{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T09:38:59Z","timestamp":1780997939498,"version":"3.54.1"},"reference-count":50,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2023,5,13]],"date-time":"2023-05-13T00:00:00Z","timestamp":1683936000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["aiche.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Bioengineering &amp; Transla Med"],"published-print":{"date-parts":[[2023,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Cyclic peptides are poised to target historically difficult to drug intracellular protein\u2013protein 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\u2010 and electrophoretic\u2010free approaches. However, the number of payloads (e.g., peptides) and\/or concentrations delivered via microfluidic mechanoporation is limited by having to pre\u2010mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (\n                    <jats:italic>t<\/jats:italic>\n                    <jats:sub>1\/2<\/jats:sub>\n                    \u2009=\u20091.1\u20132.8\u2009min) after microfluidic vortex shedding (\u03bcVS) 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\u2010microfluidic vortex shedding (D\u03bcVS), that integrates a \u03bcVS chip with inline microplate\u2010based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on\/off dispense valve, and an x\u2010y motion platform in a software\u2010driven feedback loop. Using this system, we were able to deliver low microliter\u2010scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96\u2010well plate filled in &lt;2.5\u2009min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry\u2010 and NanoBRET\u2010based cell permeability assay in 96\u2010well format, with robust delivery across the full plate. Furthermore, we demonstrated that D\u03bcVS could be used to identify functional, low micromolar, cellular activity of otherwise cell\u2010inactive MDM2\u2010binding peptides using a p53 reporter cell assay in 96\u2010 and 384\u2010well format. Overall, D\u03bcVS can be combined with downstream cell assays to investigate intracellular target engagement in a high\u2010throughput manner, both for improving structure\u2013activity relationship efforts and for early proof\u2010of\u2010biology of non\u2010optimized peptide (or potentially other macromolecular) tools.\n                  <\/jats:p>","DOI":"10.1002\/btm2.10542","type":"journal-article","created":{"date-parts":[[2023,5,13]],"date-time":"2023-05-13T09:30:32Z","timestamp":1683970232000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A high\u2010throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell\u2010based assays"],"prefix":"10.1002","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7431-6915","authenticated-orcid":false,"given":"Stephen H.","family":"Kasper","sequence":"first","affiliation":[{"name":"Merck &amp; Co., Inc.  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