A method for quantifying cellular uptake of fluorescently labeled siRNA.
Efficient intracellular delivery of siRNA is a significant hurdle to its therapeutic success. For biological studies on the efficiency of carrier-mediated uptake of siRNA, quantitative determination of the amount of internalized siRNA is required. In this study, when the apparent uptake of fluorescently labeled siRNA, formulated in different lipo- and polyplexes, was examined using different techniques, major differences were observed. Additional experiments showed that these differences could be explained by quenching phenomena that were dependent on interactions between siRNA and carrier and their intracellular environment. Differences in fluorescent signal of complexed siRNA due to quenching could be overcome by measuring the fluorescent signal after lysing the transfected cells in lysis buffer that contained 2% SDS to dissociate siRNA from the complexes. This method offers a simple approach for quantifying cellular uptake of siRNA, which might help in the development of more efficient delivery systems.
Vader
Pieter
P
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
van der Aa
Leonardus J
LJ
Department of Biomedical Chemistry, MIRA Institute for Biomedical Technology, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
Engbersen
Johan F J
JFJ
Department of Biomedical Chemistry, MIRA Institute for Biomedical Technology, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
Storm
Gert
G
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
Schiffelers
Raymond M
RM
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands. Electronic address: R.M.Schiffelers@uu.nl.
D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006801:Humans; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000493:pharmacokinetics*
D005456:Fluorescent Dyes; D034741:RNA, Small Interfering