Cell membrane permeabilization by 12-ns electric pulses: Not a purely dielectric, but a charge-dependent phenomenon

Silve, Aude; Leray, Isabelle; Leguèbe, Michael; Poignard, Clair; Mir, Lluis M.

doi:10.1016/j.bioelechem.2015.06.002

Cited by 32 publications

(24 citation statements)

References 38 publications

(60 reference statements)

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“…We have previously demonstrated that the nanoelectroablation effect could be observed for field strengths greater than 10 kV/cm [25]. By varying the conductivity of the medium, we have confirmed the initial observation by Silve, et al [26, 27] that no significant cell death is observed from nanosecond pulsed electric field application when the current density flowing across the cells is below a threshold level. There is very little cell death in response to 100 pulses, 100 ns long and 10–20 kV/cm when only 0 to 20 A/cm 2 is flowing across the cells.…”

Section: Resultssupporting

confidence: 85%

“…When the current density is reduced to zero by increasing medium resistivity, cell viability increases dramatically to the point where no cells die after exposure to 16 J/mL. This increased viability in low conductivity media is somewhat controversial since some previous reports have observed that for low pulse numbers, the cell viability decreases in low conductivity media [26, 27] Possible explanations of these contradictory observations include: 1) Ionic dependence of membrane charging requirements to generate nanopores; 2) Maxwell stress tensor inducing a stretching force on the membrane that increases in low conductivity or, simply 3) The dependence of membrane properties on medium conductivity [13]. The fact is that these low conductivity solutions are definitely not physiological and can have many effects on cells.…”

Section: Discussionmentioning

confidence: 99%

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Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

Nuccitelli

McDaniel

Anand

et al. 2017

j. immunotherapy cancer

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BackgroundWe have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1.MethodsAfter treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA.ResultsThe initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15–20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL.ConclusionNano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells.

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Section: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

Nuccitelli

McDaniel

Anand

et al. 2017

j. immunotherapy cancer

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“…The other study was published recently by our group and indicated an opposite tendency: reversible permeabilization induced by 12-ns pulses of moderate magnitude 3.2 MV/m on DC3-F cells was detected in a medium with an external conductivity of 1.5 S/m and not when the conductivity was lowered to 0.1 S/m 22 . We hypothesized that the longer charging time of the cell’s membrane in a low conductivity medium did not allow sufficient membrane charging in order to trigger permeabilization, as discussed in detail in reference 22 .…”

mentioning

confidence: 99%

Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses

Silve

Leray

Poignard

et al. 2016

Sci Rep

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The impact of external medium conductivity on the efficiency of the reversible permeabilisation caused by pulsed electric fields was investigated. Pulses of 12 ns, 102 ns or 100 μs were investigated. Whenever permeabilisation could be detected after the delivery of one single pulse, media of lower conductivity induced more efficient reversible permeabilisation and thus independently of the medium composition. Effect of medium conductivity can however be hidden by some saturation effects, for example when pulses are cumulated (use of trains of 8 pulses) or when the detection method is not sensitive enough. This explains the contradicting results that can be found in the literature. The new data are complementary to those of one of our previous study in which an opposite effect of the conductivity was highlighted. It stresses that the conductivity of the medium influences the reversible permeabilization by several ways. Moreover, these results clearly indicate that electropermeabilisation does not linearly depend on the energy delivered to the cells.

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“…In this work, we present experimental data on extracellular medium conductivity effects during electroporation in the submicrosecond range (100 ns-900 ns) using mouse colon carcinoma MC38/0 cell line as a model. We superposition our data with available studies 10,11 in the nanosecond range using 25 ns pulses of 60 kV/cm. To maximize the consolidation of knowledge and to provide a reference for the described efficacies, we also cover the conventional microsecond range (100 μs × 8) pulses.…”

mentioning

confidence: 99%

Effects of extracellular medium conductivity on cell response in the context of sub-microsecond range calcium electroporation

Novickij

Rembiałkowska

Staigvila

et al. 2020

Sci Rep

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In the present study, we report the effects of extracellular medium conductivity on cell response in the context of sub-microsecond range (100 ns-900 ns) electroporation, calcium electroporation and cell size. The effects of 25 ns and microsecond range (100 μs) pulses were also covered. As a model, three different cancer cell lines of various size (C32, MCF-7/DX and MC38/0) were used and the results indicated different size-dependent susceptibility patterns to the treatment. The applied pulsed electric field (PEF) protocols revealed a significant decrease of cell viability when calcium electroporation was used. The dependence of calcium ion transport and finally the anticancer effect on the external medium conductivity was determined. It was shown that small differences in conductivity do not alter viability significantly, however, mostly affect the permeabilization. At the same, MC38/0 cell line was the least susceptible to calcium electroporation, while the C32 line the most. In all cases calcium electroporation was mostly dependent on the sensitivity of cells to electroporation and could not be effectively improved by the increase of CaCl 2 concentration from 2 mM to 5 mM. Lastly, sub-microsecond PEF stimulated aquaporin-4 and VDAC1/Porin immunoreactions in all treated cells lines, which indicated that cell water balance is affected, ions exchange is increased and release of mitochondrial products is occurrent.Electroporation is a phenomenon of pulsed electric field (PEF) initiated permeabilization of biological cell plasma membrane, which serves as a basepoint of many successful biomedical and biotechnological methodologies 1,2 . The mechanism of electroporation is dependent on cell polarization in PEF and transient charge accumulation on the cell membrane (known as transmembrane potential, TMP) 3,4 . When a threshold TMP is reached, the permeability of cell membrane is increased due to occurrence of transient hydrophilic pores 5 . It is believed that oxidative effects can be a separate mechanism responsible for the formation of pores during electroporation 6,7 , nevertheless, polarization of the cell is a primary trigger.Polarization of the cell depends on the parameters of PEF, however, also on the permittivity and conductivity of both the cells and the medium 3,8 . Therefore, the effects of extracellular medium conductivity on electroporation efficiency have been focused for decades 9-12 . Absolute majority of the scientific works focus the micro-millisecond range of pulses, while a new modality of shorter (nanosecond range) pulses was introduced and is gaining popularity 13,14 . Currently, there are two contributions 10,11 experimentally focusing the 12-102 ns range of pulses in the context of extracellular medium conductivity, while the sub-microsecond range is not covered in literature. Both papers are published by the same group Silve et al., however the upmost interest lies in the peculiar cell response to 12 ns pulses. According to these studies, the same DC3-F cell line indicated an opposite respons...

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Cell membrane permeabilization by 12-ns electric pulses: Not a purely dielectric, but a charge-dependent phenomenon

Cited by 32 publications

References 38 publications

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses

Effects of extracellular medium conductivity on cell response in the context of sub-microsecond range calcium electroporation

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