BACKGROUND: Targeting to integrin receptor alpha(nu)beta(3) by RGD peptides seems to be a promising approach for gene delivery to proliferating endothelial cells of tumor metastases. PEGylation of cationic polymers offers a reduction of non-specific binding to cell surfaces. However, little knowledge exists on the influence of charge shielding by PEGylation on targeted gene delivery. Therefore, a variety of RGD peptide-polyethylenimine (PEI) conjugates with different degrees of substitution, with or without poly(ethylene glycol) (PEG) spacer, were synthesized. Influence of degree of substitution and PEG spacer on physicochemical properties as well as on integrin targeting of DNA/polymer complexes was evaluated. METHODS: The tetrapeptide RGDC was coupled to PEI with or without a PEG spacer. Complex formation with DNA was monitored by ethidium bromide (EtBr) fluorescence quenching. Hydrodynamic diameters of complexes and zeta-potential were assessed using a Zetasizer. Fluorescence correlation spectroscopy (FCS) was used to determine peptide binding to living cells. Transfection efficiency was evaluated employing a luciferase reporter gene. Binding of complexes to Mewo cells was monitored by flow cytometry. RESULTS: Polyplexes of RGD-PEI or RGD-PEG-PEI and DNA showed reduced quenching of EtBr fluorescence compared with PEI. All RGD conjugates formed small polyplexes (approximately 100 nm in diameter at a nitrogen/phosphate (N/P) ratio of 6.7). At N/P = 6.7, the zeta-potentials of RGD-PEI complexes were similar to PEI complexes (25-30 mV), while RGD-PEG-PEI formed neutral complexes. FCS showed saturable binding of RGD peptide to Mewo human melanoma cells and only low binding to A549 human lung carcinoma cells. A degree of substitution of 4.6% with SPDP as coupling reagent yielded a conjugate showing 50 times higher luciferase expression in Mewo cells than unmodified PEI at low N/P ratios around 3.3, while a degree of substitution of 1.6% only led to a moderately increased transfection efficiency. Flow cytometry experiments suggest that this effect is partly caused by increased attachment of complexes to cell surfaces. No improvement in transfection efficiency was found in alpha(nu)beta(3)-negative A549 cells. RGD-PEG-PEI complexes showed reasonable transfection efficiencies at high N/P ratios; however, no targeting effect could be found. CONCLUSIONS: Coupling of the tetrapeptide RGDC without a PEG spacer improved transfection efficiency of PEI in integrin-expressing Mewo cells by 1-2 orders of magnitude, especially at low N/P ratios. The use of a PEG spacer seems to impair targeting, possibly by not only shielding PEI, but also the RGD ligand. Copyright 2003 John Wiley & Sons, Ltd.
BACKGROUND: Targeting to integrin receptor alpha(nu)beta(3) by RGD peptides seems to be a promising approach for gene delivery to proliferating endothelial cells of tumor metastases. PEGylation of cationic polymers offers a reduction of non-specific binding to cell surfaces. However, little knowledge exists on the influence of charge shielding by PEGylation on targeted gene delivery. Therefore, a variety of RGD peptide-polyethylenimine (PEI) conjugates with different degrees of substitution, with or without poly(ethylene glycol) (PEG) spacer, were synthesized. Influence of degree of substitution and PEG spacer on physicochemical properties as well as on integrin targeting of DNA/polymer complexes was evaluated. METHODS: The tetrapeptide RGDC was coupled to PEI with or without a PEG spacer. Complex formation with DNA was monitored by ethidium bromide (EtBr) fluorescence quenching. Hydrodynamic diameters of complexes and zeta-potential were assessed using a Zetasizer. Fluorescence correlation spectroscopy (FCS) was used to determine peptide binding to living cells. Transfection efficiency was evaluated employing a luciferase reporter gene. Binding of complexes to Mewo cells was monitored by flow cytometry. RESULTS: Polyplexes of RGD-PEI or RGD-PEG-PEI and DNA showed reduced quenching of EtBr fluorescence compared with PEI. All RGD conjugates formed small polyplexes (approximately 100 nm in diameter at a nitrogen/phosphate (N/P) ratio of 6.7). At N/P = 6.7, the zeta-potentials of RGD-PEI complexes were similar to PEI complexes (25-30 mV), while RGD-PEG-PEI formed neutral complexes. FCS showed saturable binding of RGD peptide to Mewo humanmelanoma cells and only low binding to A549 humanlung carcinoma cells. A degree of substitution of 4.6% with SPDP as coupling reagent yielded a conjugate showing 50 times higher luciferase expression in Mewo cells than unmodified PEI at low N/P ratios around 3.3, while a degree of substitution of 1.6% only led to a moderately increased transfection efficiency. Flow cytometry experiments suggest that this effect is partly caused by increased attachment of complexes to cell surfaces. No improvement in transfection efficiency was found in alpha(nu)beta(3)-negative A549 cells. RGD-PEG-PEI complexes showed reasonable transfection efficiencies at high N/P ratios; however, no targeting effect could be found. CONCLUSIONS: Coupling of the tetrapeptide RGDC without a PEG spacer improved transfection efficiency of PEI in integrin-expressing Mewo cells by 1-2 orders of magnitude, especially at low N/P ratios. The use of a PEG spacer seems to impair targeting, possibly by not only shielding PEI, but also the RGD ligand. Copyright 2003 John Wiley & Sons, Ltd.