Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Jan;74(1):98–108. doi: 10.1016/S0006-3495(98)77771-9

Mechanism of electroporative dye uptake by mouse B cells.

E Neumann 1, K Toensing 1, S Kakorin 1, P Budde 1, J Frey 1
PMCID: PMC1299366  PMID: 9449314

Abstract

The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.

Full Text

The Full Text of this article is available as a PDF (227.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Budde P., Bewarder N., Weinrich V., Frey J. Biological functions of human Fc gamma RIIa/Fc gamma RIIc in B cells. Eur J Cell Biol. 1994 Jun;64(1):45–60. [PubMed] [Google Scholar]
  3. Dimitrov D. S., Sowers A. E. Membrane electroporation--fast molecular exchange by electroosmosis. Biochim Biophys Acta. 1990 Mar;1022(3):381–392. doi: 10.1016/0005-2736(90)90289-z. [DOI] [PubMed] [Google Scholar]
  4. Domenge C., Orlowski S., Luboinski B., De Baere T., Schwaab G., Belehradek J., Jr, Mir L. M. Antitumor electrochemotherapy: new advances in the clinical protocol. Cancer. 1996 Mar 1;77(5):956–963. doi: 10.1002/(sici)1097-0142(19960301)77:5<956::aid-cncr23>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
  5. Evans E. A. Bending resistance and chemically induced moments in membrane bilayers. Biophys J. 1974 Dec;14(12):923–931. doi: 10.1016/S0006-3495(74)85959-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gass G. V., Chernomordik L. V. Reversible large-scale deformations in the membranes of electrically-treated cells: electroinduced bleb formation. Biochim Biophys Acta. 1990 Mar 30;1023(1):1–11. doi: 10.1016/0005-2736(90)90002-6. [DOI] [PubMed] [Google Scholar]
  7. HODGKIN A. L., KATZ B. The effect of sodium ions on the electrical activity of giant axon of the squid. J Physiol. 1949 Mar 1;108(1):37–77. doi: 10.1113/jphysiol.1949.sp004310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Heller R., Jaroszeski M. J., Glass L. F., Messina J. L., Rapaport D. P., DeConti R. C., Fenske N. A., Gilbert R. A., Mir L. M., Reintgen D. S. Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy. Cancer. 1996 Mar 1;77(5):964–971. doi: 10.1002/(sici)1097-0142(19960301)77:5<964::aid-cncr24>3.0.co;2-0. [DOI] [PubMed] [Google Scholar]
  9. Hibino M., Itoh H., Kinosita K., Jr Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential. Biophys J. 1993 Jun;64(6):1789–1800. doi: 10.1016/S0006-3495(93)81550-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kakorin S., Stoylov S. P., Neumann E. Electro-optics of membrane electroporation in diphenylhexatriene-doped lipid bilayer vesicles. Biophys Chem. 1996 Jan 16;58(1-2):109–116. doi: 10.1016/0301-4622(95)00090-9. [DOI] [PubMed] [Google Scholar]
  11. Kinosita K., Jr, Tsong T. Y. Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature. 1977 Aug 4;268(5619):438–441. doi: 10.1038/268438a0. [DOI] [PubMed] [Google Scholar]
  12. Kinosita K., Jr, Tsong T. Y. Survival of sucrose-loaded erythrocytes in the circulation. Nature. 1978 Mar 16;272(5650):258–260. doi: 10.1038/272258a0. [DOI] [PubMed] [Google Scholar]
  13. Mir L. M., Tounekti O., Orlowski S. Bleomycin: revival of an old drug. Gen Pharmacol. 1996 Jul;27(5):745–748. doi: 10.1016/0306-3623(95)02101-9. [DOI] [PubMed] [Google Scholar]
  14. Montané M. H., Dupille E., Alibert G., Teissié J. Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields. Biochim Biophys Acta. 1990 May 9;1024(1):203–207. doi: 10.1016/0005-2736(90)90227-f. [DOI] [PubMed] [Google Scholar]
  15. Neumann E., Kakorin S., Tsoneva I., Nikolova B., Tomov T. Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation. Biophys J. 1996 Aug;71(2):868–877. doi: 10.1016/S0006-3495(96)79288-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Neumann E., Schaefer-Ridder M., Wang Y., Hofschneider P. H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1982;1(7):841–845. doi: 10.1002/j.1460-2075.1982.tb01257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Parsegian A. Energy of an ion crossing a low dielectric membrane: solutions to four relevant electrostatic problems. Nature. 1969 Mar 1;221(5183):844–846. doi: 10.1038/221844a0. [DOI] [PubMed] [Google Scholar]
  18. Sersa G., Cemazar M., Miklavcic D. Antitumor effectiveness of electrochemotherapy with cis-diamminedichloroplatinum(II) in mice. Cancer Res. 1995 Aug 1;55(15):3450–3455. [PubMed] [Google Scholar]
  19. Sowers A. E., Lieber M. R. Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts. FEBS Lett. 1986 Sep 15;205(2):179–184. doi: 10.1016/0014-5793(86)80893-6. [DOI] [PubMed] [Google Scholar]
  20. Sowers A. E. The long-lived fusogenic state induced in erythrocyte ghosts by electric pulses is not laterally mobile. Biophys J. 1987 Dec;52(6):1015–1020. doi: 10.1016/S0006-3495(87)83294-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Teissie J., Tsong T. Y. Electric field induced transient pores in phospholipid bilayer vesicles. Biochemistry. 1981 Mar 17;20(6):1548–1554. doi: 10.1021/bi00509a022. [DOI] [PubMed] [Google Scholar]
  22. Tekle E., Astumian R. D., Chock P. B. Electro-permeabilization of cell membranes: effect of the resting membrane potential. Biochem Biophys Res Commun. 1990 Oct 15;172(1):282–287. doi: 10.1016/s0006-291x(05)80206-2. [DOI] [PubMed] [Google Scholar]
  23. Tekle E., Astumian R. D., Chock P. B. Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4230–4234. doi: 10.1073/pnas.88.10.4230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tekle E., Astumian R. D., Chock P. B. Selective and asymmetric molecular transport across electroporated cell membranes. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11512–11516. doi: 10.1073/pnas.91.24.11512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tsong T. Y. Electroporation of cell membranes. Biophys J. 1991 Aug;60(2):297–306. doi: 10.1016/S0006-3495(91)82054-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES