Evaluation of MB Delivery of Etoposide, by MTT Assay and Trypan Blue Exclusion

Since MBs were the fastest MNP, experiments were designed to determine whether or not they could be used to deliver a chemotherapeutic drug (etoposide), in the absence of direct conjugation to the drug. Etoposide was purchased from Cayman Chemical Company (Ann Arbor, MI), and stored at −20°C. Etoposide dilutions were made from a stock solution of 1 mg/mL in DMSO. The direct cytotoxic effects of different concentrations of etoposide (ranging from 1 to 100 µM) were studied prior to MB testing. Baseline (static) studies were performed in 12-well culture plates, with direct treatment of cells (U138 and H2122) in monolayer culture, using 1) etoposide alone, 2) MBs alone, or 3) a combination of MBs with etoposide, in comparison to untreated control cells. In these static studies, no magnet was used.

Standard MTT assays were performed in 12-well plates, as previously described.39 Cells were seeded at a density of 10⁵ cells per well and grown for 24 hrs then treated for 24 hrs. Thiazolyl blue tetrazolium bromide powder (MilliporeSigma, Burlington, MA) was dissolved in 1X PBS at a concentration of 5 mg/mL to make the MTT working solution, which was diluted in media at 10% v/v. After cells had grown for 24 hrs, media was aspirated, cells were gently washed 3 times with 200 μL PBS, fresh media was added, and a 100 μL treatment volume was applied. After incubation at 37°C in 5% CO₂ for 24 hrs, media was aspirated, MTT-media was added, and cells were incubated at 37°C (5% CO₂) for 4 hrs. The MTT-media was aspirated, DMSO was added, and cells were incubated at 37°C for 30 mins. The resulting solution was transferred to 96-well plates in triplicate and read with a SPECTRAmax 340PC (Molecular Devices, San Jose, CA) at 590 nm in triplicate.

Translational (i.e. dynamic) experiments were then performed to determine whether or not etoposide could be transported 10 cm down the length of the MIRT tray by MB clusters in response to the mini-MED. The straight lane MIRT tray in the 20 cm pull position was used. Sixty μL of MBs alone and 60 µL of MBs plus 100 μM etoposide were loaded at the starting points of the tray. The mini-MED was turned on (“activated”) for 5 mins to ensure that all MBs had traveled the complete distance of the tray. Four hundred μL of MB solution was removed from the end of each lane and transferred to an Eppendorf tube. One hundred μL of this solution was applied to wells of a pre-seeded 12-well culture tray (U138 and H2122; 10⁵ cells/well, 24 hrs), in triplicate. After a treatment time of 2 hrs at 37°C in 5% CO₂, the supernatant (including any dead cells) was transferred into a 15 mL Falcon tube (Corning, Inc., Corning, NY) and 1 mL of media was added per well in order to harvest the remaining cells. Cells were detached with gentle use of a cell scraper and added to the 15 mL tube, which was centrifuged at 1800 rpm for 5 mins. Cell pellets were resuspended in 1 mL of media. One hundred μL of cell suspension was added to 400 μL of trypan blue. Ten μL of trypan blue cell suspension was then transferred to a hemocytometer, and live and dead cells were counted, with counts being performed 5 times per group.