Enhanced gene transfection in vivo using magnetic localisation of ultrasound contrast agents: Preliminary results

In previous work we demonstrated that microbubble mediated gene delivery can be enhanced in vitro through simultaneous exposure of cells to ultrasound and magnetic fields in the presence of magnetically loaded microbubble ultrasound contrast agents. The aim of this preliminary study was to investigate the feasibility of the technique for in vivo applications. Phospholipid coated microbubbles loaded with a hydrocarbon suspension of magnetic nanoparticles were prepared through sonication and sized using optical microscopy (concentration 1.4 x 108 bubbles/ml). Plasmid pGL4.13, which encodes for firefly luciferase, was prepared at a concentration of 4 μg/μl in endotoxin-free water. A Siemens Acuson Sequioa clinical imaging system with a 26 mm linear array transducer (15L8) was used throughout the investigation. 20, 6-8 week old CD1 female mice were injected with of 150 μl of microbubble suspension and 50 μl plasmid intra-venously through the tail vein. Mice were anaesthetized using isoflurance and imaged with the transducer above the left lung (14 MHz, 0.06 MI) to locate the thoracic region. Immediately following injection, a NdFeB permanent magnet was positioned over the right lung and the acoustic output was increased (H7MHz, 1.7 MI, focal depth 7.5 mm). Exposure to ultrasound and/or magnetic field was maintained for two minutes. 20 mice were exposed to ultrasound and magnetic field, two to ultrasound only and two to magnetic field only. On the third day post treatment, luciferase substrate (D-luciferin) was administered through intra-peritoneal injection and allowed to catalyse the transfected substrate for 10 minutes before animals were sacrificed and their organs recovered for individual bioluminescence imaging (IVIS 100, Xenogen) and quantification (Living Image Software, Xenogen). Animals treated with both ultrasound and the magnetic field showed transfection in the right lung, while no animals showed transfection in the contralateral organs. Of the 20 mice treated, 17 showed transfection at a level greater than for ultrasound alone and 12 greater than that of magnetic field alone. The results of this preliminary study indicate that microbubbles which include magnetic nanoparticles within their shells may be used to control the location of transfection in vivo. Further work is required to improve microbubble formulations and magnetic array design to allow more accurate targeting of transfection.