Efficient Microbubble- and Ultrasound-Mediated Plasmid DNA Delivery into a Specific Rat Liver Lobe via a Targeted Injection and Acoustic Exposure Using a Novel Ultrasound System

Abstract

To develop efficient gene delivery in larger animals, based on a previous mouse study, we explored the luciferase reporter gene transfer in rats by establishing a novel unfocused ultrasound system with simultaneous targeted injection of a plasmid and microbubble mixture into a specific liver lobe through a portal vein branch. Luciferase expression was significantly enhanced over 0–30 Vol% of the Definity® microbubbles, with a plateau between 0.5 and 30 Vol%. The increase of gene delivery efficiency also depended on the acoustic peak negative pressure, achieving over 100-fold enhancement at 2.5 MPa compared with plasmid only controls. Transient, modest liver damage following treatment was assessed by transaminase assays and histology, both of which correlated with gene expression induced by acoustic cavitation. In addition, pulse-train ultrasound exposures (i.e., with relatively long quiescent periods between groups of pulses to allow tissue refill with microbubbles) produced gene expression levels comparable to the standard US exposure but reduced the extent of liver damage. These results indicated that unfocused high intensity therapeutic ultrasound exposure with microbubbles is highly promising for safe and efficient gene delivery into the liver of rats or larger animals.

Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery

“…US targeted MB destruction (UTMD)-mediated gene delivery has emerged as a promising non-invasive alternative that can address these issues. Therapeutic application of this system has demonstrated spatial and temporal control [1–5] in addition to being well-tolerated in animal experiments [16–18]. However, it has yet to show sufficient gene delivery efficiency [7], and improvements are required for clinical translation.…”

“…More detailed description of the surgical procedure is mentioned previously [16]. Briefly, six-week old SAS SD male rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA), and were kept in a specific pathogen-free (SPF) vivarium for at least three days for acclimation.…”

“…Despite its attractive potential for clinical applications, US-mediated gene delivery has thus far shown limited success compared to viral vectors and other methods [7]. Previous studies by our group and others have explored different ways to improve this technology, including varying drug or DNA dosages [10–15], customizing US systems [5, 12, 16–18] and improving surgical techniques in animal models [16–18]. These investigations suggested that US-mediated gene therapy can be significantly improved for potential therapeutic applications.…”

“…Therapeutic US-mediated MB destruction have already been applied in a multitude of ways, including delivery of DNA, siRNA [19–21], drugs [7, 22], stem cells [23] to different cell types such as tumor [24, 25], cardiovascular [26, 27], and skeletal muscle [1, 28], and organs such as liver [16–18], brain [2, 3, 22], and kidney [29, 30]. Given that the effectiveness of MBs depends on several variables such as their acoustic behaviors, size, stability, and compositions [31, 32], it is imperative to generate MBs that are tailored for their specific functions, instead of using generic MBs, such as the commercially available Definity ® that is approved as a contrast agent for echocardiographic imaging.…”

High intensity ultrasound transducer used in gene transfection

“…A 400-fold enhancement in gene expression was observed in rats using the H-158. 2 The H-158 transducer was designed using an unfocused multilayer piezoelectric stack with a 16 mm active area. In agreement with Raleigh diffraction calculations, the natural focus measured 47 mm past the radiating surface and a side lobe pressure pattern was produced outside of the Full Width Half Maximum (FWHM) at the radiating surface.…”