Resealing Time Research Articles

Monitoring optoporated process on mammalian cells by real-time measurement of membrane resealing time.

Resealing time based loading efficiency of optoporation is the key parameter for drug or gene delivery. This work describes a comparatively simple optical approach to directly measure the cell membrane resealing time of the gold nanoparticle mediated photoporation. To establish a membrane potential detection optical system, which can provide a direct measurement of resealing time of the optoporated cells. Voltage sensitive dye has been used to label the gold nanoparticle covered cell before laser activation and the resealing time was estimated from the voltage change due to the fluorescence light intensity change before and after laser activation. The approach has been validated by the simulated data based on diffusion model and Monte Carlo simulation and the experimental data obtained from a flow cytometry analysis. The measured resealing time after perforation varied from 28.6 to 163.8s on Hela cells when the irradiation fluence was increased, with a correlation coefficient () of 0.9938. This result is in agreement with the resealing time (1-2min) of photothermal porated Hela cells measured by electrical impedance method. The intracellular delivery efficiency of extracellular macromolecular under the same irradiation fluence depends mainly on diffusion velocity rather than pore size. The method described here can be used to directly measure resealing time of optoporated cells for accurately estimating the loading efficiency on discovering the mechanism of optoporation.

Membrane Repairing Capability of Non-Small Cell Lung Cancer Cells Is Regulated by Drug Resistance and Epithelial-Mesenchymal-Transition.

The plasma membrane separates the interior of the cells from the extracellular fluid and protects the cell from disruptive external factors. Therefore, the self-repairing capability of the membrane is crucial for cells to maintain homeostasis and survive in a hostile environment. Here, we found that micron-sized membrane pores induced by cylindrical atomic force microscope probe puncture resealed significantly (~1.3–1.5 times) faster in drug-resistant non-small cell lung cancer (NSCLC) cell lines than in their drug-sensitive counterparts. Interestingly, we found that such enhanced membrane repairing ability was due to the overexpression of annexin in drug-resistant NSCLC cells. In addition, a further ~50% reduction in membrane resealing time (i.e., from ~23 s to ~13 s) was observed through the epithelial-mesenchymal-transition, highlighting the superior viability and potential of highly aggressive tumor cells using membrane resealing as an indicator for assessing the drug-resistivity and pathological state of cancer.

A dynamic and quantitative biosensing assessment for electroporated membrane evolution of cardiomyocytes

The electrophysiological study is an essential approach to perform the biology and basic medicine research. To achieve the intracellular electrophysiological investigation, electroporation is introduced as an effective and convenient strategy to achieve the intracellular access of electrogenic cells and obtain high-fidelity action potentials. However, seldom platform could provide a quantitative and dynamic strategy to assess the electroporation-induced membrane perforation and recovery during intracellular electrophysiological investigation. Here we develop a high-throughput, sensitive, and stable biosensing platform to assess the evolution of electroporated cell membrane dynamically and quantitatively based on the recorded intracellular electrophysiological signals of cardiomyocytes. Following the electroporation, the extracellular action potentials transiently convert to the intracellular action potentials, whose amplitude rapidly increases to the maximum and then gradually decays. The intracellular action potentials finally convert back to the extracellular action potentials. This biosensing platform can dynamically explore and characterize the evolution procedures of perforation, stabilization, and resealing of the cell membrane by intracellular recordings. Moreover, the effect of electroporation voltages on the cell membrane is segmentally and quantitatively analyzed, demonstrating that a higher electroporation voltage induced a longer resealing time within the safe range of electroporation voltage. We believed that this dynamic and quantitative electroporated membrane evolution biosensing assessment platform will be a promising tool to pave a new avenue to bridge the electrophysiology and electroporated membrane evolution.

Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics.

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.

Characterizing the malignancy and drug resistance of cancer cells from their membrane resealing response.

