Prolonging pulse duration in ultrasound-mediated gene delivery lowers acoustic pressure threshold for efficient gene transfer to cells and small animals

Abstract

While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 μs–36 ms and PNPs spanning 0.5–2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 μs–22 ms and PNPs spanning 0.5–2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage 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, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.

Enhanced Radiosensitization for Cancer Treatment with Gold Nanoparticles through Sonoporation

“…In the present study, we combined a much longer pulse duration of 200 µs with a conservative PRF of 200 Hz (compared to a pulse duration of 30 µs with a PRF of 100 Hz in vitro) for a mouse model, based on the rationale of the presence of greater attenuation and the lack of immediate MB-cell contact. This approach is similar to that used previously in a study addressing gene delivery in mice [44].…”

“…Although some investigations have claimed that vascular disruption played a role during high-dose irradiation, the actual contributions from endothelial cells to the tumor response to radiation remain controversial [42,43]. Besides, a PRF as high as 3 kHz is not usual in research on ultrasound-guided drug delivery involving animal models [44][45][46][47], in which PRFs ranging from 1 to 100 Hz have been reported most frequently. In the present study, we combined a much longer pulse duration of 200 µs with a conservative PRF of 200 Hz (compared to a pulse duration of 30 µs with a PRF of 100 Hz in vitro) for a mouse model, based on the rationale of the presence of greater attenuation and the lack of immediate MB-cell contact.…”

Ultrasound-Mediated Gene Therapy in Swine Livers Using Single-Element, Multi-lensed, High-Intensity Ultrasound Transducers

“… 13 In addition, our lab has recently demonstrated that prolonging the pulse duration can decrease the pressure threshold required for efficient gene transfer, which can further decrease the potential tissue damage. 25 …”

Transcutaneous Ultrasound-Mediated Nonviral Gene Delivery to the Liver in a Porcine Model

“…Our recent studies achieved significant gene delivery enhancement in the liver using UMGD via a laparotomic procedure in small- and large-animal models 8, 20, 21, 22. Furthermore, we improved gene transfection efficiency using novel US transducer designs and beam patterns, as well as US treatment safety by modifying US protocols using longer pulse durations and lower PNPs 9 . In order to translate our recent findings into a clinically relevant minimally invasive approach, we developed an interventional radiological procedure to facilitate transcutaneous UMGD.…”

“…Ultrasound (US)-mediated gene delivery (UMGD) has emerged as an effective gene transfer approach with great clinical relevancy and translational potential to treat various diseases 1, 2, 3, 4, 5, 6. The technique has been applied to deliver genes and therapeutic agents to liver7, 8, 9 and various other tissues that are notoriously difficult to access, such as brain,10, 11, 12, 13 bone,14, 15 myocardium, 16 skeletal muscle,17, 18 and salivary glands 19 . Other gene delivery methods such as DNA-packaged nanoparticle delivery face the challenge of crossing the nuclear envelope for DNA transcription.…”