5th EuropEan MolEcular IMagIng MEEtIng - ESMI

88
WarSaW, poland May 26 – 29, 2010
Evaluation of the temporal window for drug delivery following ultrasound mediated
membrane permeability enhancement
Yudina A. (1) , Lepetit-Coiffé M. (1) , Moonen C. (1) .
Laboratoire IMF CNRS UMR 5231 / Université Bordeaux 2, France
anna@imf.u-bordeaux2.fr
Introduction: Ultrasound (US)-mediated delivery
is a new therapeutic option to locally facilitate the
passage of drugs through cell plasma membrane
by reversibly altering its permeability[1] possibly
via the formation of pores that are known to spontaneously
reseal within a short time – seconds to
minutes[2]. However under certain conditions the
effects of US for drug delivery last for hours after
the exposure[3]. The present work is the first in
vitro attempt to confirm and quantitatively assess
the temporal window for the US-mediated intracellular
drug delivery by live-cell imaging.
Methods: Cell-impermeable optical chromophores
with fluorescence intensity increasing 100-1000
fold upon intercalation with nucleic acids served
as smart agents for reporting cellular uptake. Opticell
chambers with a monolayer of C6 cells were
subjected to ultrasound in the presence of microbubbles
followed by varying delays between
0 and 24 hours before addition of Sytox Green
optical contrast agent. Micro- and macroscopic
fluorescence imaging was used for qualitative and
quantitative analysis.
Results: Strong enhancement of the fluorescence
signal upon binding to nucleic acids allowed efficient
visualization of the local effect of US on internalization
of cell-impermeable intercalating dyes.
Up to 25% of viable cells showed uptake of contrast
agent with a half time of 8 hours, with cellular uptake
persisting even at 24 hours (Figure 1). Only
cells exposed to ultrasound showed the effect.
imaging life
Conclusions: Optical imaging showed that temporal
window of increased membrane permeability is
much longer than previously suggested. This may
have important repercussions for in vivo studies in
which membrane permeability may be temporally
separated from drug administration to better adapt
to pharmacokinetic / pharmacodynamic properties
of the drug or drug carrier.
Acknowledgement: This work is supported by the
EC-project FP7-ICT-2007-1-213706 SonoDrugs and
Foundation InNaBioSanté-project ULTRAFITT. Microscopy
was performed in the Bordeaux Imaging
Center of the Neurosciences Institute of the University
of Bordeaux II.
References:
Figure 1. In the absence of US
only occasional cells with the
compromised membrane show
the uptake of cell-impermeable
Sytox green (A). However the
effects of the US on membrane
permeability can last up to
24h (B), with the fluorescence
intensity increasing as the time
between US application and
fluorophore administration
shortens (C-F).
1. Hernot S, Klibanov AL; Adv Drug Deliv Rev. 60(10):1153-
66 (2008)
2. van Wamel A et al.; J Control Release. 112(2):149-55
(2006)
3. Hancock HA et al. Ultrasound Med Biol. 35(10):1722-36
(2009)