Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization
- 185 Downloads
Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 μs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.
KeywordsCalcium electrotransfer Pulsed electric fields Permeabilization angle Transmembrane potential HeLa S3 cells
Pulsed electric fields
Minimum essential medium
Phosphate buffer saline
Ethylene diamine tetra acetic acid
Fetal bovine serum
Hank’s balanced salt solution
Metal oxide semiconductor field effect transistor
Voltage-dependent calcium channels
The authors would like to thank Ms. M. Ota, Mr. R. Matsushima, and Mr. Mr. N. Ohnishi for their help in conducting the experiments.
This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17K06163).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Guionet A. La décontamination bactérienne de l’eau par impulsions électriques ultracourtes [Internet] [phd]. Université de Toulouse, Université Toulouse III—Paul Sabatier; 2014 [cited 2015 Oct 9]. Available from: http://thesesups.ups-tlse.fr/2519/
- 13.Frandsen SK, Gibot L, Madi M, Gehl J, Rols M-P. Calcium Electroporation: Evidence for differential effects in normal and malignant cell lines, evaluated in a 3D spheroid model. PLoS ONE [Internet]. 2015 [cited 2017 Jan 4];10. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669124/
- 22.Yamashita K, Hatanaka T, Akiyama H, Sakugawa T. Study of fast rise time pulse power generator using SiC-MOSFET and FRD. IEEE Pulsed Power Conf PPC. 2015;2015:1–4.Google Scholar
- 27.Neumann E, Sowers AE, Jordan CA. Electroporation and electrofusion in cell biology. Electroporation electrofusion cell biology. New York: Plenum; 1989.Google Scholar
- 44.Orio J, Bellard E, Baaziz H, Pichon C, Mouritzen P, Rols M-P, et al. Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer. J RNAi Gene Silenc Int J RNA Gene Target Res. 2013;9:479–85.Google Scholar
- 54.Pakhomov AG, Miklavcic D, Markov MS. Advanced electroporation techniques in biology and medicine. CRC Press; 2010.Google Scholar