PURPOSE: Inefficient cellular uptake and endosomal entrapment are among the obstacles impeding the therapeutic use of oligonucleotides (ONs). The objectives of this study are to investigate the feasibility of utilizing a synthetic import peptide as a drug carrier for cytoplasmic delivery of ONs and to study its transport mechanisms. METHODS: A molecular conjugate consisting of a signal import peptide (IP) derived from Kaposi fibroblast growth factor (K-FGF) and a polycationic ON linker, polylysine (PL), was synthesized and complexed with 5' fluorescently-labeled ON. Complex formation was verified by spectral shift assay and cellular uptake of the ON complex was studied fluorometrically. Microscopic studies were performed to visualize the intracellular distribution of the ON. RESULTS: Cells treated with the ON:IP-PL complex exhibited a dose-dependent increase in ON uptake over free ON-treated controls. The uptake of the complex was shown to occur via an energy-independent, non-endocytic, process since metabolic and endocytic inhibitors and low temperature did not prevent the uptake. Microscopic studies revealed a non-punctate fluorescence pattern, consistent with the non-endocytic transport process. Intense nuclear fluorescence was observed in cells treated with the complex but not with free ON, suggesting enhanced cytoplasmic delivery and nuclear accumulation of the ON by the conjugate. Efficient complex uptake was shown to require both the ON-binding moiety PL and the IP moiety. The delivery system was found to be non-toxic at the concentrations used. CONCLUSIONS: The peptide carrier was effective in promoting the cellular uptake of ON. The mechanism by which the peptide facilitates ON uptake appears to involve a direct translocation of ON via a non-endocytic process. The peptide carrier has the potential to overcome the problem of ON endosomal entrapment and degradation.
PURPOSE: Inefficient cellular uptake and endosomal entrapment are among the obstacles impeding the therapeutic use of oligonucleotides (ONs). The objectives of this study are to investigate the feasibility of utilizing a synthetic import peptide as a drug carrier for cytoplasmic delivery of ONs and to study its transport mechanisms. METHODS: A molecular conjugate consisting of a signal import peptide (IP) derived from Kaposi fibroblast growth factor (K-FGF) and a polycationic ON linker, polylysine (PL), was synthesized and complexed with 5' fluorescently-labeled ON. Complex formation was verified by spectral shift assay and cellular uptake of the ON complex was studied fluorometrically. Microscopic studies were performed to visualize the intracellular distribution of the ON. RESULTS: Cells treated with the ON:IP-PL complex exhibited a dose-dependent increase in ON uptake over free ON-treated controls. The uptake of the complex was shown to occur via an energy-independent, non-endocytic, process since metabolic and endocytic inhibitors and low temperature did not prevent the uptake. Microscopic studies revealed a non-punctate fluorescence pattern, consistent with the non-endocytic transport process. Intense nuclear fluorescence was observed in cells treated with the complex but not with free ON, suggesting enhanced cytoplasmic delivery and nuclear accumulation of the ON by the conjugate. Efficient complex uptake was shown to require both the ON-binding moiety PL and the IP moiety. The delivery system was found to be non-toxic at the concentrations used. CONCLUSIONS: The peptide carrier was effective in promoting the cellular uptake of ON. The mechanism by which the peptide facilitates ON uptake appears to involve a direct translocation of ON via a non-endocytic process. The peptide carrier has the potential to overcome the problem of ON endosomal entrapment and degradation.