Enhancement Of Cell Membrane Permeability By Single Microbubble Sonoporation

Ultrasound application has been demonstrated can transiently increase the cell membrane permeability. With the presence of ultrasound contrast agent (micro bubbles), ultrasound energy mechanically creates pores on the membrane of cell and increase the porosity of cell plasma membrane, allow intracellular uptake of drugs, macromolecules and gene which usually couldn't through the intact cell membrane,this mechanism was called sonoporation.However, the mechanism and process of sonoporation has not been fully understood due to the lack of control and real-time monitoring methods for single bubble sonoporation at cellular level. There are three disadvantage of previous researches: 1)Can not provide controllable sonoporation with single cavitation bubble and individual cell; 2)Can not real time monitor the sonoporation dynamics ; 3) Can not real time record and analysis the response of single cell in sonoporation.In this research we study the following: 1)Spatial control of single micron-sized bubbles in a non-contact fashion;2)The mechanism of enhancement of cell membrane permeability by controlled single microbubble sonoporation。In this study, we investigated the dynamic process of bubble generation and trapping by self-focused femto-second laser pulses using high frequency ultrasound imaging and fast-frame video microscopy. We quantitative investigated the restoring force of laser beam by measuring the transverse force using an acoustic radiation force technique. We develop a method to spatial control single micron-sized bubbles in a non-contact fashion by optical and acoustic technology.The acoustic radiation was utilized to push the originally trapped bubble toward the cell membrane and the distance could be controlled precisely(withμm scale). Cavitation (collapse) of the bubble was generated near the cell membrane by a short high energy intensity ultrasound exposure. The permeability of ell membrane was enhanced with sonoporation. Four methods were utilized to real time monitor the process including: 1)M-mode ultrasound imaging, 2)B-mode ultrasound imaging, 3)ultra fast optical imaging , 4)voltage clamp techniques. We reveal various the correlation of parameters (ultrasound energy intensity, Pulse repetition frequency, pulse cycle number and the distance between microbubble and cell membrane) and the outcome of sonoporation. High speed camera and voltage clamp system were adapted to real time record the dynamic process synchronized.In summary, this study revealed: 1) The dynamics of laser induced optical breakdown (LIOB), the microbubble generation and trapping by a self-focusing femtosecond laser; 2) Develop a technique to manipulate a single microbubble in a non-contact fashion; 3) Correlate sonoporation with acoustic cavitation activities at single cell level;4) Quantitative analysis the effective distance between microbubble and cell membrane in sonoporation;5)To real time investigate the mechanism of sonoporation and the optimal sonoporation parameters.