Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields

Abstract

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Long-term physical evolution of an elastomeric ultrasound contrast microbubble

“…Focusing on air-filled elastomeric PVA-shelled MBs, we demonstrated here that such agents when dispersed in water medium are found to become strongly stable over time in terms of morphology and structure, as compared to lipid-shelled MBs (months vs. hours). The system showed a good echogenicity in a range of frequencies centred at around 10 MHz, complementary to that of MBs with a lipid shell and useful to minimise the biological risks related to high-resolution imaging [11,20,57,58]. This should facilitate their use as UCAs as well as in derivatisation protocols for drug delivery and theranostics [1,7,18].…”

Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level

“…According to Domenici et al . 18 , such exposure conditions allow internalisation of particles with size of tens of nanometers. After SP treatment a restoring time of 10 minutes was scheduled for the resealing of the pores in the cell membrane 18 .…”

“…Immediately after incubation, the solution was dispersed in Hepes (final volume of 2 ml) and centrifuged. The cell pellets were re-dispersed in 400 μl of the binding buffer and just before flow cytometry reading, 10 μl of PI stock solution (100 μg/ml in PBS) was added 18 . The experiments have been conducted in triplicate.…”

“…Herein we focused on the well-established murine fibroblasts cell line, NIH-3T3, being fibroblast cells predominant in connective tissue. This cell line has been successfully employed in studying US-induced bioeffects, and this can help us to compare and to interpret our results reliably 17,18,22 .…”

“…We recently pointed out the experimental conditions to obtain reversible plasma membrane permeability enhancement on NIH-3T3 fibroblast and HaCaT keratinocytes cells at very low intensity megasonic fields (in the subcavitation regime) 17,18,23 . According to the BiLayer Sonophore model 2 , these effects were related to transient alterations of the plasma membrane 17,23 allowing efficient internalisation of molecules with Stokes diameters of the order of magnitude of 10 nm, observing no severe cell damage 18 . Taking advantage of this latter finding, the internalisation of nanomaterials in vitro by SP has been considered herein.…”