. The EGFR-targeted PEGylated immunoliposomes (PILs) were finally constructed by conjugation of Cetuximab to PEG termini on the surface of liposomes. At the same time, the prepared PEGylated cationic liposomes were conjugated with the same antibody molecules and then complexed with the plasmid DNA, becoming PEGylated immunolipoplexes (PILPs). The formulation stability of the immunoliposomes and immunolipoplexes was confirmed by a gel retardation assay. The resulting Cetuximab-coupled immunoliposomes and immunolipoplexes were able to selectively bind to the various types of EGFR-overexpressing cancer cells and effectively deliver the reporter gene. According to in vitro transfection tests, the immunolipoplex formulation was more efficient than the immunoliposomes in terms of gene delivery capability. This result was confirmed by competitive inhibition of target-mediated gene delivery. In addition, in vivo target-directed transfection was successfully examined by intravenous administration of anti-EGFR PILs or anti-EGFR PILPs to mice carrying tumors overexpressing EGFR. According to the in vivo gene expression data, the anti-EGFR PILPs were more efficient than anti-EGFR PILs in terms of target-directed in vivo gene transfection. These results suggest that the anti-EGFR nanoparticles, specially the immunolipoplex formulation, would be useful for targeted gene delivery to EGFR-overexpressing tumor cells. A noble modality of efficient tumor-specific gene delivery system for cancer gene therapy can be suggested based on further in vivo researches with these delivery systems.