5 research outputs found
Versatile Cellular Uptake Mediated by Catanionic Vesicles: Simultaneous Spontaneous Membrane Fusion and Endocytosis
International audienc
Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer.
International audienceLow biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide: The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFP-Rab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets
Versatile Cellular Uptake Mediated by Catanionic Vesicles: Simultaneous Spontaneous Membrane Fusion and Endocytosis
Lactose-derived catanionic vesicles
offer unique opportunities
to overcome cellular barriers. These potential nanovectors, very easy
to formulate as drug delivery systems, are able to encapsulate drugs
of various hydrophilicity. This article highlights versatile interaction
mechanisms between these catanionic vesicles, labeled with hydrophilic
and amphiphilic fluorescent probes, and a mammalian cell line, Chinese
Hamster Ovary. Confocal microscopy and flow cytometry techniques show
that these vesicles are internalized by cells through cellular energy
dependent processes, as endocytosis, but are simultaneously able to
spontaneously fuse with cell plasma membranes and release their hydrophilic
content directly inside the cytosol. Such innovative and polyvalent
nanovectors, able to deliver their content via different internalization
pathways, would positively be a great progress for the coadministration
of drugs of complementary efficiency