Sequential intracellular delivery of genetic coding molecules using an acoustic electric microfluidic platform

Intracellular delivery of genetic coding molecules is a critical step in both fundamental cell biology and the development of cell and gene therapies. With the emergence of diverse genetic coding molecules, next-generation cell engineering increasingly relies on delivering multiple types of payloads into target cells to add, remove, or modify specific cellular functions. Most existing technologies rely on co-transfection, where target molecules are mixed and delivered simultaneously. However, a major limitation of co-transfection is reduced transfection efficiency and precision due to interactions and/or competition between cargos during intracellular delivery. Here, we present the use of acoustic microstreaming for efficient sequential intracellular transfection of genetic coding molecules. In this microfluidic platform-termed acoustic-electric shear orbiting poration (AESOP)-arrays of acoustic microstreaming vortices are generated via oscillating air-liquid interfaces to trap hundreds of thousands of cells and enable the sequential delivery of target molecules into the chip without the need for external pumping. The cells are then transfected using a combination of gentle, tunable mechanical shearing and electric fields. As a proof of concept, we demonstrate that sequential transfection of plasmid DNA and Cas9 ribonucleoprotein (RNP) via AESOP increases the transfection efficiency up to 7-fold compared to co-transfection methods by eliminating the competition between cargos. Without this obstacle engendering the limitations of co-transfection, AESOP-enabled sequential delivery supports the advancement of cell and gene therapies that depend on multiplex intracellular delivery, including cancer immunotherapy, mRNA therapies, and CRISPR-Cas9 gene editing.