Involvement of cytoskeleton in sonoporation and drug delivery
Résumé
The mechanisms by which sonoporation increases the
native plasma membrane permeability are still unknown but
various hypotheses have been suggested including pore formation
and endocytosis. We have shown recently that caveolae-mediated
endocytosis plays a major role during sonoporation. In addition,
some studies have reported the clear participation of actin in
mammalian cell endocytosis. Thus, this study aims to investigate
the effect of sonoporation on actin microfilaments and
microtubules and to identify the role of both cytoskeletons on
sonoporation-mediated membrane permeabilization. Adherent
U-87 MG cells were insonated at 1MHz, 1 W/cm2, 20% duty cycle
for 60 s, in the presence of BR14® microbubbles. SYTOX® Green
was used to assess the membrane permeabilization, by flow
cytometry. The cells were incubated with phalloidin-TRITC to
stain actin microfilaments and tubulin antibody Alexa Fluor® 555
to stain tubulin. The ultrastructural changes of plasma
membrane were monitored by scanning electron microscopy. To
inhibit the polymerization of actin and tubulin cytoskeleton, the
cells were treated with cytochalasin D (cytoD) and nocodazole
(Noco), respectively. Immunofluorescence results show
alteration of actin and tubulin cytoskeleton, immediately after
sonoporation while control cells present a filamentous
cytoskeleton with polygonal shape. However, the disorganization
of the cytoskeleton network is reversible since 60 min postsonoporation,
only few cells show a tubulin (8%) and actin (25%)
cytoskeleton disruption. Moreover based on SEM study reveals
that the treatment of the cells with both cytoD and Noco induced
a strong decrease in the number of TPS (transient and permeant
structures): 98.5 ± 0.2% and 96 ± 0.6%, respectively. Moreover,
flow cytometry results showed that cytoD and Noco lead to a
decrease in the membrane permeabilization rate: 58% and 87%,
respectively. In conclusion, this study demonstrates the transient
alteration of actin and tubulin cytoskeleton following
sonoporation. It suggests that cytoskeleton plays a role during
sonoporation, as cytoskeleton inhibitors provoke a decrease in
the cell permeabilization rate. Its implication could occur during
both the entry and transport of endocytosed molecules.
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