Sonoporation: Past, Present, and Future

Abstract

A surge of research in intracellular delivery technologies is underway with the increased innovations in cell-based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high-throughput delivery that is critical for providing the desired cell quantity for cell-based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and has been demonstrated for delivering a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble-based sonoporation methods usually require special contrast agents. Bubble-based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non-bubble-based sonoporation mechanisms are under development. This review will cover both the bubble-based and non-bubble-based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.

Keywords: acoustics; acoustofluidics; intracellular delivery; physical permeabilization; sonoporation.

Figure 1.

Schematics for illustrating the bubble-based sonoporation mechanisms. They illustrate mechanisms based on (a) the jetting effect caused by a bubble undergoing inertial cavitation, (b) the shock waves generated from a collapsing bubble, (c) the acoustic streaming induced by a stable oscillating bubble, (d) the pushing effect on the cell membrane induced by bubble expansion during oscillation, (e) the pulling effect on the cell membrane induced by bubble contraction during oscillation, (f) the force applied on the cell membrane by a bubble that is pushed through the membrane under an acoustic radiation force.

Figure 2.

Conventional bubble-based sonoporation technologies. (a-c) Schematics for illustrating the typical setups, which usually have a sonicating bath and a chamber containing cells and bubbles. (a) The typical setup with an ultrasonic transducer at the bottom of the bath and a chamber (e.g., sealed petri dish) with cells adhered on the chamber wall. (b) The typical setup with an ultrasonic transducer at the bottom of the bath and a chamber (e.g., Eppendorf tube) with cells in suspension. (c) The typical setup having a chamber (e.g., customized sealed chamber) with suspension/adherent cells at the bottom of the bath and an ultrasonic transducer at the top of the bath. The transducers in (a) to (c) can either be planar or focused ultrasonic transducers. (d) Experimental results showing that bubbles in acoustic fields can have different dynamic behaviors, such as limited oscillations with small deformation at the acoustic pressure of 0.5 MPa (top row), asymmetric compression/expansion oscillations at 0.8 MPa (middle row), and acoustic wave-induced shell cracking at 1 MPa (bottom row). White arrows point to the bubbles with gas release induced by shell cracking. (e) The histories of normalized bubble areas for quantitatively characterizing different dynamic behaviors observed in (d). (f) The response frequencies of bubbles showing nonlinear components. (g) Quantitative characterization of sonoporation induced propidium iodide (PI) intensity changes in the cytoplasm and nucleus with respect to time. The experimental results were acquired using endothelial cells, an acoustic frequency of 2 MHz, and a peak-negative acoustic pressure of 0.9 MPa. (d-g) Reproduced with permission.^[102] 2017, Ultrasound in Medicine and Biology.

Figure 3.

Recent sonoporation technologies that fuse bubble-based sonoporation and microfluidics. (a) A schematic of a device that can trap an array of microbubbles along the sidewalls of microfluidic channels and apply acoustic waves to induced stable cavitation (i.e., oscillation of trapped bubbles). The bubble oscillation further generates vortex streaming, which can trap cells near the bubble shells and apply shear forces to increase the bubble permeability. (b) A microscopic image showing vortex streaming caused by oscillating bubbles. (c) Sonoporation results of MDA-MB-231 cells by using the bubble-based microfluidic device. Calcein-AM (green) is used to show viability and propidium iodide (PI, red) is used to indicate the membrane permeability change. (a-c) Reproduced with permission.^[115] 2019, Advanced Science. (d) A schematic of a setup that fuses the conventional sonicating bath and a microfluidic channel for continuous sonoporation. (e, top and bottom) A photo and a schematic of a spiral microfluidic channel for cells to flow through. The spiral configuration allows for controlling the cell residence time in the microfluidic channel so that cells experience sufficient treatment time. (d-e) Reproduced with permission.^[131] 2020, Biomicrofluidics. (f) A schematic of a setup that leverages surface acoustic wave-based acoustic tweezers to precisely move a microbubble to a target cell and then apply an acoustic pulse to enable inertial bubble cavitation at the cell location for sonoporation. (g, left) A stacked image showing a bubble can be translated following a complex path to a target cell. (g, right) A captured fluorescence image showing that PI (red) can successfully enter the transiently porous cell, after inertial bubble cavitation. (f-g) Reproduced with permission.^[132] 2014, Applied Physics Letters.

