Electroporation-Induced Cell Modifications Detected with THz Time-Domain Spectroscopy
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Electroporation (electropermeabilization) increases the electrical conductivity of biological cell membranes and lowers transport barriers for normally impermeant materials. Molecular simulations suggest that electroporation begins with the reorganization of water and lipid head group dipoles in the phospholipid bilayer interface, driven by an externally applied electric field, and the evolution of the resulting defects into water-filled, lipid pores. The interior of the electroporated membrane thus contains water, which should provide a signature for detection of the electropermeabilized state. In this feasibility study, we use THz time-domain spectroscopy, a powerful tool for investigating biomolecular systems and their interactions with water, to detect electroporation in human cells subjected to permeabilizing pulsed electric fields (PEFs). The time-domain response of electroporated human monocytes was acquired with a commercial THz, time-domain spectrometer. For each sample, frequency spectra were calculated, and the absorption coefficient and refractive index were extracted in the frequency range between 0.2 and 1.5 THz. This analysis reveals a higher absorption of THz radiation by PEF-exposed cells, with respect to sham-exposed ones, consistent with the intrusion of water into the cell through the permeabilized membrane that is presumed to be associated with electroporation.
KeywordsElectroporation THz time-domain spectroscopy Human monocytes MM-6 cells Pulsed electric fields Water content
Short, intense pulsed electric fields (PEFs) increase the permeability of cell membranes, enabling the transport of normally impermeant molecules into or out of the cell. This manipulation of the membrane barrier function, called electroporation or electropermeabilization (EP), is the basis for a number of biomedical, industrial, and environmental applications . Electrochemotherapy, for example, combines EP with the administration of cytotoxic drugs to increase the efficacy of chemotherapy for solid tumors , and innovative strategies are under development to utilize EP for biomass processing in industrial and environmental applications .
Optimization, expansion, and development of EP-based technologies and treatments will be facilitated by a more complete understanding of the mechanisms of membrane-permeabilizing interactions between external electric fields and cellular structures, and a more quantitative and rigorous characterization of electropermeabilized cell membranes. Experimental investigations and electrophysical models provide a generalized, schematic view of EP, and the importance of pulse electrical parameters, exposure conditions, and the specific cell type and physiological state [4, 5, 6, 7]. Molecular simulations show in atomic detail the formation of water defects and ion-conductive pores in the phospholipid bilayer. Although these simulations presently take into account the complexity of living cell membranes in only a limited way, they demonstrate the primary role played by water in membrane electropore formation .
Direct experimental validation of the molecular model of electric field-driven water intrusion and pore formation requires a method sensitive enough to detect molecular-level signatures of EP. Common methods for detecting EP include measurement (usually relative or qualitative) of transport of normally impermeant compounds into or out of the cell , quantification of changes in cellular morphology, like swelling and blebbing [10, 11, 12]. Recent confocal Raman microspectroscopy results show changes in cellular protein and lipid signatures following PEF exposure [13, 14].
THz spectroscopy has been demonstrated to be an effective method for the observation of the properties of water in biological systems, and for distinguishing interfacial or hydration water from bulk water. Because many of the vibrational features that are characteristic of bound water lie within the picosecond and sub-picosecond time scale, THz spectroscopy can be used to probe the translational and rotational dynamics of water in cells in a label-free way [15, 16]. In this paper, we describe a procedure for evaluating the effectiveness of THz spectroscopy as a tool for detecting plasma membrane EP in cells subjected to PEFs.
In our experiments, samples of Mono-Mac 6 (MM-6) human monocytes were exposed/sham-exposed to 8 pulses of 100 μs duration, 100 kV/m electric field amplitude, 5 kHz repetition rate, and, following an ad hoc sample preparation procedure, a THz time-domain spectroscopy analysis was carried out. Using a commercial THz spectroscopy system, time-domain signals were acquired and the optical material parameters, absorption coefficient and refractive index, were derived with a post-processing procedure. Our observation that PEF-exposed cells absorb more THz radiation than sham-exposed cells is compatible with water intrusion into electropermeabilized cells.
