Selectivity And Mechanism Study On Tumor Cell Ablation By High-voltage Nanosecond Pulsed Electric Fields

Cancer seriously endangers the lives and health of Chinese people.With the increasing number of new cancer cases and deaths,the treatment situation of malignant tumors is becoming more and more serious.At present,surgery and chemotherapy are still some of the mainstream treatment methods for most patients with advanced cancer.However,patients will still be insensitive to conventional treatment methods due to the drug resistance of tumor cells after receiving treatment,resulting in poor prognosis and easy recurrence,leading to treatment failure.In recent years,pulsed electric field tumor therapy,as a new physiotherapy method,has attracted the attention and research of many researchers at home and abroad for its unique cell regulation function.Irreversible electroporation(IRE)caused cell death by targeting the cell membrane and destroying the internal and external physiological balance.And shorter nanosecond pulsed electric field technology will be targeted to action to the tumor,including the nucleus,mitochondria and other organelles membrane,electric induced cell treatment effect and immunogenicity induced cell death,can activate the body tumor patients-immune circulation,stimulate the antitumor immune response,high in the treatment of malignant tumor,displayed the great advantage to improve the prognosis.In this context,it is of great significance to study the killing effect and mechanism of High-voltage nanosecond pulsed electric fields on drug-resistant tumor cells,and to compare the response difference of High-voltage nanosecond pulsed electric fields with the corresponding tumor cells and normal cells,in order to overcome the difficulty of chemotherapy resistance.Based on this,we used human immortalized keratinocytes(Ha Ca T),human melanoma cells(A375)and their wilofenib Resistance(1?g/m L)cells(A375/R,R denoted Resistance,Human lung epithelial cells(BEAS-2B),human lung cancer cell line(A549)and cisplatin-resistant(1 ?g /m L)lung cancer cells(A549/R)were used as the research objects.Around the High-voltage nanosecond pulsed electric fields for different types of tumor cells response differences and internal response mechanism,measuring the different cell types in the geometry size,electrical parameters and biochemical composition,physical and chemical parameters,such as comparison and analysis of the High-voltage nanosecond pulsed electric fields under the action of killing effect and ablation threshold,and combined with simulation and theoretical calculation,the selective effect of High-voltage nanosecond pulsed electric fields on tumor cells was further explained.The main achievements include:(1)The geometrical size,electrical parameters,biochemical components and other physical and chemical parameters of human normal cells,corresponding tumor cells and drug-resistant tumor cells were measured.The differences of nucleoplasmic ratio,membrane capacitance,and nuclear membrane capacitance were analyzed.The drugresistant tumor cells had the largest nucleoplasmic ratio,and the membrane capacitance and nuclear membrane capacitance were larger than those of homologous tumor cells.It is used for the follow-up simulation analysis of cell killing sensitivity under the action of High-voltage nanosecond pulsed electric fields.Meanwhile,the distribution of protein,lipid,nucleic acid and other biological macromolecules in a single living cell was explored,which laid a foundation for the follow-up study on the mechanism of cell death induced by High-voltage nanosecond pulsed electric fields.(2)A five-layer dielectric model of a single cell was established to study the dynamic development of electroporation in the inner and outer membrane of cells under Highvoltage nanosecond pulsed electric fields.Scanning simulation of parameters such as geometric size and electrical parameters was carried out to clarify the influence of physical and chemical parameters on the degree of electroporation.According to the measured geometric parameters and electrical parameters characteristics of cells,the difference of nuclear membrane electroporation degree between normal cells,tumor cells and drug-resistant tumor cells under High-voltage nanosecond pulsed electric fields was analyzed by simulation.The drug-resistant tumor cells had the largest perforation area and number.At the same time,the confocal Raman platform was used for detection,and the Raman spectral information of the cell nucleus and cell membrane after pulse treatment was extracted.By analyzing the Raman signal of biological macromolecules,it was revealed that the changes of biological macromolecules induced by the bioelectric effect in cells after the high voltage nanosecond pulsed electric field had A dosedependent effect on the electric field.Among them,the changes of skeletal phosphate group adenine A,guanine G and phospholipid C-C structure are closely related to the mechanism of cell apoptosis.(3)High-voltage nanosecond pulsed electric fields experimental research was carried out,and an experimental platform for simulating tissue with hydrogel was established.The survival rate,ablation area and ablation threshold of homologous cells were detected and calculated according to the cell suspensions and three-dimensional hydrogel scaffolds.Then the sensitivity of human normal cells,tumor cells and corresponding drug-resistant tumor cells to High-voltage nanosecond pulsed electric fields was analyzed,drug-resistant tumor cells had the lowest survival rate,the largest ablation area and the smallest ablation threshold,the most sensitive electrical response to High-voltage nanosecond pulsed electric fields.To sum up,in this paper,impedance spectroscopy,Raman spectroscopy,as well as three-dimensional hydrogel scaffolds ablation research of the normal cells and tumor cells and the corresponding drug-resistant tumor cells were studied.We found that under the action of the High-voltage nanosecond pulsed electric fields,drug resistant tumor cells have the strongest sensitivity,with normal cells sensitivity to the minimum.The study in this paper is helpful to further understand the coupling mechanism of High-voltage nanosecond pulsed electric fields on tumor cells,and provide experimental support and theoretical basis for the development of an electrical therapy to overcome the resistance of tumor radiotherapy and chemotherapy.It also provides some solutions for the clinical application of High-voltage nanosecond pulsed electric fields combined with drug therapy for tumor in the future.