Jerry W. Shan

Quantification of propidium iodide delivery using millisecond electric pulses: Experiments

The transport mechanisms in electroporation-mediated molecular delivery are experimentally investigated and quantified. In particular, the uptake of propidium iodide (PI) into single 3T3 fibroblasts is investigated with time- and space-resolved fluorescence microscopy, and as a function of extracellular buffer conductivity. During the pulse, both the peak and the total integrated fluorescence intensity exhibit an inverse correlation with extracellular conductivity. This behavior can be explained by an electrokinetic phenomenon known as Field-Amplified Sample Stacking (FASS). Furthermore, the respective contributions from electrophoresis and diffusion have been quantified; the former is shown to be consistently higher than the latter for the experimental conditions considered. The results are compared with a compact model to predict electrophoresis-mediated transport, and good agreement is found between the two. The combination of the experimental and modeling efforts provides an effective means for the quantitative diagnosis of electroporation.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

Scaling Relationship and Optimization of Double-Pulse Electroporation

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis inxa0delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.

Continuous-flow, electrically-triggered, single cell-level electroporation

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 (1kV/cm for 5ms), where the viability dropped to 67.1%. These studies can assist in determining critical permeabilization and molecular delivery parameters while preserving viability.

Quantifying the Effects of Extracellular Conductivity on Transport During Electroporation

Electroporation is an effective means to permeabilize the cell membrane and deliver biologically active molecules (such DNA, RNA, dyes, etc…) into the cell cytoplasm, while maintaining cell viability and functionality [1]. Despite extensive research, electroporation still suffers from major drawbacks such as high cell death and low delivery efficiency. In the past, studies focused mainly on permeabilization of the membrane during electroporation while transport of molecules from one side of the membrane to the other has been overlooked. Previous experimental work demonstrated an inverse relation between the electrical conductivity of the extracellular buffer and total concentration delivered into cells [2]. This inverse correlation suggests that additional molecular transport mechanisms, besides diffusion, govern the delivery into cells.Copyright

Extreme Elongation of Vesicles Under DC Electric Fields

Electrodeformation refers to the deformation of cell or vesicle lipid membranes under the application of an electric field. Such a phenomenon often accompanies electroporation processes, and also can be leveraged to detect pathological changes in cells. Recent studies have suggested that the electrical conductivity difference across the lipid membrane is a dominant factor in determining the characteristics of deformation, and various regimes of deformation were observed. Using a vesicle model system, the current work is the first report of extreme elongation of vesicles of high conductivity ratio under DC electric fields. The results suggest that the osmolarity difference between the encapsulated and bathing solutions may contribute to such abnormal deformation behavior.Copyright