In this report, we showed that two tumor cell characteristics, namely the malignancy and drug-resistance status can be evaluated by their membrane resealing response. Specifically, membrane pores in a number of pairs of cancer and normal cell lines originated from nasopharynx, lung and intestine were introduced by nano-mechanical puncturing. Interestingly, such nanometer-sized holes in tumor cells can reseal ~2–3 times faster than those in the corresponding normal cells. Furthermore, the membrane resealing time in cancer cell lines exhibiting resistance to several leading chemotherapeutic drugs was also found to be substantially shorter than that in their drug-sensitive counterparts, demonstrating the potential of using this quantity as a novel marker for future cancer diagnosis and drug resistance detection. Finally, a simple model was proposed to explain the observed resealing dynamics of cells which suggested that the distinct response exhibited by normal, tumor and drug resistant cells is likely due to the different tension levels in their lipid membranes, a conclusion that is also supported by direct cortical tension measurement.

Membrane Perforation and Recovery Dynamics in Microbubble-Mediated Sonoporation

Transient sonoporation can essentially be epitomized by two fundamental processes: acoustically induced membrane perforation and its subsequent resealing. To provide insight into these processes, this article presents a new series of direct evidence on the membrane-level dynamics during and after an episode of sonoporation. Our direct observations were obtained from anchored fetal fibroblasts whose membrane topography was imaged in situ using real-time confocal microscopy. To facilitate controlled sonoporation at the single-cell level, microbubbles that can passively adhere to the cell membrane were first introduced at a 1:1 cell-to-bubble ratio. Single-pulse ultrasound exposure (1-MHz frequency, 10-cycle pulse duration, 0.85-MPa peak negative pressure in situ) was then applied to trigger microbubble pulsation/collapse, which, in turn, instigated membrane perforation. With this protocol, five membrane-level phenomena were observed: (i) localized perforation of the cell membrane was synchronized with the instant of ultrasound pulsing; (ii) perforation sites with temporal peak area <30 μm(2) were resealed successfully; (iii) during recovery, a thickened pore rim emerged, and its temporal progression corresponded with the pore closure action; (iv) membrane resealing, if successful, would generally be completed within 1 min of the onset of sonoporation, and the resealing time constant was estimated to be below 20 s; (v) membrane resealing would fail for overly large pores (>100 μm(2)) or in the absence of extracellular calcium ions. These findings serve to underscore the spatiotemporal complexity of membrane-level dynamics in sonoporation.

Electrical Impedance Monitoring of Photothermal Porated Mammalian Cells

To transfer large cargo into mammalian cells, we recently provided a new approach called a photothermal nanoblade. Micron-sized membrane pores generated by the nanoblade are surprisingly well repaired with little cell death, suggesting rapid membrane-resealing dynamics. Here, we report the resealing time of photothermal porated mammalian cell plasma membranes using an electrical impedance sensor. Cell membrane pores were generated by high-speed cavitation bubbles induced by laser pulsing of metallic microdisks on a pair of transparent indium tin oxide electrodes. Electrical responses from the sensor electrodes were obtained with a signal voltage of 500 mV and a frequency at 500 kHz. Real-time impedance measurements show that membrane resealing and impedance recovery take a surprisingly long 1 to 2 min after laser pulsing. A nonrecovering impedance shift is also detected for cells after high-energy laser pulsing. This impedance response is also confirmed by a separate experiment in which thin-film gold electrodes are used to trigger cavitation bubbles for opening transient membrane pores on cells cultured on electrodes. Overall, our study platform provides new insight for micron-sized membrane defect repair dynamics to maintain cell viability.

Kinetics of skin resealing after insertion of microneedles in human subjects

Over the past decade, microneedles have been shown to dramatically increase skin permeability to a broad range of compounds by creating reversible microchannels in the skin. However, in order to achieve sustained transdermal drug delivery, the extent and duration of skin's increased permeability needs to be determined. In this study, we used electrical impedance spectroscopy to perform the first experiments in human subjects to analyze the resealing of skin's barrier properties after insertion of microneedles. Microneedles having a range of geometries were studied in conjunction with the effect of occlusion to test the hypothesis that increasing microneedle length, number, and cross-sectional area together with occlusion leads to an increase in skin resealing time that can exceed one day. Results indicated that in the absence of occlusion, all microneedle treated sites recovered barrier properties within 2 h, while occluded sites resealed more slowly, with resealing windows ranging from 3 to 40 h depending on microneedle geometry. Upon subsequent removal of occlusion, the skin barrier resealed rapidly. Longer microneedles, increased number of needles, and larger cross-sectional area demonstrated slower resealing kinetics indicating that microneedle geometry played a significant role in the barrier resealing process. Overall, this study showed that pre-treatment of skin with microneedles before applying an occlusive transdermal patch can increase skin permeability for more than one day, but nonetheless allow skin to reseal rapidly after patch removal.

