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.

Ultrasound-activated microbubbles as a novel intracellular drug delivery system for urinary tract infection.

The development of new modalities for high-efficiency intracellular drug delivery is a priority for a number of disease areas. One such area is urinary tract infection (UTI), which is one of the most common infectious diseases globally and which imposes an immense economic and healthcare burden. Common uropathogenic bacteria have been shown to invade the urothelial wall during acute UTI, forming latent intracellular reservoirs that can evade antimicrobials and the immune response. This behaviour likely facilitates the high recurrence rates after oral antibiotic treatments, which are not able to penetrate the bladder wall and accumulate to an effective concentration. Meanwhile, oral antibiotics may also exacerbate antimicrobial resistance and cause systemic side effects. Using a human urothelial organoid model, we tested the ability of novel ultrasound-activated lipid microbubbles to deliver drugs into the cytoplasm of apical cells. The gas-filled lipid microbubbles were decorated with liposomes containing the non-cell-permeant antibiotic gentamicin and a fluorescent marker. The microbubble suspension was added to buffer at the apical surface of the bladder model before being exposed to ultrasound (1.1 MHz, 2.5 Mpa, 5500 cycles at 20 ms pulse duration) for 20 s. Our results show that ultrasound-activated intracellular delivery using microbubbles was over 16 times greater than the control group and twice that achieved by liposomes that were not associated with microbubbles. Moreover, no cell damage was detected. Together, our data show that ultrasound-activated microbubbles can safely deliver high concentrations of drugs into urothelial cells, and have the potential to be a more efficacious alternative to traditional oral antibiotic regimes for UTI. This modality of intracellular drug delivery may prove useful in other clinical indications, such as cancer and gene therapy, where such penetration would aid in treatment.