A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays

A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays

Bioengineering & Translational Medicine

10.1002/btm2.10542

http://dx.doi.org/10.1002/btm2.10542

Stephen H. Kasper, Stephanie Otten, Brian Squadroni, Cionna Orr‐Terry, Yi Kuang, Lily Mussallem, Lan Ge, Lin Yan, Srinivasaraghavan Kannan, Chandra S. Verma, Christopher J. Brown, Charles W. Johannes, David P. Lane, Arun Chandramohan, Anthony W. Partridge, Lee R. Roberts, Hubert Josien, Alex G. Therien, Erik C. Hett, Bonnie J. Howell, Andrea Peier, Xi Ai, Jason Cassaday

13 May 2023

Kasper, S. H., Otten, S., Squadroni, B., Orr‐Terry, C., Kuang, Y., Mussallem, L., Ge, L., Yan, L., Kannan, S., Verma, C. S., Brown, C. J., Johannes, C. W., Lane, D. P., Chandramohan, A., Partridge, A. W., Roberts, L. R., Josien, H., Therien, A. G., Hett, E. C., … Cassaday, J. (2023). A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays. Bioengineering & Translational Medicine, 8(5). Portico. https://doi.org/10.1002/btm2.10542

AbstractCyclic 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 (t1/2 = 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.

Attribution 4.0 International (CC BY 4.0)

This research is supported by core funding from: Bioinformatics Institute (BII)
Grant Reference no. : NA

https://oar.a-star.edu.sg/communities-collections/articles/20668

2380-6761

Bioinformatics Institute

https://doi.org/10.1002/btm2.10542