Experiments And Mechanism Research On Killing Tumor Cells By Irreversible Electroporation With High-frequency Nanosecond Pulse Bursts

High-frequency nanosecond pulse bursts comprise a new form of pulsed electric field proposed in recent years for tumor ablation.Compared with traditional microsecond and nanosecond pulsed electric fields,high-frequency nanosecond pulse burst therapy technology has significant advantages in effectively killing tumor cells,reducing muscle contraction,and improving electrical safety.However,the biophysical mechanism of high-frequency nanosecond pulse bursts inducing electroporation to kill tumor cells is still unclear.In particular,the cumulative and irreversible electroporation effects reflected in the experiments require in-depth research for better understanding.In order to explain the mechanism of this new pulse form for killing tumor cells,experiments with high-frequency nanosecond pulse bursts were systematically carried out to verify their effect of killing tumor cells and provide a realistic basis for a subsequent irreversible electroporation model.A novel electroporation mathematical model that considers irreversible electroporation could then be established by introducing the influence of electric field on the surface tension of a cell membrane and setting an irreversible pore radius threshold value.Combined with the mesh transport network(MTN)method,the single-cell electroporation and molecular transport under the application of high-frequency nanosecond pulse bursts were simulated,and the correctness of the model and the electroporation characteristics were indirectly verified through PI(propidium iodide)fluorescence dye experiments.The main work and results of this paper are as follows:(1)Taking the human melanoma cell A375 as the research object,the effectiveness of the high-frequency nanosecond pulse burst treatment method and the main mode of cell death were first studied through macro-killing effect experiments.Among them,the results of CCK-8 cell viability experiments showed that high-frequency nanosecond pulse bursts could effectively kill cells.The field strength and pulse width were in line with the positive correlation dose rule with the decreased cell viability rate,while the intraburst frequency had a threshold of 100 k Hz,which significantly reduced cell viability.Then,flow cytometry experiments showed that high-frequency nanosecond pulses kill tumor cells mainly by inducing cell necrosis,and the pulse dose rule was consistent with the cell viability detection experiments.Furthermore,through scanning electron microscopy experiments,we observed the microstructure of A375 cells and chicken red blood cell membranes after the application of high-frequency nanosecond pulse bursts.Clearer areas of perforations or defects were found on the membranes.This qualitatively verified the irreversible electroporation of the cell membranes and provided a realistic basis for subsequent model improvement.(2)Based on the geometric simulation model and the mathematical model of the existing mesh transport network,the pore transport model was improved,and a model of the irreversible electroporation of tumor cell membranes caused by high-frequency nanosecond pulse bursts was established.The improvement of the irreversible electroporation model is due to two aspects: the pore energy equation in the original dynamic electroporation model is enhanced by considering the additional electric fieldinduced surface tension;the dynamic irreversible electroporation radius threshold is set in the model.In addition,referring to relevant phenomenological models and theoretical assumptions,we introduced the concept of stable reversible pores to reflect the effect of the intraburst frequency on the accumulation of reversible pores.Finally,to compare and verify the simulation results with subsequent fluorescent dye experiments,the binding equation of Pr(propidium)and nucleic acid was coupled based on the original molecular electrodiffusion equation.(3)Numerical calculations were carried out on the electrical transport,pore transport,and molecular transport characteristics of a single-cell system under the application of high-frequency nanosecond pulse bursts.We analyzed the cumulative and irreversible electroporation of the cell membrane under varying pulse parameters.The results showed that the simulation could realize irreversible electroporation,and irreversible pores have various degrees of cumulative effect under different pulse parameters.Irreversible pores on the cell membrane are concentrated in the transition area where few pores are present,but the pore radius is easy to expand,mainly distributed around 8 nm.The time-domain results of molecular transport showed that the slope of the molecular concentration curve first slowed down and then remained unchanged.With the increase of the pulse parameter level,the molecular concentration in the cell increased at different times,meeting the positive correlation dose rule of the parameter.The molecular spatial distribution indicates that the molecules first entered from the positive and negative poles of the cell and then slowly diffused into the middle of the cell.(4)A fluorescent dye experiment of single cells in vitro was carried out to verify the electroporation characteristics of tumor cell membranes under the application of high-frequency nanosecond pulse bursts.Firstly,the time-domain results of molecular transport that were obtained from experiments and simulation under different pulse parameters after correction are compared.The results showed that,qualitatively,the simulated molecular transport rules under different pulse parameters were consistent with the experimental results,and all groups had an irreversible electroporation trend.Quantitatively,the difference in the binding ratios of intracellular nucleic acid was small.Then,the results of the fluorescence spatial distribution detection experiment compared with the simulation verified that the molecules first entered from the positive and negative poles of the cell and then slowly diffused into the middle of the cell.Finally,through the detection experiment of cell membrane permeability recovery,the fluorescence intensity continued to rise after the pulse was removed,which indirectly further confirmed that the cells underwent irreversible electroporation.In summary,based on the proposed model considering the irreversible electroporation effect,this paper conducted in-depth studies with experimental verification on the electrical transport,pore transport,and molecular transport characteristics of a single cell under the application of high-frequency nanosecond pulse bursts.The mechanism of killing tumor cells by irreversible electroporation with high frequency nanosecond pulse bursts was determined,providing important guidance in the field of cell irreversible electroporation.