The role of pH gradients in tissue electroporation-based treatments

MAGLIETTI F, MICHINSKI S, OLAIZ N, SUAREZ C, MARSHALL G; CASTRO M

Pacific Grove

Conferencia; BIOEM 2015 Conference; 2015

IntroductionTreatments based on electroporation (EP) induce the formation of pores in cell membranes due to the application of pulsed electric fields. The circulation of electric current through a tissue will produce electrolysis of water molecules that lead to extreme pH changes near the electrodes. We present experimental evidence of the existence of pH gradients emerging from both electrodes during treatments based on tissue EP, for conditions found in many studies, and that these fronts are immediate and substantial.Materials and methodsElectroporator: We used a BTX ECM 830 square wave electroporator (Harvard Apparatus, Massachusetts, USA). It is a very common device capable for many kinds of treatments based on electroporation. Please add range of values used for pulse width, amplitude and frequency usedElectrodes: Stainless steel needles.Samples: Muscular tissue samples of 3x2x1 cm were sliced from fresh chicken (no ethical approval was required since the ex vivo experiments where performed with chicken muscular tissue acquired from a retailer).pH indicator dye: phenolphthalein, C20H14O4, transition pH range 8.0?9.6, from colorless to red. When the pH goes back to 8 it again changes to colorless. Imaging procedure: Using a CASIO EXFH25 high-speed camera videos were recorded at 30 fps with a 1280x720 pixel resolution. Imaging analysis techniques: Only 1 frame per second in the green channel was used (ImageJ, http://rsbweb.nih.gov/ij). In each frame a hard fuzzy-c-means algorithm was used in order to separate the intensity of the pixels into 4 categories (MIPAV, http://mipav.cit.nih.gov). Using an Octave script (GNU OCTAVE 3.6.2) the number of pixels matching the category of the dye was counted. We define evanescence time (ET) as the time when 90% of the dye changed from red to colorless after the end of the pulse delivery (90% reduction in the pixel count). This ET, which is an indirect measure of the amount of hydroxyls produced, is estimated by the time needed for the tissue buffer to turn the pH value to less than 8 (nearly neutral).Pulse parameters: Different pulse parameters were obtained from literature. A surrogate of the Coulomb dose is calculated as electric current density times pulse duration times number of pulses delivered. IRE: 90 pulses of 1500 V/cm, 100 µsec at 1 Hz; ECT: 8 pulses of 1000 V/cm, 100 µsec at 10 Hz; GET standard: 4 pulses of 80 V/cm, 100 ms at 5 Hz. We also tested GET doubling and tripling each parameter (voltage, pulse length and number of pulses).Results and discussionWhen pulse parameters of electrochemotherapy (ECT)[1] or irreversible electroporation (IRE)[2] were used, the ET is short. This means that the pH change induced is rather small compared to the neutralizing capacity of the natural buffers present in the tissue. These extreme pH changes lead to tissue damage that in the case of treating a tumor is not relevant, but when the safety margin is treated, normal tissue will be damaged. On the contrary when pulse parameters typical for gene electrotransfer (GET) [3] are used, the ET is much longer. In this case, detrimental effects are more significant since cell loss, and plasmid denaturation [4] contribute to reduce the gene expression. When using typical GET pulses and pulse number, voltage or pulse length is doubled or tripled the ET grows and, remarkably, when any is halved, the ET drops significantly.Higher coulomb doses correlate with stronger pH changes and thus with longer evanescence times. A linear regression analysis shows a linear law of ET as a function of Coulomb dose, i.e. ET = 289 D1.16 with an R-squared of 0.985Choosing the right pulse parameters that minimize pH changes in the tissue has relevant implications for GET treatment efficiency, due to a substantial reduction of plasmid damage and cell loss.Conclusion In this work we showed that pH gradients are immediate and substantial even in tissues where natural buffers are present to neutralize them. The evanescence time could be a useful indicator of the magnitude of these changes. If they are taken into account for treatment planning better results can be expected, especially in GET protocols.