Ultrasound and microbubble mediated generation of transient pores on cell membranes in vitro

The therapeutic efficiency of biologically active molecules that exert their effect within the cell is ultimately constrained by the inability of complex molecules to cross the plasma membrane. The application of ultrasound increases cell membrane permeability and allows molecules to cross the otherwise-impermeable plasma membrane and enter the intracellular space, a phenomenon referred to as sonoporation. This work investigates the mechanism underpinning sonoporation. The objectives of this study are to directly observe bioeffects on the surface of cell membranes induced by ultrasound and microbubbles with electron microscopy and correlate them with flow cytometry data on cell permeability. KHT-C cells in suspension were exposed to ultrasound pulses of 500 kHz centre frequency, 570 kPa peak negative pressure, 32 ?;s pulse duration, 3 kHz pulse repetition frequency and 5 s insonation time in the absence and presence of Definity microbubbles (3.3% v/v). Cell permeabilisation was assessed with a permeability marker (FITC-dextran, 70 kDa) added 60 s before and 60 s after the termination of the ultrasound treatment. Following treatment, reversible permeability (PR) and cell viability (VPI) were measured with flow cytometry. PR is defined as the number of cells stained with FITC-dextran and unstained with propidium iodide (PI). VPI is defined as the number of cells unstained with PI. Cell membrane morphology was observed using electron microscopy. PR's of 71% was achieved with ultrasound and microbubbles with the FITC-dextran added before ultrasound exposure. PR's similar to control (~0.5%) were achieved with ultrasound alone, and with FITC-dextran added 60s following termination of the treatment. Untreated cells exhibited continuous plasma membrane morphology as assessed with electron microscopy. Ultrasound and microbubble treated cells demonstrated generation of membrane disruption generally within 30 to 100 nm and as large as 400 nm, immediately following treatment. The pores resealed within one minute following the termination of the ultrasound and microbubble treatment. No pores were observed on cells exposed to ultrasound alone. Pore generation on cell membranes was congruent with intracellular uptake of FITC-dextran molecules indicating that the mechanism underpinning sonoporation is the generation of transient pores on cell membranes induced with ultrasound-activated microbubbles. This study may be applied to guide the development of sonoporation-mediated therapies.