| Backgrounds:Breast cancer is one of the most common malignant tumors in women and the main cause of death from cancer.In the past decades,the incidence of breast cancer has been rising rapidly worldwide.Although the large-scale popularization of early screening and the continuous development of effective adjuvant chemotherapy have significantly reduced the recurrence and mortality of breast cancer,there are still many challenges in clinical treatment,such as lack of targeted destruction of chemotherapeutic drugs,severe systemic toxicity and the production of acquired multidrug resistance(MDR),etc.Especially at present,there is no therapeutic drug for triple negative breast cancer,and its prognosis is still very poor.Therefore,it has been a common concern for clinicians and researchers to find a safe and effective treatment with few side effects and strong targeting.Ultrasound combined with microbubbles(USMB)technology has the advantages of safety,high efficiency and high targeting for targeted and locally specific delivery of cancer drugs.It has developed rapidly in the past decade,and its main mechanism is USMB-mediated sonoporation effect.Sonoporation effect means that microbubbles oscillate under the action of ultrasound,resulting in local shear stress,expansion and collapse,thus forming temporary holes on the surface of cell membrane,through which genes or drugs are transferred into target cells.This USMB-mediated sonoporation effect can increase cell membrane permeability and enhance the delivery efficiency of chemotherapeutic drugs,so as to effectively improve the efficacy of tumor and reduce the toxic and side effects of chemotherapeutic drugs.At present,most studies on sonoporation effect are mainly focused on the transfection of USMB-loaded drugs or genes into cells and the interaction between microbubbles and cells,however,in the process of USMB-mediated gene and drug transfection,inappropriate USMB parameters may lead to cell damage,even cell death or transmission efficiency reduction,resulting in unsatisfactory clinical effect.Therefore,the reasonable setting of USMB parameters is particularly important to improve the sonoporation efficiency.The effects of microbubble parameters(including microbubble size,contrast agent concentration,shell properties)and ultrasound pulse parameters(including pulse repetition frequency,sound intensity,irradiation time,pulse wavelength and peak negative sound pressure)on sonoporation efficiency have been studied in some previous literatures,however,most of these studies focus on the effects of single-factor changes on sonoporation efficiency,and there is a lack of comprehensive research on the influence of USMB multi-parameter and multi-level combination on sonoporation efficiency.Additionally,the effect of different cell lines from a certain tumor tissue on sonoporation efficiency was seldom studied,and there are few comparative studies on the effects of tumor cell lines and drug-resistant cell lines on sonoporation efficiency.Objectives:1.The combination and optimization of different parameters of USMB at different levels were studied,the effects of different parameters on different subtypes of cell lines were compared and analyzed,and the characteristics of the best combination of USMB parameters of different cell lines were observed.Under the condition of the optimal combination of USMB parameters,the maximum sonoporation efficiency of different subtypes of cell lines was observed to explore the effects of different subtypes of tumor cell lines(MDA-MB-231,MDA-MB-468,MCF-7)from the same tissue on the sonoporation efficiency.2.The effects of different USMB parameters on drug-resistant cell lines and drug-sensitive cell lines were observed and analyzed,and the optimal USMB parameter combinations of cell lines were observed.The maximum sonoporation efficiency of drug-resistant and drug-sensitive cell lines was observed under the optimal USMB parameter combination mode to explore the effects of drug-sensitive cell lines and drug-resistant cell lines(MCF-7,MCF-7/ADR)from the same tissue on sonoporation efficiency.Methods:1.In this study,three drug-sensitive breast cancer cell lines MDA-MB-231,MDA-MB-468,and MCF-7 were selected.Three factors,microbubble concentration,ultrasound intensity and irradiation time,were selected to design L9(33)orthogonal array experiment.The parameters of three variables were selected respectively(microbubble contrast agent concentration:10%,20%,30%,ultrasound intensity:0.5W/cm2,1.0W/cm2,1.5W/cm2,irradiation time:30s,60s,90s).Then three cell lines were irradiated by ultrasound combined with microbubbles according to the established experimental scheme to mediate intracellular transport of the model drug fluorescein isothiocyanate-dextran(FD500).The sonoporation efficiency and cell survival rate of each cell line were used as indexes,and the experimental results were recorded.2.The range analysis of the experimental results of L9(33)orthogonal array is carried out.The m-level test index and Kjm of the j column of each parameter and the average value of the test index of each parameter(kjm)are calculated respectively to calculate Kjm and kjm to determine the optimal level of each parameter.At the same time,the range R value(kmax-kmin)was calculated,and the primary and secondary effects of the three parameters of each subtype of breast cancer