In-vitro and in-vivo siRNA transfection by an ultrasonic confocal device
Résumé
The internalization of genetic materials, molecules or tracer particles in cells aims at understanding the intracellular mechanisms or can be considered for new therapies. The sonoporation is more and more used for such objective. The goal of this work is to assess the transfection potential of an ultrasonic device which combined 2 focused transducers (1.1MHz; Diameter and radius of curvature: 50mm) placed confocally. In this study in-vitro transfection tests were first carried out. 2ml tubes (Eppendorf) containing 0.5ml of cellular medium (follicular lymphoma RL; RPMI1640+10%SVF; 2x106cells/ml) and 45µl/ml of siRNA (20nMol; Qiagen; Allstar Neg Control) were placed at the device focal point. Transfection and mortality rates were evaluated according to the duration of irradiation (8s to 60s), the pulse repetition rate (≤500Hz) and the duty cycle (0.12 to 1%) for the peak intensity Isppa=10.8kW/cm2. Maximal transfection rates of 60% associated with very low mortality (<5%) were measured. Preliminary tests of tumor transfection (RL; Volume~1cm3) implanted subcutaneously in mouse CB17-SCID were performed. SiRNA (7.5 µg/ml) were injected into the tumor before ultrasonic irradiation (PRF=250Hz; DC=1%; Scanning rate 1mm/s; Isppa=10.8kW/cm2). The transfection rate was determined by flow cytometry after tumor removal and fragmentation. In-vivo transfection rate reached 16% of the studied samples.
Domaines
Acoustique [physics.class-ph]
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