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Nanotheranostics 2024; 8(3):285-297. doi:10.7150/ntno.90516 This issue Cite

Research Paper

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

Bin Qin¹, Xucai Chen¹, Jianhui Zhu¹, Jonathan Kopechek¹, Brandon Helfield¹, Francois Yu¹, Jissy Cyriac¹, Linda Lavery¹, Jennifer R. Grandis², Flordeliza S. Villanueva^1✉

1. Center for Ultrasound for Molecular Imaging and Therapeutics, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, PA, USA.
2. Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco, CA, USA.

✉ Corresponding author: Flordeliza S. Villanueva, MD, Professor of Medicine, Heart and Vascular Institute, 200 Lothrop Street, South Tower E354.4, Pittsburgh, PA 15213; 412 647 5840; villanuevafsedu.

Abstract

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 C₄F₁₀ 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.

Keywords: Ultrasound Targeted Microbubble Cavitation, Squamous Cell Carcinoma, Tumor Growth Inhibition, Head and Neck Cancer

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