The Electrophysiological Effect of Fibroblast to the Defibrillation in Aged or Diseased Heart

Background: The effect of fibroblast to electroporation in the aged or diseased heart was unclear. This study used a mathematical model to investigate the fibroblast effect to the myocardium under large premature electrical shock. Methods: The latest 2007 Luo-Rudy ventricular dynamic model was used. The electroporation was described as a transmembrane pore, mimics the reversible breakdown of the cell membrane. 50 times constant basic stimulation was applied to the cell model and the pore formation was carried out at the 50th action potential plateau phase. The pore size was set to occupy 0.15% to 0.25% of the total cell membrane during 10 ms shock. The pore was also assumed to decrease exponentially with a 100–1400 ms time constant after shock to simulate the resealing process. Two different fibroblast models were used: the passive fibroblast which connected the model parallel and the active fibroblast with similar membrane capacitance and other ionic currents. Results: Early afterdepolarization and trigger activity were significant increased while increasing either the pore size or resealing time constant. After adding the fibroblast model, the early afterdepolarization was more easily to be induced even with smaller pore size and shorter time constant. Conclusion: Fibroblast electrophysiologically influence the effect of electroporation in ventricular cell, explained the poorer defibrillation outcome in aged or diseased heart.

Nonlinear current response of micro electroporation and resealing dynamics for human cancer cells

This paper presents a novel method to measure the dynamic process of membrane permeability during electroporation (EP) on microchips for human cancer cells. Micro EP chips with three-dimensional gold electrodes accommodating a single cell in between were fabricated with a modified electroplating process. Electrochemical impedance spectroscopy (EIS) was carried out with an electrochemistry analyzer on micro EP chips and a nonlinear equivalent circuit model was proposed to describe the dynamic response of the whole system. Using such a method, micro EP current was isolated from undesired leakage current to study the corresponding electroporation dynamics under different input voltages. In addition, cell membrane recovery dynamics after electroporation was also studied and the resealing time constants were determined for different pulse treatments.

Mechanism for the conductivity changes caused by membrane electroporation of CHO cell-pellets

Electric field pulses, applied to densely packed pellets of Chinese hamster ovary (CHO) cells of mean radius āc = 7.5 ± 0.7 μm, cause major electric conductivity changes, described by three kinetic normal modes. The first mode reflects Wien effects of ionic atmosphere perturbations and ion pair dissociations (cell surfaces). Using Maxwell’s conductivity equation, the second and third mode are converted to the respective membrane conductivity modes. Electrothermodynamic analysis in terms of structural transitions from closed (C) to porated (P) membrane states of very different lifetimes, according to the scheme (C ⇌ C1) ⇌ (P2 ⇌ P3), yields the mean pore radii 2 = 1.00 ± 0.05 nm (P2) pores and 3 = 1.5 ± 0.1 nm (P3) at T = 293 K (20 °C). The relaxation time τ2 (P2-formation) reflects the rate limiting step (C ⇌ C1), associated with the activation dipole moment of Δm1 = 63 × 10−30 C m (or 19 Debye units), suggesting orientational changes of dipolar lipid head groups, in the solution membrane interfaces preceding the actual pore formations. Besides: the field-dependencies of the pore fractions f2 and f3 (order of 10−3), the field reduction factors fλ,i≤ 1 and the membrane voltage, we obtain the zero-field pore conductivities λ0p,2 = 1.7 × 10−2 mS cm−1 (P2) and λ0p,3 = 0.10 mS cm−1 (P3) and the membrane conductivity λm0 = 3.2 μS m−1. The post-field conductivity changes, due to the long-lived P3-pores, are analyzed in terms of time- (and field-) dependent efflux coefficients. The characteristic post-field pore resealing time τR = τ03 = 45 ± 3 s is independent of the field strength of the causative pulse and independent of the distance between the two electrodes. These results are an essential part for the optimization of the electrical pulse parameters, also for the clinical electrotransfer of bioactive substances into aggregated biological cells (tissue).

