Ultrasound-Mediated Plasmid Delivery by Stable Oscillation of Linear Array Microbubbles in Microfluidic Channels

Intracellular delivery is among the biotechnological methods used for studying cell therapy, drug delivery, and biomanufacturing. Currently, ultrasound-mediated intracellular delivery with microfluidics has the advantage of being controllable and efficient. However, the conventional method of sonoporation through the generation of microbubbles in cavities faces challenges in maintaining optimal microbubble size and shape. In this article, we present a device based on linear array microbubbles to induce acoustic streaming, which can regulate cell membrane permeability in a stable and controllable method. To solve these challenges, high-resolution photosensitive resin 3-D printing is used to fabricate vertical array T-junction structures with significant aspect ratios for the generation of microbubbles. When microbubble volume changes due to environmental influences, the size of the microbubble cap remains constant due to the constraints of microchannel width, thus reducing the adverse effects of microbubble size variations and ensuring stable oscillation. Then cells are captured and trapped on the surface of microbubbles under the combined effects of oscillating microbubble-induced secondary acoustic radiation force and acoustic streaming, ultimately leading to alterations in the permeability of the cell membrane under shear stress. The experiments show that the delivery efficiency of 293T cells is 93.2% <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\pm ~2.3$ </tex-math></inline-formula>% while maintaining high cell viability at a driving voltage of 30 V. Furthermore, we have successfully delivered a plasmid encoding green fluorescent protein (8.2 kbp) to the cells, and the experimental results show a transfection efficiency of 30.7% <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\pm ~2.8$ </tex-math></inline-formula>%. This novel acoustofluidic method achieves rapid, reusable intracellular delivery, and may have utility in microfluidic chip applications.