Optimization of cavitation-mediated mRNA delivery to cancer and non-cancer cells in vitro and in vivo

Abstract

The use of mRNA provides several compelling benefits. Cavitation-mediated delivery of mRNA has yet to be fully explored, especially in the context of achieving delivery of non-encapsulated, “free” mRNA. We present data that explore the relationship between cavitation and the transfer of, and protein expression from, free, fluorescently labelled mRNA. The protein expression exhibited in various cell lines in vitro was further tested in vivo, using sub-micron sized cavitation nuclei and the effects on transfection efficiency and expression time were examined. Acoustic exposure parameters were carefully selected, using a focused 0.5 MHz ultrasound transducer: pressure was varied from 0.3 to 2.1 MPa and 100–50 000 cycles at 5% duty cycle were used to achieve different cavitation regimes. A difference in expression time and transfection rate was observed between different cavitation nuclei and acoustic parameters. A luciferase reporter gene assay was used to characterise events in vitro. Expression due to cavitation-mediated delivery was further examined in vivo using the In Vivo imagining system and quantified using excised homogenised tumour tissue. These results provide us with a measurement window for maximum expression, exploration of how time points change across cell lines and lay the foundations for cavitation-mediated delivery and transfection of free mRNA.