Pulsed electric fields and currents for cancer therapy.
Thesis DisciplineElectrical Engineering
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Incidence of cancer is growing in modern society, especially in New Zealand and the U.K. where cancer is now the leading cause of death, motivating development and research into new therapies. The ultimate goal of the work in this thesis is to offer novel electrical methods in destroying cancer cells. Literature suggests that there are certain physical means of producing more consistent electroporation among a variety of different cells in a tumour, such as the use of higher frequency pulsing electric fields, and these means need to be further investigated. The goal of this thesis was to design apparatus that can be used to investigate these new means of improving electroporation, including producing more uniform electroporation among a range of different cell sizes and morphologies as is typically present in malignant tumours, and to validate this apparatus. A novel electroporator apparatus that uses a dual stage cascaded multilevel inverter topology with high voltage RF MOSFETs has been designed and used successfully for the experiments in this study. The electroporator meets the broad specifications of being adjustable or programmable in terms of voltage (output ranging from 100 V to 1250 V peak), frequency (monopolar pulses of up to 500 μs and bipolar pulses ranging from 10 kHz to 800 kHz), and pulse burst regime (programmable number of pulses, with programmable inter-pulse burst intervals). The electroporator offers a variety of output waveforms and is also capable of producing bipolar or monopolar pulses. It has demonstrated reasonable performance with an output slew rate of up to 2 x 1010 V/s for frequencies up to 1 MHz pulses into a load of 80 Ω in parallel with 235 pF from a dual isolated supply of 1600 VDC total, despite substantial coupled noise in the circuit. A robust cuvette, that produces a uniform electric field between its electrodes, has been developed for the experiments in this study and has withstood the rigors of all the electroporation work done successfully. Cell culture and electroporation protocols have been developed for the experiments undertaken. A substantial body of work was carried out involving pulsed electric field electroporation of Ishikawa human cancer cells (a commercial human endometrial adenocarcinoma cell line) in suspension. Two methods were employed including fluorescence microscopy and particularly flow cytometry. Microscopic analysis proved less productive than flow cytometric analysis and also lacked fluorescence resolution. Differences between bipolar and monopolar pulses were examined and characterised in detail. DC (monopolar) pulses appear to have more detrimental effects on cells compared to AC (bipolar) pulses for an equivalent mean fluorescence. Also, monopolar fields do not allow loading of cells with non-permeable small molecules (can only rapidly penetrate cells when they are porated) to the same high concentration as for bipolar fields in the case where high viability is desirable. Thus bipolar fields show a clear advantage over monopolar fields if the aim is not to kill cells directly. No direct evidence could be found whether higher frequency (ranging from 10 kHz to 800 kHz) bipolar fields produce better normalisation of the electroporation effect across a range of cell sizes (6 μm to 43 μm, ±10 %) for isolated cells in suspension. Higher frequencies also tend to demand higher amplitude electric fields to produce the same degree of electroporation than at lower frequencies due to inherent membrane time constant and relative slew rate limitations of the MOSFET switches. If the aim is to kill cells over loading of cells with external molecules (in this case fluorescent dye), monopolar pulses may offer the best result so long as the electric field can be adequately applied to the cells. The added complexity of cells embedded within a tissue structure, which may yield different results regarding comparative electroporation between monopolar and bipolar pulses, has not been investigated here. Radio frequency ablation is used in cancer therapy treatment motivating development of a radio frequency ablator. The general H-bridge topology was employed for this radio frequency ablator as it allows for flexibility of ablation frequencies. Also, a practical radio frequency ablation probe has been developed and a low power ablation protocol has been established for the low power experimental work on ex-vivo animal liver at room temperature. The radio frequency ablator system was used to ablate fresh animal liver successfully and reliably with very similar characteristics to standard commercial radio frequency ablation systems, creating an average lesion of 2.7 cm3 for a 2 cycle 20 W ramped ablation protocol lasting 5 minutes. The ablator compared well with the literature, where low power protocols were used on healthy animal liver as a convenient model. The system performed less electrically efficiently at high frequencies (excessive heat was produced in the circuit due to the particular topology used). It could not be established whether higher frequencies (above 460 kHz) are capable of producing better ablation performance as could be expected when considering that tissues tend to be more conductive at higher frequencies due to inherent tissue capacitance causing the impedance to drop. Several novel contributions have been made to the fields of electroporation, electrochemotherapy and radio frequency ablation techniques and technologies, as identified above. For future work, further investigation of electroporation effects across a wider range of electric field pulse frequencies, burst parameters, cell types, and various tissues is required. Lastly, radio frequency ablation of tumours at higher frequencies (than the normal 450 kHz used for the commercial sinusoidal output radio frequency ablators) of pulsed bipolar electric currents is still required. Higher frequencies may produce more uniform ablation within the highly inhomogeneous tissue of unhealthy liver. A multimodal apparatus has been described and proposed, combining electroporation with radio frequency ablation, as a candidate for non-invasive deep tissue tumour therapy, and for other applications including gene therapy, genome editing and electrofusion. In conclusion, a general H-bridge topology and its extension using a cascaded multilevel inverter arrangement has been employed to design and implement practical circuits that produce pulsatile output voltages for various uses, including an application in electroporation and an application in radio frequency ablation. A non-conventional method for analysing the effect of variable electroporation electric field pulses across a frequency range of 10 kHz to 800 kHz (bipolar pulses) and for single monopolar pulses, across a range of electric fields from 0.5 x 105 V/m to 3.5 x 105 V/m, based on flow cytometry, has been studied and presented. This study demonstrates how these various parameters affect Ishikawa cells in suspension, in terms of loading of exogenous molecules, and survival, and may promote the use of specific sets of parameters for specific applications. These applications may include the use of bipolar electric field pulses for biotechnology applications where survival is important, and monopolar or low frequency bipolar electric field pulses used in combination with very high fields for irreversible electroporation and may be used to ablate undesirable tissue such as tumours in the presence of other important surrounding tissues such as blood vessels. It has also been shown that pulsed AC electric currents (as opposed to sinusoidal alternating currents) may be used to ablate liver tissue, similar to commercial radio frequency ablators.