Cancer is a devastating disease that takes the lives of hundreds of thousands of people every year. Due to disease heterogeneity, standard treatments, such as chemotherapy or radiation, are effective in only a subset of the patient population. Tumors can have different underlying genetic causes and may express different proteins in one patient versus another. This inherent variability of cancer lends itself to the growing field of precision and personalized medicine (PPM). There are many ongoing efforts to acquire PPM data in order to characterize molecular differences between tumors. Some PPM products are already available to link these differences to an effective drug. It is clear that PPM cancer treatments can result in immense patient benefits, and companies and regulatory agencies have begun to recognize this. However, broader changes to the healthcare and insurance systems must be addressed if PPM is to become part of standard cancer care.

Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m 2). The energy of the pulse application primarily dictated cell viability, with Mg 2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg 2+-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg 2+ also hinders eTE compared to K +-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.

Electroporation creates transient openings in the cell membrane, allowing for intracellular delivery of diagnostic and therapeutic substances. The degree of cell membrane permeability during electroporation plays a key role in regulating the size of the delivery payload as well as the overall cell viability. A microfluidic platform offers the ability to electroporate single cells with impedance detection of membrane permeabilization in a high-throughput, continuous-flow manner. We have developed a flow-based electroporation microdevice that automatically detects, electroporates, and monitors individual cells for changes in permeability and delivery. We are able to achieve the advantages of electrical monitoring of cell permeabilization, heretofore only achieved with trapped or static cells, while processing the cells in a continuous-flow environment. We demonstrate the analysis of membrane permeabilization on individual cells before and after electroporation in a continuous-flow environment, which dramatically increases throughput. We have confirmed cell membrane permeabilization by electrically measuring the changes in cell impedance from electroporation and by optically measuring the intracellular delivery of a fluorescent probe after systematically varying the electric field strength and duration and correlating the pulse parameters to cell viability. We find a dramatic change in cell impedance and propidium iodide (PI) uptake at a pulse strength threshold of 0.87 kV/cm applied for a duration of 1 ms or longer. The overall cell viability was found to vary in a dose dependent manner with lower viability observed with increasing electric field strength and pulse duration. Cell viability was greater than 83% for all cases except for the most aggressive pulse condition (1[Formula: see text]kV/cm for 5[Formula: see text]ms), where the viability dropped to 67.1%. These studies can assist in determining critical permeabilization and molecular delivery parameters while preserving viability.

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