Abstract
Treatments based on electroporation (EP) induce the formation of pores in cell membranes due to the application of pulsed electric fields. We present experimental evidence of the existence of pH fronts emerging from both electrodes during treatments based on tissue EP, for conditions found in many studies, and that these fronts are immediate and substantial. pH fronts are indirectly measured through the evanescence time (ET), defined as the time required for the tissue buffer to neutralize them. The ET was measured through a pH indicator imaged at a series of time intervals using a four-cluster hard fuzzy-c-means algorithm to segment pixels corresponding to the pH indicator at every frame. The ET was calculated as the time during which the number of pixels was 10% of those in the initial frame. While in EP-based treatments such as reversible (ECT) and irreversible electroporation (IRE) the ET is very short (though enough to cause minor injuries) due to electric pulse characteristics and biological buffers present in the tissue, in gene electrotransfer (GET), ET is much longer, enough to denaturate plasmids and produce cell damage. When any of the electric pulse parameters is doubled or tripled the ET grows and, remarkably, when any of the pulse parameters in GET is halved, the ET drops significantly. Reducing pH fronts has relevant implications for GET treatment efficiency, due to a substantial reduction of plasmid damage and cell loss.
Highlights
Cancer is one of the leading causes of death worldwide, accounting for 7.6 million deaths in 2008 and it is projected to continue rising, with an estimate of 13.1 million deaths in 2030 [1,2]
As an extension of our previous results [39], the aim of this paper is to show that pH fronts generated by EP-based techniques produce non negligible pH changes in a tissue regardless of the presence of natural buffers. pH fronts are measured through the evanescence time (ET), that is, the time required for the tissue buffer to neutralize them
The gene electrotransfer (GET) test consists of one high voltage (HV) pulse followed by four low voltage (LV) pulses
Summary
Cancer is one of the leading causes of death worldwide, accounting for 7.6 million deaths in 2008 and it is projected to continue rising, with an estimate of 13.1 million deaths in 2030 [1,2]. To improve efficiency and reduce side effects, during the last decades a considerable number of new therapies have been presented, many of which are currently under study. Theoretical and experimental studies about EP-based techniques were conducted in the 1970s [7,8,9] and Neumann et al [10] reported for the first time a successful gene transfer into murine cells using EP. After the development of EP devices this technique became widespread for delivering molecules inside the cell [11,12,13]. Since many tumor treatment modalities using EP, delivering genes or drugs, have been developed [15,16,17,18,19]
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