cell line were determined.And the optimal combination of USMB parameters of each breast cancer cell line was obtained by combining Kjm,kjm and R values.3.Under the optimal combination of USMB parameters,ultrasound combined with microbubble irradiation was used to mediate the intracellular transport of FD500 in three different subtypes of breast cancer cell lines,and the experimental results were recorded.Under the optimal combination of USMB parameters,the maximum sonoporation efficiency and cell survival rate of three different breast cancer cell lines were compared,and the effects of different subtypes of cell lines from the same tissue source on USMB-mediated sonoporation efficiency were studied.4.According to the R value obtained by the above range analysis,the influence degree of each parameter on sonoporation efficiency and cell survival rate of different cell lines was determined(transverse analysis),and then the influence level order of different parameters of each cell line was obtained.According to the results of range analysis,the response curve of cells to different influencing factors was drawn,and the changes of sonoporation efficiency and cell survival rate of each cell line with the change of single parameter level were observed(longitudinal analysis).5.Breast cancer drug-sensitive cell line MCF-7 and drug-resistant cell line MCF-7/ADR were selected,and the parameters of three variables were selected respectively(microbubble contrast agent concentration:10%,20%,30%,ultrasound intensity:0.5W/cm2,1.0W/cm2,1.5W/cm2,irradiation time:30s,60s,90s)to design L9(33)orthogonal array experiment.According to the orthogonal test scheme,the above two cell lines were irradiated with USMB respectively to mediate the intracellular transport of the model drug FD500.The cell survival rate and ultrasonic sonoporation efficiency were used as indexes,and the experimental results were recorded.6.According to the above L9(33)orthogonal array experimental results,the Kjm,kjm and R values were calculated,and the optimal USMB parameter combinations of drug-resistant and drug-sensitive breast cancer cell lines were obtained respectively.The optimal USMB parameter combinations of the two cell lines were compared.Moreover,under the optimal USMB combination conditions,ultrasound combined with microbubble irradiation was used to mediate the intracellular transport of FD500 in the two cell lines.The sonoporation efficiency and cell survival rate of drug-resistant cell lines under the optimal USMB parameters were observed and compared to study the effects of drug-sensitive and drug-resistant breast cancer cell lines from the same tissue source on USMB-mediated sonoporation efficiency.7.The range of R value of each parameter combination was calculated by range analysis,and the influence degree of each parameter on the above two indexes of drug resistant cell line was judged(horizontal analysis).According to the results of range analysis.the response curves of cells to different influencing factors were drawn,and the changes of sonoporation efficiency and cell survival rate of drug-resistant and drug-sensitive cell lines were observed with the change of different parameters.The effects of various parameters on cell survival rate and sonoporation efficiency of drug-sensitive and drug-resistant cell lines were judged.Results:1.USMB parameters optimization of different breast cancer cell linesThrough the range analysis of the orthogonal test results,we obtained the K,k and R values.Through the further calculation,observation and comparison of the K,k and R values,the optimum combination of USMB irradiation parameters for three groups of cells was summarized as follows:MDA-MB-231:microbubble concentration 10%,ultrasound intensity 1.5W/cm2,irradiation time 90 seconds;MDA-MB-468:microbubble concentration 20%,ultrasound intensity 0.5W/cm2.irradiation time 30 seconds;MCF-7:microbubble concentration 30%,ultrasound intensity 0.5W/cm2.irradiation time 30 seconds.2.The maximum sonoporation efficiency and cell survival rate of each cell line under the optimal combination of USMB parametersUnder the optimal combination of USMB parameters,the maximum sonoporation efficiencies of MDA-MB-231,MDA-MB-468 and MCF-7 were 46.70±5.79%,53.44±5.69%and 59.88±5.53%respectively,while the cell survival rates of MDA-MB-231:84.24±5.83%,MDA-MB-468:81.03 ±3.43%and MCF-7:80.48±3.22%,respectively.The results of one-way ANOVA and LSD-t test showed that,under the optimum conditions,there were significant differences in sonoporation efficiency between any two different cell lines(P<0.05),but there was no significant difference in cell survival rate(P>0.05).3.Effects of different parameter levels on sonoporation efficiency and cell survival rate of each cell lineAccording to the intuitive analysis results of different parameter combinations in orthogonal experiments on the efficiency and viability of several kinds of cells,the effects of three factors(including microbubble concentration,sound intensity and irradiation time)on the efficiency and viability of the cells were determined by comparing the R values of the intuitive analysis results.The effects of each parameter on the efficiency of each cell line were sorted(MDA-MB-231:microbubble concentration>irradiation time>sound intensity;MDA-MB-468:sound intensity>irradiation time>microbubble concentration;MCF-7:sound