Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

Background: The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance: Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired

Cationic peptide exposure alone influenced membrane permeability to PI.

<p><b><i>A</i></b>: Cells were exposed to a peptides composed of neutral residues at neutral pH (polyasparagine, at left) and peptides with cationic residues at neutral pH (polyarginine, at right). These two peptides were of the same molecular weight. When 250 μg/ml of neutral polyasparagine or cationic polyarginine was added to cell media at −60 seconds, cells either fluoresced at baseline levels (<b><i>B</i></b>) or internalized PI in delayed, accelerating, and high levels (<b><i>C</i></b>). <b><i>D</i></b>–<b><i>G</i></b>: Reducing the cationic peptide concentration reduced the number of cells that internalized PI during the course of the experiment, and increased the delay time observed for abrupt, accelerated, high-level PI uptake. In <b><i>B</i></b>–<b><i>G</i></b>, the number of cells examined was 15, 18, 16, 14, 11, and 22, respectively.</p

PI uptake versus time signatures from every experiment from each cell fell into one of four categories, as defined here.

<p><b><i>A</i></b>: The NEP signature is assigned to cells that remain at baseline PI fluorescence levels of <25 million molecules per cell during the 1400 second experimental time course. <b><i>B</i></b>: The TEP signature is assigned to cells that internalize between 25 and 200 million PI molecules per cell during the 1400 second time course. <b><i>C</i></b>: The delayed IEP signature is assigned when cells internalize more than 200 million PI molecules per cell between 200 and 1400 seconds after PEF stimulation. <b><i>D</i></b>: The immediate IEP signature is assigned when cells internalize more than 200 million PI molecules within the first 200 seconds after PEF exposure. <b><i>E</i></b>: A table summarizing the names, qualities, and quantitative definitions of each signature. In <b><i>A</i></b>–<b><i>D</i></b>, grey regions on plots are provided to indicate the widows defining the uptake signature in terms of PI uptake amount and timeframes.</p

When used in concert with an externally applied PEF, cationic peptides can be used to enhance membrane disruption at lower PEF intensities at the anode-facing cell membrane.

<p>When a cell rests in an ionic medium containing membrane impermeable entities (<b><i>A</i></b>) and cationic peptides are added to that medium (<b><i>B</i></b>), a PEF can be used to accumulate those cationic peptides about the anode-facing membrane (<b><i>C</i></b>) where the anode-facing membrane can experience a local increases in electrostatic potential near individual peptides (<i>V</i>_p) plus enhanced electrostatic potential due to the ion relocation due to the applied field (<i>V</i>_e). These local enhancements in transmembrane electrostatic potentials can result in anodally preferenced electroporation at lower PEF intensities (<b><i>D</i></b>) and subsequent internalization of normally membrane-impermeable entities through the anode-facing membrane (<b><i>E</i></b>).</p

A microcuvette with a microchannel was used to expose cells to PEFs while imaging them over time under fluorescence and bright-field microscopy.

<p><b><i>A</i></b>: The microcuvette was designed to be placed on the platform of an inverted microscope. <b><i>B</i></b>–<b><i>D</i></b>: The microcuvette has an 80 mm microchannel where cells can be exposed to PEFs between two parallel electrodes and monitored under microscopy. <b><i>E</i></b>: FEM analysis results showing field heterogeneity within the microcuvette’s microchannel when the electrodes are excited with a nominal electric field value of <i>E</i>_n = 12.5 kV/cm (as calculated by the excitation voltage divided by the electrode gap). HL60 s resting in region I would be exposed to 0.87<i>E</i>_n to <i>E</i>_n while cells resting in region II would be exposed to <i>E</i>_n to 1.27<i>E</i>_n. However, because of the steep field gradient in region II, only cells in region I were analyzed for this study. Thus, for a given experiment, the range in PEF intensities to which a given cell was exposed was known. Part <b><i>E</i></b> was adapted from Kennedy et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092528#pone.0092528-Kennedy1" target="_blank">[18]</a> with authors’ permission.</p

Experiments confirmed that cationic peptide exposure results in PI internalization, preferentially through the anode-facing membrane, particularly at time points immediately following PEF administration.

<p>Examples at 2.7/cm (<b><i>A</i></b>), 3.4 kV/cm (<b><i>B</i></b>), and 3.8 kV/cm (<b><i>C</i></b>), comparing real time fluorescence images during experiments revealed that cationic-peptide-exposed cells internalize PI asymmetrically (bottom fluorescence microscopy sequences, anode is at left) and non-peptide-exposed cells internalize PI symmetrically (top image sequences). This anodally preferenced PI uptake was most apparent at earlier time points (bottom graphs in each subfigure of the spatial distribution in PI fluorescence across the cellular axis). Top graphs in each subfigure show PI fluorescence axial distributions at later time points (40–240 seconds). The vertical line in each graph represents the point of maximum fluorescence at 240 seconds and is intended to estimate the cell center line. In each image and graph, the anode is at right.</p

When no peptides were used, use of progressively more intense PEFs resulted in PI uptake signatures that were indicative of progressively higher degrees of membrane disruption.

