Ultrasound enhances cellular lipid trafficking: Implications for lipid therapy

Lipidomics (the system-level analysis of lipids and their interacting moieties) is an emerging field in biomedical research that seeks to identify lipids as molecular targets for diagnosis and therapy. However, the delivery of lipids to pathological cells depends on the biochemical characteristics of the lipid including the overall charge, size of the polar head groups and length of fatty -acid side chains (Struck and Pagano, 1980). We have reported previously that focused ultrasound energy produced by clinical phase array imagers (2 MHz, 1.9 MI) can augment lipid delivery to cancer cells in vitro with the use of perfluorocarbon nanoparticles as non-cavitating carriers, which comprise a perfluorocarbon core and an outer lipid surfactant monolayer (Crowder et al, 2005). Accordingly, we now seek to establish the role of non-cavitational ultrasound in facilitating the delivery of lipids with a surrogate bulky polar head group. PFOB nanoparticles were synthesized by incorporating a bulky (1.4 nm) polar head group nanogold-DPPE (Nanoprobes, NY) at 0.172 mol% in the lipid monolayer. C-32 melanoma cells were grown on cell culture inserts and incubated with nanogold particles for 30 min followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa) and further incubation for 1 hour at 37 °C. The cells were then prefixed with glutaraldehyde, silver enhanced with HQ silver Enhancement Kit (Nanoprobes, NY) and fixed with sodium thiosulphate. After postfixation with osmium tetroxide the cells were treated with uranyl acetate, embedded in epoxy resin, thin-sectioned and stained with lead citrate. Sections 60 nm thick were examined with a Hitachi H-600 electron microscope. C-32 cells not exposed to ultrasound showed silver-enhanced gold particles partitioned only in the outer bilayer of the cell membrane and no evidence of intracellular nanogold. However, the cells exposed to ultrasound showed numerous nanogold-DPPE components inside the cell polarized to one end of intracellular vesicles. These observations are consistent with a mechanism that suggests ultrasound is capable of stimulating lipid internalization, sorting and intracellular trafficking. We surmise that non-cavitational ultrasound may facilitate the lipid uptake even under conditions not conducive to lipid transport by increasing the lipid diffusion coefficients. Therefore, adjunctive ultrasound could be used to increase the range of lipids delivered from perfluorocarbon nanoparticles to targeted cells, which might otherwise remain confined to the outer bilayer leaflet due to biophysical constraints.