intensity>micro bubble concentration>irradiation time).The effects of these factors on cell viability were MDA-MB-231 and MCF-7:sound intensity>irradiation time>microbubble concentration;MDA-MB-468:microbubble concentration>sound intensity>irradiation time.According to the response curves of cells to different factors,with the increase of microbubbles concentration,the vocal efficiency of MCF-7 and MDA-MB-468 cells increased gradually,while the vocal efficiency of MDA-MB-231 cells increased slightly at the beginning,and decreased at higher microbubble concentration(30%)in the set range.On the other hand,with the increase of acoustic wave intensity,the efficiency of all cell lines increased.Finally,the sonoporation efficiency of MDA-MB-468 cells increased with the prolongation of irradiation time,while the permeability of MDA-MB-231 cells decreased significantly,while that of MCF-7 cells increased slightly at first and then decreased slightly,but increased slightly as a whole.The cell survival rate of the three cell lines decreased in varying degrees with the increase of microbubble concentration,sound intensity and irradiation time.4.Optimal USMB parameter combination of drug-resistant and drug-sensitive breast cancer cell linesThrough the range analysis of the results of orthogonal test,we obtained the K,k and R values of drug-resistant and drug-sensitive cell lines.Through further calculation,observation and comparison of K,k and R values,the optimal USMB parameter combinations of the two cell lines were obtained as follows,MCF-7:microbubble concentration 30%,ultrasound intensity 0.5W/cm2,irradiation time 30 seconds;MCF-7/ADR:microbubble concentration 30%,ultrasound intensity 1.0W/cm2,irradiation time 60 seconds.5.Effects of drug-resistant and drug-sensitive breast cancer cell lines on FD500 delivery under the optimal combination of USMB parameters.Under the optimal combination of USMB parameters,compared with MCF-7 cell line,the maximum sonoporation efficiency of MCF-7/ADR was significantly increased(65.39±4.01%vs 59.88±5.53%,P=0.026),however,there was no significant difference in the survival rate between the two cells irradiated by the optimal combination of USMB parameters.6.Comparative analysis of the effects of USMB parameters on the sonoporation efficiency and cell survival rate of sensitive and drug-resistant MCF-7 cell linesAmong the three parameter variables,sound intensity was the most important factor affecting the vocal efficiency of MCF-7 and MCF-7/ADR cell lines.According to the analysis of single factor response curve,at the beginning,the efficiency of the two cell lines increased with the increase of acoustic intensity,but when the acoustic intensity continued to increase to 1.5W/cm2,the permeability of drug-resistant cells decreased.On the other hand,with the increase of the concentration of microbubbles,the vocal efficiency of both groups increased gradually.Finally,the sonoporation efficiency of MCF-7/ADR cells increased with the prolongation of irradiation time,while the permeability of MCF-7 cells did not change significantly.It can be seen that for MCF-7 cells,the effects of various factors on the sonic pore efficiency are sound intensity>microbubble concentration>irradiation time,and for MCF-7/ADR,sound intensity>irradiation time>microbubble concentration.Meanwhile,with the increase of microbubble concentration,sound intensity and irradiation time,the overall survival rate of the two kinds of cells decreased,while when the microbubble concentration was 30%,the cell survival rate of MCF/7 showed a slight upward trend.Conclusion:1.The orthogonal test confirmed that the optimal parameter combinations of different breast cancer cell lines under the action of USMB were different,and the range analysis indicated that each parameter had different effects on sonoporation efficiency and cell survival rate in different cell lines.2.Under the condition of respective optimal combination of USMB parameters,the maximum sonoporation efficiency of different cell subtypes from unified tissue was different,and the difference was statistically significant.3.The best combination of USMB parameters for the best FD500 transfection efficiency of drug-sensitive and drug-resistant breast cancer cell lines(MCF-7 and MCF-7/ADR)is different.4.Under the condition of the best combination of USMB parameters,there was a significant difference in the maximum sonoporation efficiency between drug-sensitive and drug-resistant breast cancer cell lines(MCF-7 and MCF-7/ADR).The maximum sonoporation efficiency of MCF/ADR cell line was significantly higher than that of MCF-7 cell line.This result provides more information for further exploring the feasibility and mechanism of USMB combined with anticancer drugs in reversing MDR.5.The maximum sonoporation efficiency of triple negative breast cancer subtype cell lines(MDA-MB-231,MDA-MB-468)was significantly lower than that of HER2 overexpression subtype cell line(MCF-7)and drug resistant cell line(MCF-7/ADR).6.For four kinds of breast cancer cell lines,different USMB parameters have different effects on sonoporation efficiency and cell survival rate for the same cell line.Similarly,the sonoporation efficiency and cell survival rate of the same cell line change with the same parameter level,and the response degree is different. |
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