Figure 4.

Schematics for illustrating non-bubble-based sonoporation mechanisms. To increase the membrane permeability, different mechanisms have been used, including (a) using travelling acoustic waves to eject a cell through a nozzle orifice for sonoporation, (b) using standing acoustic waves in a half wavelength resonator to push cells to the center of the resonator for sonoporation of cells flowing through the resonator, (c) using standing acoustic waves in a quarter wavelength resonator (or a thin resonator) to push cells to the wall of the resonator for sonoporation of cells flowing through the resonator, (d) using Lamb waves (or flexural waves) propagating along a thin-wall substrate for sonoporation of cells adhered on the substrate, (e) using surface acoustic waves propagating along the surface of a substrate for sonoporation of cells adhered on the substrate, (f) using travelling bulk acoustic waves for sonoporation of adherent cells in the area above the acoustic transducer, (g) using focused bulk acoustic waves with concentrated energy for localized sonoporation of a single cell adhered on a substrate, (h) using acoustic streaming induced by hyper-frequency bulk acoustic waves for sonoporation of adherent cells in a small area near the wave source, and (i) using acoustic streaming induced by focused surface acoustic waves for sonoporation of cells in a glass well plate.

Figure 5.

Sonoporation technologies based on low-frequency bulk acoustic waves and Lamb waves. (a) A schematic of a bulk acoustic wave resonator-based sonoporation device, composed of a glass capillary coated with cargos and a piezoelectric transducer. (b) Microscopic image showing that the acoustic radiation force can push Jurkat cells on the capillary wall coated with cargos, as the cells flow through the capillary. (c) Confocal microscope images showing that Cy3-labeled DNA (TRITC channel, orange) can be delivered to a Jurkat cell. The nucleus is stained with 4,6-diamidino-2-phenylindole (DAPI, blue). (c, bottom) Fluorescence intensity plots showing the distributions of DAPI and TRITC across the cell and indicating that TRITC can be delivered to the nucleus. (d) Confocal images of mouse embryonic fibroblasts that were virally transduced with a nuclear localization signal green fluorescent protein (NLS-GFP), treated with the acoustofluidic device, fixed, and stained with DAPI (red). The NLS-GFP allowed for the observation of perturbations to the cell nuclei. The acquired overlay images indicate nuclear envelope rupture, allowing for the delivery of DNA into the nucleus. (a-d) Reproduced with permission.^[45] 2020, Proceedings of the National Academy of Sciences. (e) A Schematic of a Lamb wave-based sonoporation device that consists of a surface bonded piezoelectric disk, a polydimethylsiloxane (PDMS) chamber, and a glass substrate with adherent cells. The Lamb wave generated by the transducer can propagate along the glass substrate, temporarily permeabilize the cell, and induce microstreaming to enhance both the delivery of cargos to the cell membrane and endocytosis. Reproduced with permission.^[148] 2021, Lab on a Chip.

Figure 6.

