TY - JOUR
T1 - A method for quantifying cellular uptake of fluorescently labeled siRNA
AU - Vader, Pieter
AU - van der Aa, Leonardus J.
AU - Engbersen, Johan F J
AU - Storm, Gert
AU - Schiffelers, Raymond M.
PY - 2010/11/20
Y1 - 2010/11/20
N2 - 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.
AB - 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.
KW - Fluorescence
KW - Lipoplex
KW - Polyplex
KW - Quantitative uptake
KW - Quenching
KW - SiRNA
UR - http://www.scopus.com/inward/record.url?scp=78649529300&partnerID=8YFLogxK
U2 - 10.1016/j.jconrel.2010.06.019
DO - 10.1016/j.jconrel.2010.06.019
M3 - Article
C2 - 20600397
AN - SCOPUS:78649529300
SN - 0168-3659
VL - 148
SP - 106
EP - 109
JO - Journal of Controlled Release
JF - Journal of Controlled Release
IS - 1
ER -