Gene delivery system using FOL receptor-mediated endocytosis

Abstract

Part I For efficient receptor-mediated gene transfection, a new and simple formulation method based on using polyethylenimine (PEI) and folate (FOL)-poly(ethylene glycol) (PEG)-poly(L-lysine) (PLL) conjugate was presented. Luciferase plasmid DNA and PEI were complexed to form slightly positive-charged nanoparticles, onto which FOL-PEG-PLL conjugate was surface coated. With increasing the coating amount of FOL-PEG-PLL conjugate, the FOL-PEG-PLL/PEI/DNA complexes exhibited increased surface zeta-potential values with concomitantly increased diameters, indicating that the PLL part was physically anchored on the surface of preformed PEI/DNA complexes with exposing FOL moieties outside. The formulated complexes exhibited far greater transfection efficiency against FOL receptor over-expressing KB cells than FOL receptor deficient A549 cells. This was caused by enhanced cellular uptake of the resultant complexes via a receptor-mediated endocytosis process. The formulated complexes showed higher gene expression level, even in the presence of serum, than the PEI/DNA or Lipofectamine/DNA complexes. This was attributed to the PEG chains present on the surface of complexes that could work as a protective shield layer against aggregation caused by non-specific protein adsorption. The FOL-PEG-PLL/PEI/DNA complexes also demonstrated better cell viability than the PEI/DNA complexes. Part II Recombinant reversed caspase-3 (rev-caspase-3), a pro-apoptotic gene, is capable of intracellular autocatalytic processing which induces apoptosis, leading to programmed cell death. Folate receptor-specific intracellular delivery of rev-caspase-3 plasmid DNA into folate-receptor over-expressing KB cells was explored using folate-poly(ethylene glycol)-polyethylenimine (FOL-PEG-PEI) conjugate as a novel nonviral polymeric carrier. Using luciferase as a reporter gene, various formulation conditions of DNA/polymer polyplexes were pre-optimized to attain the highest folate receptor-mediated gen...