Sonoporation technologies based on high-frequency bulk acoustic waves. (a) A schematic of a sonoporation device that utilizes lead magnesium niobate-lead titanate (PMN-PT) micropillars to generate high-frequency (~30 MHz) bulk acoustic waves for the sonoporation of cells seeded on gold electrodes above the micropillars. (b) A bright field image of fabricated 3×3 micropillars with gold electrodes as well as cells adhered on the electrodes. (c) Quantum dots (CdSe/ZnS QD) can be successfully delivered to green fluorescent protein (GFP) expressing human melanoma (LU1205) cells seeded above the micropillars. (a-c) Reproduced with permission.^[167] 2011, Biosensors and Bioelectronics. (d) A schematic of a platform that uses focused high-frequency (~150 MHz) ultrasonic waves for single-cell sonoporation. By using a stage to precisely control the ultrasonic transducer’s position, this platform can be used for the sonoporation of any target cell adhered on the petri dish. (e) An image and a schematic of the ultrasonic transducer, which relies on a curved lithium niobate (LiNbO₃) layer to generate focused ultrasonic waves with concentrated energy in a narrow ultrasonic beam. (f) Experimental results showing that two different proteins, mTurquoise2 and mCherry, that can be successfully delivered to two neighboring cells. (d-f) Reproduced with permission.^[169-170] 2016, 2017, Scientific Reports.

Figure 7.

Sonoporation technology based on acoustic streaming induced by hyper-frequency bulk acoustic waves. (a) A schematic of a setup that uses a pentagon-shaped GHz acoustic resonator to generate hyper-frequency bulk acoustic waves, which further induce acoustic streaming. The acoustic streaming can apply shear forces on cells adhered on a substrate to deform the cell for temporary membrane permeabilization. Reproduced with permission.^[178] 2021, Advanced Science. (b, left to right) A photo of a GHz acoustic resonator, a scanning electron microscopy (SEM) image of the resonant area, and a cross-section SEM image of the GHz acoustic resonator. Reproduced with permission.^{[173, 177]} 2020, ACS Applied Materials and Interfaces and 2017, Small. (c) Microscopic images showing HeLa cells with 40 kDa fluorescein isothiocyanate (FITC) dextran after sonoporation using the GHz resonator for 10 min. Reproduced with permission.^[178] 2021, Advanced Science.

Figure 8.

Surface acoustic wave-based permeabilization technologies. (a) A schematic of a platform that uses a straight interdigital transducer (IDT) on a lithium niobate (LiNbO₃) wafer to generate surface acoustic waves (SAWs) for the acoustically-mediated delivery to cells adhered on the glass bottom of a well plate. In this platform, the energy of the surface acoustic waves is coupled to the glass well plate using a fluid couplant. (b) Confocal microscopy images of HeLa cells with and without the surface acoustic wave-based sonoporation. Compared to the group without surface acoustic waves, the siRNA cargos are more efficiently delivered to the cells in the group with surface acoustic waves. (a-b) Reproduced with permission.^[149] 2018, Nanoscale. (c) A schematic of a platform that uses a focused elliptical single-phase unidirectional transducer (FE-SPUDT) on a LiNbO₃ wafer to generate focused surface acoustic waves for the permeabilization of suspension cells in a glass well plate. (d) Confocal microscopy images of Jurkat cells with and without surface acoustic wave-based sonoporation. For the group treated with surface acoustic waves, the delivered siRNA cargos are found to overlap less with the lysosomes. (c-d) Reproduced with permission.^[179] 2021, ACS Applied Bio Materials. (e) A schematic of a setup for the delivery of drug to the mucosal layer by using surface acoustic waves. (f) Microscopy images showing the distributions of fluorescein isothiocyanate (FITC) albumin (green) in multiple layers of the porcine lip tissue at different depths, after surface acoustic wave-based sonoporation. (e-f) Reproduced with permission.^[233] 2018, Lab on a Chip.

Figure 9.

Sonoporation technology relying on acoustic streaming induced by surface acoustic waves. (a) A schematic of a platform that uses acoustic streaming induced by traveling surface acoustic waves for the sonoporation of cells flowing through a microfluidic channel. (b) An acquired microscopy image showing acoustic streaming induced by traveling surface acoustic waves. (c) Experimental results showing fluorescein isothiocyanate (FITC, green) dextran can be delivered to HeLa cells. The 4,6-diamidino-2-phenylindole (DAPI, blue) stain is used for the cell nucleus. The Alexa 594 red stain is used for indicating dead cells. (a-c) Reproduced with permission.^[180] 2021, Processes.