Experiments And Mechanism Research Of Irreversible Electroporation By High Frequency Bipolar Microsecond Pulse For Tumor Ablation

Recently,the applications of pulse electric field on biomedical field have been emerged as the research focus for the scholars.Irreversible electroporation(IRE)is a relatively new focal ablation technique for the treatment of solid tumors,and the clinical results are very inspiring.IRE is a non-thermal mechanism which can be applied in close proximity to heat-sensitive structures,such as large blood vessels,nerve beds,or the ventricles.So the research and clinical application of IRE will be of benefit to treatment of tumors and people’s health.However,in clinical applications,IRE also was challenged by some problems.First,biological tissue contains a variety of cells and is anisotropic,so the distribution of electric field in the tissue is very uneven.The distribution of electric field in the tissue is very different with the simulations,which results in ablation blind area in the target zone,and leads tumor recurrence;Second,electrically induced muscle contractions are an unintended consequence which may move the electrodes during treatment,resulting in possible complications,and must be considered when treating near these sensitive structures.When improperly managed,organ translocations have been reported in responseto pulse delivery.In order to solve these problems,this paper puts forward a new type electric pulse for tumor ablation,which is hopeful to produce a uniform electric field distribution and inhibit the muscle contractions.To this end,this paper researchs the key technologies of high frequency bipolar microsecond pulse on tumor ablation,uniform electric field and inhibition of muscle contractions with the support of the Natural Science Foundation Project of CQ CSTC(grant No.cstc2014jcyjjq90001)and the Fundamental Research Funds for the Central Universities(grant No.106112015CDJZR158804).The major achievements are as follows:(1)High frequency bipolar microsecond pulse electric field was presented for solve the problems in IRE for the ablation blind area and muscle contraction in clinical application.Through the simulations,we found that the high frequency pulse can make the electric field distribution more homogeneous in cells and tissues.And it can make the action potential extend difficult with the enhancing of threshold current density,which can reduce the strength of muscle contraction.The typical five-layer dielectric model of single cell was established,and the simulation analysis of high frequency bipolar microsecond pulse dynamic process was conducted.The results show that membrane potential and rapidly rising above The threshold,and the perforated area gradually expanded and conductivity exploded.The Aperture and perforated area increases with electric field intensity,and at the same field intensity and duration pulses,the pore size and pulse width have positively proportional relationship.(2)Based on power electronic technology,a novel configuration of modular bipolar pulse generator topology base on marx generator with double power charging was designed.The generators were controlled by FPGA.Finally,a high frequency bipolar tumor ablation prototype was developed,which has the capability of producing composite pulses,in which the center frequencies of the bursts are up to 2 MHz,the inter-burst delay is 0.1~10 s,the pulse width is from 200 ns to 100 μs,the pulse amplitude is ±3 kV,and the rise time is around 30 ns.The Prototype is according to the national medical equipment standards,and has passed the commissioned test by Shanghai medical instrument Testing Center.The test contents include electrical safety,electrical properties,electromagnetic compatibility test project,etc.(3)Human cancer cells were selected as experimental subject,by single-celled DiI dyeing experiments,the effects of pulse parameters on the cell membrane damage was studied.Combined with CCK 8 dyeing experiments,indicated that the mechanism of high frequency bipolar microsecond pulse cause cell death is destroying the structure of the cell membrane.By flow cytometry and nucleus shape change detection,cell apoptosis phenomenon was detected,and along with the increase of pulse width,apoptosis percentage increase gradually.On mitochondrial membrane potential and immunohistochemical apoptosis protein detection,we determined the mechanism of endogenous model of tumor cells apoptosis pathway.(4)A large amount of experiments was conducted in vivo tissue ablation.Potato organization as the research object,and the relationship between the change of impedance and ablation effect was studied.The impedance changes can be used for judging curative effect.Plate electrodes and needle electrodes were used respectively for liver tissue ablation,and combined with the electric field simulation,we optimized the parameters of pulse,revealed the mechanism of high frequency bipolar microsecond pulse uniform electric field distribution,and exposed the tissue ablation threshold of high frequency bipolar microsecond pulse.(5)The law of muscle contraction under the high frequency bipolar microsecond pulse electric field on animals.According to muscle contraction by the plate electrodes and needle electrodes and electric field distribution,the results suggested that the muscle contraction and electrode structure has a close relationship.Then the insulated needle electrodes were developed.Through in vivo ablation experiments,the results show that the insulated needle electrode can ablate similar tissue with traditional needle electrodes with a less extent of of muscle contractions.In conclusion,this paper puts forward the new technology of high frequency bipolar microsecond pulse electric field for tumor ablation,studied the principle of electric field distribution and inhibition of muscle contractions,and analysised the mechanism of the induction of cell membrane electroporation;Based on pulse generator technology and solid-state switch technology,developed high-frequency bipolar microsecond pulsed electric field treatment prototype for biomedical experiment and clinical tumor ablation,which passed the national medical equipment certification testing;In vitro tumor cells was selected as experimental object,revealed the mechanism of the high frequency bipolar microsecond pulse induced cell death and apoptosis;Based on rabbit liver tissue ablation experiments,studied the high frequency bipolar microsecond pulse uniform electric field distribution and the mechanism of ablation threshold field strength;Comparing different parameters of high-frequency bipolar microsecond pulse and intensity of muscle contraction caused by needle electrode,a new type of insulated needle electrodes were developed to further reduce muscle contraction.These works promote the clinical application of high frequency bipolar microsecond pulse for ablation tumor and provided the necessary theoretical basis,the hardware foundation and technical support.