Preparation And Characterization Of EGFR ScFv Conjugated Liposomes For Targeted Drug And Gene Delivery

Aberrant activation and upregulation of epidermal growth factor receptor?EGFR?have been found in human cancers of various origins.Differences in the levels of EGFR expression between normal and tumor cells suggested that EGFR can be used as a target for drug delivery.Cetuximab is a human-murine chimeric anti-EGFR monoclonal antibody that's approved for clinical use in treating advanced chemo-refractory cancers.In this study,we would like to develop a EGFR targeting liposome using Cetuximab-scFv and explore its drug/gene delivery activities.The expression plasmid pET20b?+?/Cetu-scFv was constructed encoding the variable regions of Cetuximab's heavy chian and light chain connected using a?GGGGS?₃ linker.The plasmid was transfected into E.coli strain BL21-CodonPlus?DE3?-RIPL and the scFv fragments were produced and secreted led by the pelB signal peptide.The Cetu-scFv were purified and confirmed for its EGFR binding capability based on SDS-PAGE,western blot and ELISA.The purified Cetu-scFv was then conjugated to DSPE-PEG2000-Maleimide and used to prepare EGFR targeting liposomes and DNA//cationic lipoplex.The Cetu-scFv liposomes and DNA//cationic lipoplexes were tested for binding to EGFR over-expressing tumor cells in vitro.The Cetu-scFv antibody fragment were produced with more than 90%purity and retain specific binding activities against EGFR.Cell-based ELISA and flow cytometry analysis showed specific binding of purified Cetu-scFv to EGFR high-expressing A549cells.The Cetu-scFv conjugated liposomes and DNA/cationic lipoplex were also prepared and tested for binding in vitro.The result showed that Cetu-scFv conjugated liposomes and DNA/cationic lipoplex can bind to A549 more efficiently compared to non-targeted liposomes and DNA/cationic lipoplex.Furthermore,Cetu-scFv conjugated liposomes loaded with doxorubicin were prepared and tested for cytotoxic effects to A549 cells.Both targeting and nontargeting liposomes showed considerable inhibitory effects on cell growth,and in a doxorubicin concentration range of 3¹00ug/ml,cell viability decreased more significantly for Cetu-scFv conjaguted liposomes than nontargeting liposomes,which indicated that the EGFR immunoliposomes are more cytotoxic than nontargeting liposomes in A549 cells.Besides Cetu-scFv conjugated pGL3-luciferase/lipoplex showed better tranfection effect in A549 than non targeting lipoplex in vitro.The Cetu-scFv conjugated liposomes may be used for targeted delivery of varies drugs and genes to EGFR expression cells for cancer therapy.