Electroporation of cell membranes supporting penetration of photodynamic active macromolecular chromophore dextrans

The aim is to demonstrate that macromolecular chromophore dextrans (Cibacron-dextran) acting as photosensitizers can be transported easily into cancer cells by electroporation of their membranes (short electric pulses on cell suspension between electrodes). There are two possibilities, either:(A)irradiation starts with the electropulse—showed with easily penetrating thiopyronin—yielding nearly 100% dead cells;(B)irradiation starts after a resealing time of membrane pores during which macromolecular photosensitizers can penetrate into cells. In this way, fractions of Cibacron-dextran with molecular weights (Mw) 3300, 10,900 and 500,000 are now able to kill.This combination of bioelectrochemistry and photobiology will be suitable also for other biopolymers, connected with photodynamic active chromophores (e.g. chromopeptides) to transport them through cell walls and membranes into cells and tissues. The human cancer cells U-935 and K-562 (pulsed by 1.15 kV/cm field strength) additionally or synergistically reach high rates of necrotic cells (colored by trypan blue) by this combination.

INFLUENCE OF POLYPEPTIDES AND PROTEINS ON ELECTROFUSION OF HUMAN CANCER CELLS

The electrofusion of human cancer U937 and K562 cells was studied under the influence of a selected group of polypeptides and proteins adsorbed at their membranes. For poly-L-lysine, we found that the higher the molecular mass (Mm) the higher the relative fusion yield (Fr), whereas poly-L-arginine and poly-L-ornithine showed toxicity. As recently measured for barley protoplast fusion, the same rule of dependence on the isoelectric point (pI) could be verified:which means 0.5 < Fr < 4 in 0.3 M mannitol solution (pH 6). If the pH of the 0.3 M mannitol solution is the same as the pI of the protein, its influence on the membrane nearly disappears. Bridging by Ca ions increases Fr from 0.5 (bovine serum albumin) to Fr ∼ 1 by charge compensation. The value of Fr is commensurate with the pore resealing time τ—determined by trypan blue staining—in such a way that for pI > 8, τ is longer and for pI < 6, τ is shorter than the control without protein content. Qualitatively, plant and animal membranes studied show equal behavior. According to our technique, the adsorption of biopolymers and other substances can be detected by this bioelectrochemical method, which is also a tool for analysis of electric stress mechanisms.

The resealing process of lipid bilayers after reversible electrical breakdown

The resealing process of lipid bilayer membranes after reversible electrical breakdown was investigated using two voltage pulses switched on together. Electrical breakdown of the membranes was induced with a voltage pulse of high intensity and short duration. The time course of the change in membrane conductance after the application of the high (short) voltage pulse was measured with a longer voltage pulse of low amplitude. The decrease in membrane conductance during the resealing process could be fitted to a single exponential curve with a time constant of 10-2 μs in the temperature range between 2 and 20°C. The activation energy for this exponential decay process was found to be about 50 kJ/mol, which might indicate a diffusion process. Above 25°C the resealing process is controlled by two exponential processes. The data obtained for the time course of the resealing process can be explained in terms of pore formation in the membranes in response to the high electrical field strength. A radius of about 4 nm is calculated for the initial pore size. From the assumed exponential change of the pore area with progressive resealing time a diffusion constant of 10 −8 cm 2/s for lateral lipid diffusion can be estimated.

Dielectric breakdowm in the membranes of itValonia utricularis. The role of energy dissipation

The electrical properties of the membranes of Valonia utricularis were investigated using intracellular electrodes. Using short (0.5–1.0 ms) current pulses it was found that at a critical membrane potential difference of 0.85 V there was a large and discontinuous decrease in the membrane impedance and the slope resistance beyond this potential was virtually zero. The electrical breakdown of the membranes did not lead to global damage of the cells and after a resealing time of approx. 5 s could be repeated with identical results. Experiments with long current pulses and long bursts of pulses repeated at 1 kHz are described which show that the electrical breakdown is not due to thermal damage arising from localized heating in the membrane. Thus a dissipation of some 10 3–10 5 times the energy normally dissipated during the onset of breakdown did not lead to breakdown itself unless the critical membrane potential was exceeded. The results also show that punch-through and avalanche ionization are not likely to be important in the breakdown mechanism. The results are consitent, however, with there being a critical instability in the electro-mechanical stresses set up in the membrane at large electric field strengths.