Abstract
Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.
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Selected References
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- Bartoletti D. C., Harrison G. I., Weaver J. C. The number of molecules taken up by electroporated cells: quantitative determination. FEBS Lett. 1989 Oct 9;256(1-2):4–10. doi: 10.1016/0014-5793(89)81707-7. [DOI] [PubMed] [Google Scholar]
- Bazile D., Mir L. M., Paoletti C. Voltage-dependent introduction of a d[alpha]octothymidylate into electropermeabilized cells. Biochem Biophys Res Commun. 1989 Mar 15;159(2):633–639. doi: 10.1016/0006-291x(89)90041-7. [DOI] [PubMed] [Google Scholar]
- DeLeo F. R., Jutila M. A., Quinn M. T. Characterization of peptide diffusion into electropermeabilized neutrophils. J Immunol Methods. 1996 Oct 30;198(1):35–49. doi: 10.1016/0022-1759(96)00144-5. [DOI] [PubMed] [Google Scholar]
- Essand M., Grönvik C., Hartman T., Carlsson J. Radioimmunotherapy of prostatic adenocarcinomas: effects of 131I-labelled E4 antibodies on cells at different depth in DU 145 spheroids. Int J Cancer. 1995 Nov 3;63(3):387–394. doi: 10.1002/ijc.2910630315. [DOI] [PubMed] [Google Scholar]
- Gift E. A., Weaver J. C. Observation of extremely heterogeneous electroporative molecular uptake by Saccharomyces cerevisiae which changes with electric field pulse amplitude. Biochim Biophys Acta. 1995 Mar 8;1234(1):52–62. doi: 10.1016/0005-2736(94)00258-q. [DOI] [PubMed] [Google Scholar]
- Heller R, Gilbert R, Jaroszeski MJ. Clinical applications of electrochemotherapy. Adv Drug Deliv Rev. 1999 Jan 4;35(1):119–129. doi: 10.1016/s0169-409x(98)00067-2. [DOI] [PubMed] [Google Scholar]
- Kubiniec R. T., Liang H., Hui S. W. Effects of pulse length and pulse strength on transfection by electroporation. Biotechniques. 1990 Jan;8(1):16–20. [PubMed] [Google Scholar]
- Liang H., Purucker W. J., Stenger D. A., Kubiniec R. T., Hui S. W. Uptake of fluorescence-labeled dextrans by 10T 1/2 fibroblasts following permeation by rectangular and exponential-decay electric field pulses. Biotechniques. 1988 Jun;6(6):550-2, 554, 556-8. [PubMed] [Google Scholar]
- Mir LM, Orlowski S. Mechanisms of electrochemotherapy. Adv Drug Deliv Rev. 1999 Jan 4;35(1):107–118. doi: 10.1016/s0169-409x(98)00066-0. [DOI] [PubMed] [Google Scholar]
- Neumann E., Toensing K., Kakorin S., Budde P., Frey J. Mechanism of electroporative dye uptake by mouse B cells. Biophys J. 1998 Jan;74(1):98–108. doi: 10.1016/S0006-3495(98)77771-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prausnitz M. R., Lau B. S., Milano C. D., Conner S., Langer R., Weaver J. C. A quantitative study of electroporation showing a plateau in net molecular transport. Biophys J. 1993 Jul;65(1):414–422. doi: 10.1016/S0006-3495(93)81081-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prausnitz M. R., Milano C. D., Gimm J. A., Langer R., Weaver J. C. Quantitative study of molecular transport due to electroporation: uptake of bovine serum albumin by erythrocyte ghosts. Biophys J. 1994 May;66(5):1522–1530. doi: 10.1016/S0006-3495(94)80943-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rols M. P., Teissié J. Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. Biophys J. 1990 Nov;58(5):1089–1098. doi: 10.1016/S0006-3495(90)82451-6. [DOI] [PMC free article] [PubMed] [Google Scholar]