Drug Delivery
Sydney Riemer
Wake Forest University Student
Wake Forest University
Winston-Salem, North Carolina, United States
Erin Henslee
Assistant Professor
Wake Forest University, United States
Ulrich Bierbach
Professor of Chemistry
Wake Forest University, United States
Cellular functions, identity, and responses to various conditions are characterizable by a cell’s electrophysiological properties and how these properties change. Dielectrophoresis (DEP) is a technique that derives cytoplasmic conductivity, membrane conductance, and membrane capacitance from a cell’s distinctive pattern of movement when exposed to various electric frequencies. This label-less cell identification has many applications, from characterization to sorting and beyond.
This novel research assesses the DEP response of cancer cells exposed to P8A1, a highly cytotoxic platinum-acridine (PA) cancer drug. Our initial dose response results saw an increase in membrane conductance and cytoplasmic conductivity after 3 hours of 100nM treatment, indicating DEP’s ability to functionally monitor drug uptake (0-6 hours post treatment) prior to cell death that occurs after 6 hours of drug exposure.
For future work, we will use DEP to track cellular responses to other drug treatments to distinguish between distinct processes of cell death. By correlating dielectric property changes with both drug uptake and cell death, this research—which bridges ideas within synthetic chemistry and engineering—aims to both study and quantify the rate of P8A1 uptake by the MATE1 antiporter and identify whether cell death occurs through apoptosis, ferroptosis or another mechanism of cell death. This will help establish conditions under which the uptake of our drugs can be epigenetically enhanced.
Cell Preperation:
The lung cancer line A549 was cultured in RPMI under standard conditions. Prior to 3DEP experiments, populations of A549 were grown to confluence and subject to their respective drug treatment concentration and treatment time. We treated cells with 100 nM P8A1 for 1.5, 3, and 6 hours. Concentration and timing were determined from previous IC50 and uptake experiments. Cells released from adherence with trypsin and centrifuged at 250 g for 5 minutes. Pellets were resuspended in isosmotic 3DEP media, consisting of 8.5% (w/v) sucrose, 0.5% (w/v) dextrose, 100 µM CaCl2 and 250 µM MgCl2 and adjusted with PBS to 10 mS/m conductivity. Cell viability and radius are measured with the Countess cell counter.
3DEP Analysis:
Approximately 80 mL A549 cells, suspended in 3DEP media, were loaded into the 20-well 3DEP chip. The chip was covered with a glass cover slip and loaded into the 3DEP reader. The 3DEP program was run, energizing cells with a frequency of 1kH-20MHz for 30 seconds. The program produced a spectrum from the dielectrophoretic responses across all 20 wells (blue data points in Figure 1). Approximately 5 technical replicates of 3DEP spectra were produced for each A549 sub-population being tested.
Data Extraction:
The 3DEP system fit each dataset to a single-shell model of the Clausius-Mossoti factor and derived values for cytoplasmic conductivity and membrane conductivity and capacitance (yellow curve on Figure 1). Two-way ANOVA tests were used to identify significance in dielectrophoretic property changes between control and P8A1 treatment groups across experimental timepoints.
Preliminary 3DEP experimental data provides insight into the effect of P8A1 drug treatment on A549 cell electrophysiology, tracked across the 6-hour period during which significant intracellular drug accumulation occurs. Cells cultured in media that contained 100 nM P8A1 experienced a significant increase in membrane conductance following 3- and 6-hour drug treatments. This is indicative of an increase in ion flow across the cellular membrane. MATE1, the transporter protein that has been identified as responsible for P8A1 uptake, is an antiporter proton pump that takes in a single charged platinum-acridine drug molecule in exchange for two H+ ions that it pumps out of the cell. Therefore, we correlate this shift in conductance to the uptake of the 2+ charged P8A1 molecule and corresponding expulsion of two protons from the cell.
Present data shows a notable but insignificant increase in cytoplasmic conductivity across timepoints and no meaningful increase in membrane capacitance. Further experimental trials at 100 nM and higher drug concentrations are required to confirm P8A1’s lack of effect on these properties. Cells will also be co-treated with pyrimethamine and P8A1 in future trials. Pyrimethamine binds to MATE1 and acts as a competitive inhibitor of P8A1’s interaction with the transporter. This will provide further evidence that P8A1 uptake via MATE1 is responsible for changes in cellular electrophysiological properties.
Future experiments will also aim to identify the mechanism of cell death that occurs after P8A1 treatment. Alongside P8A1, cells will be treated with cisplatin, a cancer drug that is known to induce apoptosis, and RSL3, which induces ferroptosis. Cells will be treated with the IC90 concentration of each drug and results will be analyzed with the goal of identifying whether platinum-acridine-based cancer drugs induce cellular death via an apoptotic or ferroptotic pathway.