Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex

Purpose: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency. Methods: The peptide was conjugated to branched PEI (25kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS)-PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using 1H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay. Results: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI. Conclusions: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.