356. Ultrasound Power and Pulse Interaction for Effective Gene Delivery in Cells and Animals

Abstract

While historically ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research has shown that this is not a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions for UMGD, close to the MB inertial cavitation threshold. These conditions were focused towards further increasing gene transfer efficiency and reducing associated cell damage.To test for optimal UMGD conditions in vitro, we created a matrix of 21 conditions (n = 3/cond.) in 293T cells. In an anechoic water bath, we US-treated cells for 60 s in the near field of a 16 mm disc transducer (H158) at 1 MHz with 14 Hz PRF, using pulse durations spanning 18 μs–36 ms and PNPs spanning 450–2370 kPa. Cells were suspended and mixed with pGFP plasmid and MBs in acoustically transparent 1.7 mL TPX tubes during treatment. Using this system, we also performed endpoint studies of MB cavitation using tunable resistive pulse sensing (TRPS) and flow cytometry. Our best 293T conditions yielded up to a 900-fold increase in expression (MFI × % GFP+) relative to sham. We also identified a local maxima with a -3 dB falloff approximately bounded by PNPs 450–1000 kPa and pulses 2–22 ms. Interestingly, in contrast to murine results, the 18 μs, 2480 kPa condition produced no elevated expression relative to sham. Our early mechanistic data showed increased destruction and aggregation of MBs for increasing pulse durations under conditions normalized by energy output. This suggests a different mode of cavitation or higher cavitation efficiency for those conditions.We next tested a similar set of 17 conditions (n = 4/cond) in 8-week old C57BL/6 mice using pulse durations 50 μs–22 ms and PNPs 480–2200 kPa. H158 was applied directly to the liver surface with simultaneous injection of MBs and luciferase plasmid for 60s. These conditions were compared against a short pulse, high-intensity positive control (18 μs, PNP 2540 kPa) which previously yielded the best transgene expression. We found a similar maximum in treated mice, with a -3 dB falloff bounded by PNPs 480-700 kPa. Of these, two conditions (1 & 2 ms, PNP 480 kPa) showed a significant ≈2-fold decrease in ALT and AST (p < 0.005 for all) while maintaining better or comparable expression to our positive control. These results demonstrated a clear benefit of certain low-pressure, millisecond-pulse conditions versus the high-intensity control.Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo. This study also established a simple, laboratory-feasible methodology for efficient UMGD in cells at low-intensity and millisecond pulse durations. Furthermore, these conditions allow for effective transfection in murine livers with minimized tissue damage. Our ongoing research investigates whether therapeutic effects of our high-intensity controls and lower-intensity, millisecond conditions represent separate cavitation phenomena or a spectrum of a single effect.