<p><b><i>A–F</i></b>: Representative PI uptake versus time curves at the indicated ranges in PEF intensities highlighting general trends and emergence and disappearance of different uptake signatures. Light solid lines, dashed lines, dotted lines, and bold solid lines, indicate NEP, TEP, delayed IEP, and immediate IEP signatures, respectively. Under 1.3 kV/cm, cells exclusively exhibited NEP signatures (<b><i>A</i></b>) and began exhibiting TEP and delayed IEP signatures above 1.3 kV/cm (<b><i>B</i></b>). Above 1.9 kV/cm, immediate IEP signatures were first observed (<b><i>C</i></b>). Above 2.2 kV/cm, NEP signatures were no longer observed (<b><i>D</i></b>) and above 3.4 kV/cm, the TEP signatures were no longer observed (<b><i>E</i></b>). Finally, above 4.0 kV/cm, the immediate IEP signature was exclusively observed (<b><i>F</i></b>). <b><i>G</i></b>: A plot summarizing the data for all 80 experiments involving 269 cells. Data are represented as means and standard deviations. *, **, and *** indicate statistical significances of <i>p</i><0.05, 0.01, and 0.001, respectively, when comparing individual signatures to other individual signatures. $and$$ indicate statistical significance of <i>p</i><0.01 and 0.001, respectively, when comparing an individual signature to all other signatures combined within that PEF range. The number of independent experiments represented in 0–1, 1–2, 2–3, and 3–4 kV/cm bins are <i>N</i> = 3, 29, 30, 15, and 3, respectively. The total number of cells examined in 0–1, 1–2, 2–3, and 3–4 kV/cm bins were 28, 83, 106, 45, and 7, respectively.</p

Cationic peptide exposure can eventually lead to prolonged membrane disruption through electrostatic peptide-membrane interaction.

<p>A cell rests in an ionic media with membrane-impermeable entities (<b><i>A</i></b>) and cationic peptide is introduced into that media (<b><i>B</i></b>). Cationic peptides will electrostatically collect about the negatively charged plasma membrane (<b><i>C</i></b>) and increase the electrostatic potential across the membrane in the vicinity of the peptide (<i>V</i>_p). <b><i>D</i></b>–<b><i>E</i></b>: These transmembrane voltages may be sufficient for electroporation and disrupt the membrane in a manner leading to internalization of normally membrane-impermeable entities. <b><i>E</i></b>–<b><i>F</i></b>: Particularly at higher cationic peptide concentrations, the media serves as a reservoir for additional peptides to co-localize with the plasma membrane, causing prolonged membrane permeability.</p

A timeline summarizing the experimental protocol.

<p>The microcuvette was treated with an adhesion peptide so that cells would settle into the microchannel and remain immobilized during experimentation. Cells and PI were added to the microcuvette 300 seconds prior to PEF exposure and allowed to settle into the microchannel. If peptides were used, at 60 seconds before PEF exposure, they were added at twice their desired concentration so that, when combined with the cell/PI mixture in a 1∶1 ratio, they would dilute into the desired experimental concentration. Also at 60 seconds prior to PEF exposure, fluorescence imaging of the cells began (1 image every 10 seconds). At time 0, a PEF was administered and cells were imaged for 1400 seconds.</p

Different PI uptake signatures were associated with different degrees of membrane disruption.

<p>Representative images of PI fluorescence at the end of a 1400-second experiment (<b><i>A</i></b>) and TB inclusion/exclusion for those same cells immediately subsequent to TB addition 30 minutes after PEF administration (<b><i>B</i></b>). These images highlight the correlation between the relative intensity of PI fluorescence and TB inclusion verses exclusion. That is, NEP cells (cells 12, 15, 20, and 21) do not fluoresce at detectible levels and do not internalize TB. TEP cells (1, 4, 5, 9, 10, 11, and 18) moderately fluoresce and do not internalize TB. IEP cells (2, 3, 6, 7, 8, 13, 14, 16, 17, and 19) fluoresce brightly and do internalize TB. <b><i>C</i></b>: NEP, TEP, and IEP cells fluoresce at discernibly different fluorescence levels as shown by a fluorescence pixel intensity histogram. <b><i>D</i></b>: These fluorescence values differ in a statistically significant manner. In <b><i>C</i></b>–<b><i>D</i></b>, fluorescence data was obtained from 41 separate experiments involving the use of PEFs ranging in intensity from 0 to 6.7 kV/cm, wherein 974 individual cells were analyzed. In <b><i>D</i></b>, *** indicates statistical significance of <i>p</i><0.001. The data represent pixel counts of N = 1765, 2701, and 4022 for the NEP, TEP, and IEP cases, respectively.</p