Ultraschall Med 2016; 37 - PS1_08
DOI: 10.1055/s-0036-1587823

Sonoporation by microbubbles as gene therapy approach for liver cancer

L Rinaldi 1, G Franci 1, V Folliero 1, L Palomba 1, R Isticato 2, C Zannella 1, R Di Francia 3, I De Sio 1, LE Adinolfi 1, A Ascione 4, G Morelli 2, S Lastoria 3, L Altucci 1, C Pedone 2, M Galdiero 1
  • 1Second University of Naples, Naples, Italy
  • 2Federico II, Naples, Italy
  • 3Foundation C. Pascale, Naples, Italy
  • 4Fatebenefratelli Hospital, Naples, Italy

Purpose: We use an innovative method, known as sonoporation, to induce the expression of silenced gene in liver cancer cells (HepG2), such as (but nit restricted to) TRAIL in a specific manner. Aim of the project is the re-activation of silenced apoptotic pathway in liver cancer models, using diagnostic microbubble synovial as plasmidic gene delivery.

Material and methods: HepG2 ATCC were used to assess all the experiments. Microbubble (Sonovue®) were used at standard condition according to manufacturer's instructions. pEGFP-TRAIL plasmid (Plasmid #10953 addgene) and the respective control were selected and propagated in LB broth in order to obtain the necessary amount. Plasmid were purified with Invitrogen PureLink (thermo-fisher scientific Cod. K210017) kit. Transfection was mediated by Ultrasound device (Sonitron 2000, Artison corporation®) compared with standard protocol for lipofectamine 2000 (Invitrogen). GFP (Green Fluorescent Protein) was acquired via FACS excalibur DB analysis.

Results: HepG2 cells were used to achieve the TRAIL-GFP recombinant protein transfection. Cells were collected and re-suspended in PBS1X and Cell Cycle Buffer. FACS analysis was performed and results were analysed with Cell-Quest and ModIFit software. Among the several condition, cytotoxic parameters were acquired (5 MHz, 100% Duty Cycle, and 3 W/cm2, 60 s) with over than 80% cells in Pre-G1 phase; meanwhile lower parameters were not enough for gene delivery (1 MHz, 30% Duty Cycle, and 1 W/cm2 60 s). Best parameters were collected between 3 MHz, 100% Duty Cycle, and 1 W/cm2, 30 s). Data showed a dose dependent effect in terms of output energy. 30 – 50% transfection efficacy was acquired and TRAIL re-expression induced apoptotic effect.

Conclusion: Results showed the possibility to restore the expression of pro-apoptotic gene TRAIL in a liver cancer model HepG2. The future goal, in our vision, is the translation in animal model of our system, in order to evaluate the in-vivo effect of plasmidic gene therapy in hepatocarcinoma cells.