Effects of diagnostic ultrasound parameters on molecular uptake and cell viability

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Abstract
The success of drug or gene delivery is limited by the inability of those components to cross biological barriers like the cell membrane. Ultrasound (US) has shown to increase cell membrane permeability in a process known as sonoporation. So far most investigations have used acoustic settings such as continuous wave or high intensity focused ultrasound, which are well far from the diagnostic range. Since a few years now, other studies used US waves with diagnostic conditions but in combination with contrast bubbles. The purpose of our study is to determine the effect of US alone on cell uptake using diagnostic parameters, and to correlate the sonoporation mechanism on the different diagnostic conditions. Monolayers of CHO cells, fixed on a membrane, are used whereby Texas-red labeled dextran (10 nm) is used as a marker molecule. The experimental parameters were: acoustic pressure 0.2-1.4 MPa P_, pulse length (10/spl mu/s-15/spl mu/s), duty cycle (0-0.75%), total exposure time (0-6 minutes) and addition of contrast microbubbles. Transmitted frequency was 1 MHz and the temperature was kept constant at 37/spl deg/C. The control cells showed no molecular uptake. For exposure times 30, sonoporation increased transiently with increasing MI reaching a maximum at MI 0.7. Using MI between 0.2-0.7, maximum sonoporation was reached after 2 minutes of exposure. Using ultrasound with MI 1.4 during 30 sec gave the highest molecular uptake (33%) but also very high cell lysis (40%). Under stronger diagnostic conditions (MI 1.4 and total exposure time above 2 min), lysis occurred up to 65% and almost no cells were sonoporated. Increasing repetition rate resulted in a higher cell lysis and very low sonoporation. Addition of contrast microbubbles resulted in a higher cell lysis but not in a higher molecular uptake. Significant molecular uptake can be induced by diagnostic pulsed US without using contrast bubbles. For pressures >1.4 MPa and exposure time >30s, (repetition rate 0.01 sec and 0.1% duty cycle) the US treatment results in cell-lysis. Both molecular uptake and cell viability strongly depend on total exposure time, applied MI, repetition rate, duty cycle, and addition of contrast microbubbles. Addition of contrast microbubbles enhanced the ultrasound effects.