Sonoporation of Cells by a Parallel Stable Cavitation Microbubble Array

Abstract

Sonoporation is a targeted drug delivery technique that employs cavitation microbubbles to generate transient pores in the cell membrane, allowing foreign substances to enter cells by passing through the pores. Due to the broad size distribution of microbubbles, cavitation events appear to be a random process, making it difficult to achieve controllable and efficient sonoporation. In this work a technique is reported using a microfluidic device that enables in parallel modulation of membrane permeability by an oscillating microbubble array. Multirectangular channels of uniform size are created at the sidewall to generate an array of monodispersed microbubbles, which oscillate with almost the same amplitude and resonant frequency, ensuring homogeneous sonoporation with high efficacy. Stable harmonic and high harmonic signals emitted by individual oscillating microbubbles are detected by a laser Doppler vibrometer, which indicates stable cavitation occurred. Under the influence of the acoustic radiation forces induced by the oscillating microbubble, single cells can be trapped at an oscillating microbubble surface. The sonoporation of single cells is directly influenced by the individual oscillating microbubble. The parallel sonoporation of multiple cells is achieved with an efficiency of 96.6 ± 1.74% at an acoustic pressure as low as 41.7 kPa.

Keywords: acoustic radiation force; membrane permeability; sonoporation; stable cavitation; ultrasound bioeffects.

Figure 1

a) Schematic of the experimental setup. The PZT is placed adjacent to the microfluidic channels and excites microbubble oscillation. The microbubbles are generated at microcavities due to the surface tension. Inset: microstreaming is generated by the stable cavitation and the trapped cells are sonoporated by the oscillating microbubbles. b) The vibration amplitude of the glass substrate, measured by LDV, is relatively uniform. c) An optical image of an air microbubble array with the same 40.8 µm diameter. d) An optical image of microstreaming produced by each oscillating microbubble, indicating the independent oscillation of microbubbles. e) Optical image of cells trapped by a microbubble array.

Figure 2

a) Optical image of microstreaming induced by microbubble oscillations. When the PZT is excited, the 2 µm polystyrene particles around the microbubble are driven to follow the microstreaming. The trajectory of the particles is demonstrated in Movie S2 in the Supporting Information. b,c) The streamline and velocity vector of the flow field traced by PIV analysis. The streamlines and velocity vector images indicate that two symmetrical vortexes are generated in the fluid field induced by the oscillating microbubble. d) Illustration of the distribution of shear stress induced by microstreaming. The maximum shear stress occurs in the central area of the vortices.

Figure 3

Cells are trapped by the microbubble oscillations. a) Prior to the application of ultrasound, the cells are distributed randomly in the channel. The solution of harvested MDA‐MB‐231 cells is injected to the microchannel. b) Moving trajectory of the cells within the microstreaming. With the presence of ultrasound, cells are attracted to the proximity of the bubble membrane and trapped there.

Figure 4

a) Sonoporation process at various input acoustic amplitudes. b) Quantitative analysis of the PI fluorescence intensity as a function of time (n = 5). c) Membrane deformation of the oscillating microbubble when the acoustic pressure is 41.7 kPa. d) Scattering signal induced by microbubble oscillation. Higher harmonics are detected by PCD, illustrating that stable (noninertial) cavitation occurs.

Figure 5

a) With the presence of ultrasound, all cells in the microchannel are attracted toward the oscillating microbubbles in 520 ms. b) Bright‐field image of a single cell trapped at an oscillating microbubble surface. c,d) Fluorescence images of the membrane permeability of 88.89 ± 1.53% cells are enhanced by their corresponding oscillating microbubble and almost all the cells remain viable (n = 5).

Figure 6

a) Single cells trapped at the oscillating microbubble array emit green fluoresce, confirming the cell viability. b) Trapped single cells emit red fluoresce under the action of oscillating microbubble array. c) Merged fluorescence image showing that high sonoporation efficiency can be achieved by a parallel stable cavitation microbubble array. d,e) Sonoporation efficiency and cell viability at various acoustic pressures as a function of ultrasound treatment time.