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Massively-Parallelized, Deterministic Mechanoporation for Intracellular Delivery.

ABSTRACT: Microfluidic intracellular delivery approaches based on plasma membrane poration have shown promise for addressing the limitations of conventional cellular engineering techniques in a wide range of applications in biology and medicine. However, the inherent stochasticity of the poration process in many of these approaches often results in a trade-off between delivery efficiency and cellular viability, thus potentially limiting their utility. Herein, we present a novel microfluidic device concept that mitigates this trade-off by providing opportunity for deterministic mechanoporation (DMP) of cells en masse. This is achieved by the impingement of each cell upon a single needle-like penetrator during aspiration-based capture, followed by diffusive influx of exogenous cargo through the resulting membrane pore, once the cells are released by reversal of flow. Massive parallelization enables high throughput operation, while single-site poration allows for delivery of small and large-molecule cargos in difficult-to-transfect cells with efficiencies and viabilities that exceed both conventional and emerging transfection techniques. As such, DMP shows promise for advancing cellular engineering practice in general and engineered cell product manufacturing in particular.

SUBMITTER: Dixit HG

PROVIDER: S-EPMC8210888 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (μVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DμVS), that integrates a μVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DμVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DμVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

Project description:Intracellular delivery of cargo molecules such as membrane-impermeable proteins or drugs is crucial for cell treatment in biological and medical applications. Recently, microfluidic mechanoporation techniques have enabled transfection of previously inaccessible cells. These techniques create transient pores in the cell membrane by shear-induced or constriction contact-based rapid cell deformation. However, cells deform and recover differently from a given extent of shear stress or compression and it is unclear how the underlying mechanical properties affect the delivery efficiency of molecules into cells. In this study, we identify cell elasticity as a key mechanical determinant of delivery efficiency leading to the development of "progressive mechanoporation" (PM), a novel mechanoporation method that improves delivery efficiency into cells of different elasticity. PM is based on a multistage cell deformation, through a combination of hydrodynamic forces that pre-deform cells followed by their contact-based compression inside a PDMS-based device controlled by a pressure-based microfluidic controller. PM allows processing of small sample volumes (about 20 μL) with high-throughput (>10 000 cells per s), while controlling both operating pressure and flow rate for a reliable and reproducible cell treatment. We find that uptake of molecules of different sizes is correlated with cell elasticity whereby delivery efficiency of small and big molecules is favoured in more compliant and stiffer cells, respectively. A possible explanation for this opposite trend is a different size, number and lifetime of opened pores. Our data demonstrates that PM reliably and reproducibly delivers impermeable cargo of the size of small molecule inhibitors such as 4 kDa FITC-dextran with >90% efficiency into cells of different mechanical properties without affecting their viability and proliferation rates. Importantly, also much larger cargos such as a >190 kDa Cas9 protein-sgRNA complex are efficiently delivered high-lighting the biological, biomedical and clinical applicability of our findings.

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Massively-Parallelized, Deterministic Mechanoporation for Intracellular Delivery.

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