Tissue electroporation: quantification and analysis of heterogeneous transport in multicellular environments

Abstract

Although electroporation is gaining increased attention as a technology to enhance clinical chemotherapy and gene therapy of tissues, direct measurements of electroporation-mediated transport in multicellular environments are lacking. In this study, we used multicellular tumor spheroids of DU145 prostate cancer cells as a model tissue to measure the levels and distribution of molecular uptake in a multicellular environment as a function of electrical and other parameters. These measurements, and subsequent analysis, were used to test the hypothesis that cells in a multicellular environment respond to electroporation in a heterogeneous manner that differs from isolated cells in suspension due to differences in cell state, local solute concentration, and local electric field. In support of the hypothesis, molecular uptake was consistently lower for cells within spheroids than cells in dilute suspension and was spatially heterogeneous, with progressively less uptake observed for cells located deeper within spheroid interiors. Reduced uptake and heterogeneity can be explained quantitatively by accounting for the effects of cell size on transmembrane voltage and cell volume, limited extracellular solute reservoir, heterogeneous field strength due to influence of neighboring cells, and diffusional lag times.

FIGURE 1

Comparison of electroporation-mediated uptake of calcein molecules in multicellular spheroids versus isolated cells in suspensions at the same bulk electroporation conditions. Spheroid uptake responses were less than predicted values for single cells. Uptake by cells within spheroids was determined using 400-μm diameter spheroids. Uptake by cells in suspension was determined using the validated empirical correlation described by Canatella and Prausnitz (2001). Uptake by cells within spheroids was consistently less than cells in suspension. Data points each represent the average uptake by 20,000 cells from a single sample (n = 1), each at a different electroporation condition.

FIGURE 2

Effect of spheroid radius on molecular uptake. Single cells (▴) or multicellular spheroids of different sizes (•) were electroporated with a single, 38-ms exponential-decay pulse at 0.45 kV/cm bulk field strength. The asterisks indicate that uptake from the three largest spheroid sizes were significantly less than for single cells (t-test, p < 0.05). Average is mean ± SE; n ≥ 3.

FIGURE 3

Effect of radial position of cells within spheroids on molecular uptake using two different electroporation protocols that cause approximately the same level of uptake in isolated cells in suspension. Electroporation conditions used were one exponential-decay pulse of 7 ms, 0.5 kV/cm (•, dotted line) and four rectangle-wave pulses of 0.05 ms, 2.5 kV/cm, 20-s interpulse spacing (▪, dashed line). The points represent experimental data from spheroids that were 200 μm in radius. The dashed lines represent uptake levels for isolated cells in suspension, based on the correlation described by Canatella and Prausnitz (2001). Average is mean ± SE; n ≥ 3.

FIGURE 4

Effect of field strength, pulse length, and number of pulses on electroporation-mediated uptake as a function of radial position of cells within multicellular spheroids. Conditions used were (A) one 22-ms pulse with field strengths of 0.2 (▪), 0.4 (○), and 0.5 (♦) kV/cm; (B) one 0.5-kV/cm pulse with pulse lengths of 1 (Δ), 7 (▪), 20 (○), and 40 (♦) ms; and (C) one (▪), two (○), and four (♦) 20-ms, 0.5-kV/cm pulses. The points represent experimental data from spheroids. The lines represent predictions from the model developed in this study to account for changes in cell state (Eq. 1), local solute concentration (Eqs. 2–6), and local electric field (Eqs. 7–9) within spheroids (see text). Average is mean ± SE; n ≥ 3.

FIGURE 5

Effect on uptake caused by the multicellular environment within a spheroid before and during electroporation. (A) Isolated cells electroporated in suspension. Intact spheroids electroporated and analyzed as cells from the periphery (B) and interior (C) of the spheroid. Cells harvested from the periphery (D) or interior (E) of dissociated spheroids and electroporated as isolated cells in suspension. One 19-ms exponential-decay pulse of 0.46 kV/cm was used. Average is mean ± SE; n = 3.

FIGURE 6

Effect of preelectroporation incubation time on electroporation-mediated uptake as a function of radial position of cells within multicellular spheroids. Duration of spheroid incubation in calcein solution was 10 (□), 600 (▪), and 1800 s (♦). One exponential-decay pulse of 0.48 kV/cm and 38 ms was used. The points represent experimental data from spheroids. The lines represent predictions from the model developed in this study that accounts for changes in cell state (Eq. 1), local solute concentration (Eqs. 2–6), and local electric field (Eqs. 7–9) within spheroids (see text). Average is mean ± SE.