Cell-cycle-specific Biological Responses To Ultrasound Microbubble Mediated Sonoporation

In recent years,therapeutic ultrasound has received more and more attention because of its minimally invasive and non-invasive characteristics.Currently,the controlling and optimization of sonoporation have become a key focus in the application of therapeutic ultrasound,and have attracted broad interests in various clinical fields including targeted gene/drug delivery,cancer treatment,blood-brain-barrier opening,neurological diseases treatment,immuneological therapy,and so on.Microbubble-mediated sonoporation has shown its great potential in facilitating intracellular uptake of gene/drugs and other therapeutic agents that are otherwise difficult to enter cells.However,the biophysical mechanisms underlying microbubble-cell interactions remain unclear.Particularly,it is still a major challenge to get a comprehensive understanding of the impact of cell cycle phase on the cellular responses induced by microbubble mediated sonoporation.Therefore,the focus of this study is mainly on cell-cycle-specific biological resoponses to ultrasound microbubble mediated sonoporation.Firstly,efficient synchronizations were performed to arrest human cervical epithelial carcinoma(HeLa)cells in individual cell cycle phases.Flow cytometry analysis was adopted to assess the synchronization efficiency of HeLa cells.The topography and stiffness of synchronized cells were examined by using atomic force microscopy(AFM).The results show that G1-phase cells usually have the greatest height and modulus of elasticity,while S phase cells are usually the least flat and elastic cells.Secondly,the variations in cell membrane permeabilization and cytoskeleton arrangement induced by single microbubble mediated sonoporation were analyzed simultaneously by a real-time fluorescence imaging system.In the experiments,the cytoskeleton was labeled with green fluorescent GFP-a-tubulin to indicate cytoskeletal change caused by microbubble-mediated sonoporation and intracellular PI fluorescence intensity was used to indicate the changes of cell membrane permeability.Meanwhile,a finite element model was constructed to simulate the bubble-fluid-cell interaction and provide possible interpretation for the experimental results.Cells synchronized in S phase should be the most susceptible to instantaneous mechanical effect induced by single microbubble-mediated sonoporation,which resulted in the greatest enhancement of embrane permeability and the fastest cytoskeleton disassembly at the early stage after sonoporation.Consequently,the S Phase was found to be the preferred cycle for instantaneous sonoporation treatment,due to the greatest enhancement of membrane permeability and the fastest cytoskeleton disassembly at the early stage after sonoporation.At last,the cell-cycle-dependences of the membrane permeability and viability of HeLa cells undergoing multi-bubble sonoporation were evaluated using focused ultrasound exposure apparatus coupled passive cavitation detection system.The results indicated that:(1)the microbubble cavitation activity should be independent on cell cycle phases;(2)G1-phase cells with the largest Young's modulus were the most robust against microbubble-mediated sonoporation;(3)G2/M-phase cells exhibited the greatest accumulated FITC uptake with the lowest viability,which should be mainly attributed to the chemical effect of synchronization drugs;and(4)more important,S-phase cells with the lowest stiffness seemed to be the most susceptible to the long-term mechanical effect generated by multi-microbubble mediated sonporation,which resulted in the greatest enhancement in sonoporation-facilitated membrane permeabilization without further scarifying their viability.Whether it was the instantaneous effect of single microbubble-mediated sonoporation or the long-term effect of multi-microbubble mediated sonoporation,the S phase was found to be the preferred cycle for ultrasound microbubble medaited sonoporation for gene/drug delivery therapy.If combining advanced progresses in drugs developed from S-phase arrest with targeted microbubble,the current findings may enable more tailored cell-cycle-targeted therapeutic strategies for malignant tumors and other severe genetic diseases,by providing synergistic gene/drug delivery efficacy and reduced cytotoxicity both in vitro and in vivo.The present studies would be beneficial for better understanding the impact of cell cycle phase on the mechanical properties and microbubble sonoporation-induced cellular responses of HeLa cells,as well as the underlying biophysical mechanisms.The current findings may benefit ongoing efforts aiming to pursue rational utilization of microbubble-mediated sonoporation in cell cycle-targeted gene/drug delivery for cancer therapy.