Involvement of cytoskeleton in sonoporation and drug delivery

The mechanisms by which sonoporation increases thenative plasma membrane permeability are still unknown butvarious hypotheses have been suggested including pore formationand endocytosis. We have shown recently that caveolae-mediatedendocytosis plays a major role during sonoporation. In addition,some studies have reported the clear participation of actin inmammalian cell endocytosis. Thus, this study aims to investigatethe effect of sonoporation on actin microfilaments andmicrotubules and to identify the role of both cytoskeletons onsonoporation-mediated membrane permeabilization. AdherentU-87 MG cells were insonated at 1MHz, 1 W/cm2, 20% duty cyclefor 60 s, in the presence of BR14® microbubbles. SYTOX® Greenwas used to assess the membrane permeabilization, by flowcytometry. The cells were incubated with phalloidin-TRITC tostain actin microfilaments and tubulin antibody Alexa Fluor® 555to stain tubulin. The ultrastructural changes of plasmamembrane were monitored by scanning electron microscopy. Toinhibit the polymerization of actin and tubulin cytoskeleton, thecells were treated with cytochalasin D (cytoD) and nocodazole(Noco), respectively. Immunofluorescence results showalteration of actin and tubulin cytoskeleton, immediately aftersonoporation while control cells present a filamentouscytoskeleton with polygonal shape. However, the disorganizationof 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 revealsthat the treatment of the cells with both cytoD and Noco induceda strong decrease in the number of TPS (transient and permeantstructures): 98.5 ± 0.2% and 96 ± 0.6%, respectively. Moreover,flow cytometry results showed that cytoD and Noco lead to adecrease in the membrane permeabilization rate: 58% and 87%,respectively. In conclusion, this study demonstrates the transientalteration of actin and tubulin cytoskeleton followingsonoporation. It suggests that cytoskeleton plays a role duringsonoporation, as cytoskeleton inhibitors provoke a decrease inthe cell permeabilization rate. Its implication could occur duringboth the entry and transport of endocytosed molecules