Ultrasound enhanced siRNA delivery using cationic liposome-microbubble complexes for the treatment of squamous cell carcinoma.

Rationale: Microbubble ( MB ) contrast agents combined with ultrasound targeted microbubble cavitation ( UTMC ) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor ( EGFR ), to squamous cell carcinoma ( SCC ) via a novel MB-liposome complex ( LPX ). Methods: LPXs were constructed by conjugation of cationic liposomes to the surface of C4 F10 gas-filled lipid MBs using biotin/avidin chemistry, then loaded with siRNA via electrostatic interaction. Luciferase-expressing SCC-VII cells ( SCC-VII-Luc ) were cultured in Petri dishes. The Petri dishes were filled with media in which LPXs loaded with siRNA against firefly luciferase ( Luc siRNA ) were suspended. Ultrasound ( US ) (1 MHz, 100-µs pulse, 10% duty cycle) was delivered to the dishes for 10 sec at varying acoustic pressures and luciferase assay was performed 24 hr later. In vivo siRNA delivery was studied in SCC-VII tumor-bearing mice intravenously infused with a 0.5 mL saline suspension of EGFR siRNA LPX (7×108 LPX, ~30 µg siRNA) for 20 min during concurrent US (1 MHz, 0.5 MPa spatial peak temporal peak negative pressure, five 100-µs pulses every 1 ms; each pulse train repeated every 2 sec to allow reperfusion of LPX into the tumor). Mice were sacrificed 2 days post treatment and tumor EGFR expression was measured (Western blot). Other mice ( n =23) received either EGFR siRNA-loaded LPX + UTMC or negative control ( NC ) siRNA-loaded LPX + UTMC on days 0 and 3, or no treatment ("sham"). Tumor volume was serially measured by high-resolution 3D US imaging. Results: Luc siRNA LPX + UTMC caused significant luciferase knockdown vs. no treatment control, p <0.05) in SCC-VII-Luc cells at acoustic pressures 0.25 MPa to 0.9 MPa, while no significant silencing effect was seen at lower pressure (0.125 MPa). In vivo, EGFR siRNA LPX + UTMC reduced tumor EGFR expression by ~30% and significantly inhibited tumor growth by day 9 (~40% decrease in tumor volume vs. NC siRNA LPX + UTMC, p <0.05). Conclusions: Luc siRNA LPXs + UTMC achieved functional delivery of Luc siRNA to SCC-VII-Luc cells in vitro . EGFR siRNA LPX + UTMC inhibited tumor growth and suppressed EGFR expression in vivo , suggesting that this platform holds promise for non-invasive, image-guided targeted delivery of therapeutic siRNA for cancer treatment.