2.1 Cell Culture
Human monocytes, Mono-Mac 6 (MM-6), were kindly provided by Prof. Paolo Abrescia, University Federico II (Napoli, Italy). Cells were maintained under exponential growth conditions (37 °C, 95% air and 5% CO2) in RPMI 1640 growth medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1% penicillin/streptomycin. For consistency and reproducibility, MM-6 cells were sub-cultured three times per week. All reagents were from Biowhittaker, Verviers, Belgium.
2.2 Electric Pulse Exposure
MM-6 cells suspended in 200 μL of RPMI medium at a concentration of 7.5 × 106 cells/mL were exposed to permeabilizing electric fields in standard, 4-mm EP cuvettes (Sigma-Aldrich). A commercial pulse generator (Cliniporator™, IGEA, SpA, Italy) was used to deliver 8, 100 μs, 400 V (100 kV/m between the cuvette electrodes) pulses at 5 kHz. During experiments, each exposed sample was associated with a concurrent sham-exposed sample, which served as control.
2.3 EP Yield Analysis
Flow cytometry using double-staining with calcein-AM (calcein, acetoxymethyl ester) and propidium iodide, both from Sigma-Aldrich (St. Louis, MO, USA), enabled assignment of cells from experimental samples to one of three groups: viable and non-permeabilized, viable and permeabilized, and dead. Calcein-AM, a nonfluorescent, membrane-permeant ester of the anionic fluorochrome calcein, enters cells freely. In dead cells, it remains nonfluorescent. In viable cells, the ester linkage is hydrolyzed, converting the molecule to its fluorescent, membrane-impermeant form and trapping it in the cell interior. Propidium is membrane-impermeant and thus excluded from viable cells, but it passes through permeabilized membranes, and the membranes of dead cells, and fluoresces on binding to nucleic acids. Cells were exposed/sham-exposed to electric pulses in growth medium containing 40 μM of propidium. After 5 min of incubation at RT (to allow dye uptake by permeabilized cells), samples were washed and incubated for 10 min at RT with 200-nM calcein-AM. A BD FacsCalibur flow cytometer (488 nm of excitation wavelength, CELL QUEST software) was used to acquire 15,000 events per sample, and the raw fluorescence data were then quantitatively analyzed with the FlowJo analysis program (TreeStar, OR, USA). EP yield was calculated as electropermeabilized live cells/total live cells.
2.4 Cell Sample Preparation for THz Spectroscopy
2.5 THz Time-Domain Spectroscopy Measurements
A fiber-coupled, THz, time-domain system (FICO, Zomega TeraHertz Corp., NY, USA) was used for the spectroscopic measurements. The system consists of three main components: a control unit, emitter and receiver heads, and a laser source. This system generates and detects broadband THz pulses, with full waveform sampling rates up to 500 Hz in both transmission and reflection geometries. In particular, an external, fiber-coupled, 1.5-μm pulsed laser (average power > 200 mW, pulse duration < 100 fs, repetition rate in the MHz) is used as a pump source that is split into a pump and probe beam used for THz wave generation and detection. A large-aperture, photoconductive antenna is used to generate THz waves, and an electro-optic crystal is used to detect the THz waves. This system measures the far-infrared spectrum from 0.1 to 3.0 THz. The peak dynamic range is greater than 60 dB for 500 waveform averages (1-s acquisition) and greater than 34 dB at full speed (2-ms acquisition).
For the spectroscopy measurements, the transmission configuration was used. The sample holder, kept in vertical position by means of a tweezer mounted on a micro translational stage with micrometer screws, was placed between the emitter and the receiver heads in a position corresponding to the focal spot of the THz beam. The equipment and the sample mounted in transmission configuration are shown in Fig. 1b. For each sample, the time-domain pulse transmitted by air, mylar alone, and cell spot (sham or exposed) on mylar was recorded as the average of 1000 acquisitions. A total of 10 independent measurements were carried out.
