caa a22 4500 477126782 CHVBK 20180405111652.0 cr unu---uuuuu 170330e19971201xx s 000 0 eng 10.1023/A:1012188014919 doi (NATIONALLICENCE)springer-10.1023/A:1012188014919 Cellular Delivery of Oligonucleotides by Synthetic Import Peptide Carrier [Elektronische Daten] [Sujatha Dokka, David Toledo-Velasquez, Xianglin Shi, Liying Wang, Yon Rojanasakul] 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. Plenum Publishing Corporation, 1997 oligonucleotide nationallicence uptake nationallicence delivery nationallicence endocytosis nationallicence signal peptide nationallicence Dokka Sujatha Department of Basic Pharmaceutical Sciences, West Virginia University, School of Pharmacy, 26506, Morgantown, West Virginia aut Toledo-Velasquez David Rhone-Poulenc Rorer Central Research, Collegeville, 19426, Pennsylvania aut Shi Xianglin National Institute for Occupational Safety and Health, 26505, Morgantown, West Virginia aut Wang Liying Department of Basic Pharmaceutical Sciences, West Virginia University, School of Pharmacy, 26506, Morgantown, West Virginia aut Rojanasakul Yon Department of Basic Pharmaceutical Sciences, West Virginia University, School of Pharmacy, 26506, Morgantown, West Virginia aut Pharmaceutical Research Kluwer Academic Publishers-Plenum Publishers 14/12(1997-12-01), 1759-1764 0724-8741 14:12<1759 1997 14 11095 https://doi.org/10.1023/A:1012188014919 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1012188014919 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Dokka Sujatha Department of Basic Pharmaceutical Sciences, West Virginia University, School of Pharmacy, 26506, Morgantown, West Virginia aut NATIONALLICENCE 700 1- Toledo-Velasquez David Rhone-Poulenc Rorer Central Research, Collegeville, 19426, Pennsylvania aut NATIONALLICENCE 700 1- Shi Xianglin National Institute for Occupational Safety and Health, 26505, Morgantown, West Virginia aut NATIONALLICENCE 700 1- Wang Liying Department of Basic Pharmaceutical Sciences, West Virginia University, School of Pharmacy, 26506, Morgantown, West Virginia aut NATIONALLICENCE 700 1- Rojanasakul Yon Department of Basic Pharmaceutical Sciences, West Virginia University, School of Pharmacy, 26506, Morgantown, West Virginia aut NATIONALLICENCE 773 0- Pharmaceutical Research Kluwer Academic Publishers-Plenum Publishers 14/12(1997-12-01), 1759-1764 0724-8741 14:12<1759 1997 14 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer