Future technologies for biologics drug discovery
The introduction of biomolecules and functional nanomaterials into cells is a crucial task in diverse biological situations, including immunotherapy, genome editing, regenerative medicine, and fundamental biological studies. Traditionally, intracellular delivery is achieved by carrier-based or membrane-disruption-based techniques. Carrier-based approaches utilize reconstituted viruses (e.g., lentivirus or AAV), or liposome (e.g., Lipofectamine), and when optimized they offer effective delivery (e.g., DNA delivery for cell transfection). However, carrier-based approaches critically suffer from toxicity, low-throughput, and require time-consuming and/or labor-intensive preparation steps. Alternatively, membrane-disruption-based methods such as electroporation and microinjection create transient discontinuities on the cell membrane for target material diffusion. The physical cell membrane disruption is relatively independent of target and cell type, but they cause excessive damage to cells and suffer from limited throughput. To address these drawbacks, recent advancements in microfluidics and nanotechnologies have provided new solutions; however, identifying an ideal method that offers easy, low-cost, highly efficient, high-throughput, noninvasive, and cell type/target independent delivery, still remains challenging.
Here, we present a next-generation intracellular delivery platform termed “µ-Hydroporator,” which introduces macromolecules into any cell type, at high-throughput, in a single-step, without a vector or external apparatus. µ-Hydroporator is purely based on hydrodynamic cell deformation-restoration process, which opens the cell membrane and enables efficient transport of external target biomolecules or functional nanomaterials into the cell. In brief, the cell suspension mixed with target materials is injected into a T-junction microchannel with a micro-cavity where inertial vortices instantaneously deform cell. This rapid hydrodynamic cell deformation creates transient nanopores on the cell membrane, allowing the convective transport of foreign target molecules during the cell restoration process. Using µ-Hydroporator, we have successfully delivered diverse macromolecules (e.g., RNAs, Plasmids, DNAs, DNA origami, CRISPR-Cas9s, proteins, Q-dots, AuNPs, etc.) into various cell lines including difficult-to-transfect primary cell lines such as stem and immune cells, achieving highly efficient intracellular delivery (< 98%) in a high-throughput manner (~1,600,000 cells/min) while maintaining high cell viability (< 95%). Unlike traditional methods that rely on external apparatus, and/or chemical modification of target molecules, µ-Hydroporator only requires a syringe pump (not even a microscope!). This permits easy, robust and simple operation and cost-reduction from not requiring a skilled technician and instrument. We firmly believe that the reported µ-Hydroporator will establish a new paradigm in intracellular delivery, which will immensely benefit cellular engineering research and industry.