An investigation into the cytotoxic effects of microbubbles and their constituents on osteosarcoma and bone marrow stromal cells.

BACKGROUND:Ultrasound-responsive microbubbles offer a means of achieving minimally invasive, localised drug delivery in applications including regenerative medicine. To facilitate their use, however, it is important to determine any cytotoxic effects they or their constituents may have. The aim of this study was to test the hypothesis that phospholipid-shelled microbubbles are non-toxic to human bone-derived cells at biologically-relevant concentrations. METHODS:Microbubbles were fabricated using combinations of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), polyoxyethylene(40) stearate (PEG40S) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (DSPE-PEG2000). Microbubble size and concentration were measured as a function of time and temperature by optical microscopy. Effects on MG63 osteosarcoma and human bone marrow stromal cells (BMSCs) were measured for up to 72 h by assay for viability, metabolic activity and proliferation. RESULTS:DBPC:DSPE-PEG2000 microbubbles were significantly more stable than DSPC:PEG40S microbubbles under all conditions tested. Serum-containing medium had no detrimental effect on microbubble stability, but storage at 37 °C compared to at 4 °C reduced stability for both preparations, with almost complete dissolution of microbubbles at times ≥24 h. DSPC:PEG40S microbubbles had greater inhibitory effects on cell metabolism and growth than DBPC:DSPE-PEG2000 microbubbles, with PEG40S found to be the principle inhibitory component. These effects were only evident at high microbubble concentrations (≥20% (v/v)) or with prolonged culture (≥24 h). Increasing cell-microbubble contact by inversion culture in a custom-built device had no inhibitory effect on metabolism. CONCLUSIONS:These data indicate that, over a broad range of concentrations and incubation times, DBPC:DSPE-PEG2000 and DSPC:PEG40S microbubbles have little effect on osteoblastic cell viability and growth, and that PEG40S is the principle inhibitory component in the formulations investigated.