2.6 THz Spectroscopy Data Analysis
To quantify the interaction of THz radiation with the cell samples, the absorption coefficient, α(ν), and refractive index, n(ν), were calculated as a function of THz frequency ν. To this aim, the measured time-domain data were converted to frequency domain by using a fast Fourier transform, and amplitude and phase information were obtained. The following equations were then applied to calculate absorption coefficient and refractive index [17, 18]:
3 Results and Discussion
The EP efficiency in PEF-exposed MM-6 cell samples was about 90%, against less than 5% in sham-exposed samples. This outcome was expected for the pulsing conditions chosen in the experiments, which are similar to those applied in the clinical practice of electrochemotherapy, where high EP yield is obtained in a single pulse session .
The protocol for sample preparation for the THz spectroscopy measurements, the most critical part of the experimental procedure, was developed by trial and error. Centrifugation conditions were chosen to obtain a sufficiently wet and dense spot of cells, and the individual manipulations for centrifugation and mounting the samples in the spectrometer were organized and carried out so that the time from the beginning of sample preparation to the end of the THz analysis was the same for all samples. The time required to analyze one sample was about 4 min, excluding cytospin centrifugation. For each experiment, THz pulses transmitted by the different materials, i.e., air, mylar alone, and cells on mylar, were acquired in a random sequence to avoid bias of the measurement results. In order to maintain sample hydration during the measurements, the measurement chamber was not purged with dry gas.
Since this is the first description of THz spectroscopic analysis of PEF-exposed cells, and because of the complexity represented by a suspension of electropermeabilized cells, our interpretation of the results must be provisional. However, the spectral changes observed in electroporated cells are consistent with those reported for biological materials that become more hydrated, and to the dynamics of the hydration of the various molecular species present in cell membranes. For example, Chopra and co-workers extracted the refractive index and absorption coefficient in the 0.5–1.3 THz range from artificially synthesized skin cultures using fibroblast cells and collagen type I reagent. Both parameters sharply decreased when the fibroblast concentration in the collagen matrix was increased . In another case, Reid and co-workers performed THz time-domain spectroscopy of human blood between 0.25 and 2 THz, and detected a decrease in overall absorption coefficient as the water content of the blood was reduced. This decrease with water content was not observed, however, for blood clots. This finding was attributed to the specific structure of the blood clot, which traps water and impedes its ability to absorb THz radiation . More generally, the high absorption of THz radiation by polar liquids is the basis of several biomedical applications of THz spectroscopy and imaging that exploit the contrast between substances with lower or higher water content .
Electroporation-induced cell swelling in isosmotic media, a result of water influx driven by a colloid-osmotic imbalance across the electropermeabilized plasma membrane, is commonly observed [10, 11, 12, 22, 23]. One might predict based on this phenomenon alone the higher absorption of THz radiation by pulse-exposed cells relative to sham-exposed cells.
Absorption of THz radiation depends, however, not only on the amount of water present in the sample, but also on the strength of the associations of hydration water with cellular constituents, and the extent to which normal hydrogen bonding interactions among water molecules are disrupted. THz spectroscopy can be used for qualitative and quantitative analysis of pure compounds and simple mixtures by applying the complex methods of chemometrics . Similar analyses in whole-cell systems or tissues are not straightforward and will require new methods and procedures.
For the first time, THz time-domain spectroscopy has been employed to analyze cell samples subjected to electroporating microsecond PEFs. A procedure for sample preparation and data analysis has been developed to obtain good measurement sensitivity and repeatability. The results indicate that electroporation increases the terahertz absorbance of cells, which could be a consequence simply of water flux across the permeabilized cell membrane. It cannot be excluded, however that the increased absorbance arises at least in part from the electroporation-induced alteration of the relative amounts of bound and bulk water in the membrane phospholipid bilayer. Although the method described here is not sensitive enough for the identification of detailed EP spectral signatures at THz frequencies, THz spectroscopy has been demonstrated to be a non-invasive, label-free method for detecting EP in cell samples.
The support and advice of Dr. Antonio Pepe (CNR-IREA, Napoli) in the analysis of THz time-domain spectroscopy data is gratefully acknowledged.
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