A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays Article Swipe
Stephen H. Kasper
,
Stephanie Otten
,
Brian Squadroni
,
Cionna Orr‐Terry
,
Yi Kuang
,
Lily Mussallem
,
Lan Ge
,
Lin Yan
,
Srinivasaraghavan Kannan
,
Chandra 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
·
YOU?
·
· 2023
· Open Access
·
· DOI: https://doi.org/10.1002/btm2.10542
YOU?
·
· 2023
· Open Access
·
· DOI: https://doi.org/10.1002/btm2.10542
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.
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- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1002/btm2.10542
- https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/btm2.10542
- OA Status
- gold
- Cited By
- 3
- References
- 52
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4376503689
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
-
https://openalex.org/W4376503689Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1002/btm2.10542Digital Object Identifier
- Title
-
A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assaysWork title
- Type
-
articleOpenAlex work type
- Language
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enPrimary language
- Publication year
-
2023Year of publication
- Publication date
-
2023-05-13Full publication date if available
- Authors
-
Stephen H. Kasper, Stephanie Otten, Brian Squadroni, Cionna Orr‐Terry, Yi Kuang, Lily Mussallem, Lan Ge, Lin Yan, Srinivasaraghavan Kannan, Chandra 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 CassadayList of authors in order
- Landing page
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https://doi.org/10.1002/btm2.10542Publisher landing page
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https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/btm2.10542Direct link to full text PDF
- Open access
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YesWhether a free full text is available
- OA status
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goldOpen access status per OpenAlex
- OA URL
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https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/btm2.10542Direct OA link when available
- Concepts
-
Microfluidics, Intracellular, Biophysics, Nanotechnology, Chemistry, Cell, Drug delivery, Cell biology, Materials science, Biochemistry, BiologyTop concepts (fields/topics) attached by OpenAlex
- Cited by
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3Total citation count in OpenAlex
- Citations by year (recent)
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2025: 2, 2023: 1Per-year citation counts (last 5 years)
- References (count)
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52Number of works referenced by this work
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10Other works algorithmically related by OpenAlex
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| referenced_works | https://openalex.org/W2531550383, https://openalex.org/W2885020841, https://openalex.org/W3169729314, https://openalex.org/W2076547599, https://openalex.org/W4200031091, https://openalex.org/W2605040111, https://openalex.org/W2945543430, https://openalex.org/W1989243288, https://openalex.org/W3212051308, https://openalex.org/W2555116045, https://openalex.org/W4220975614, https://openalex.org/W3126683889, https://openalex.org/W2951116619, https://openalex.org/W2007736465, https://openalex.org/W2134178604, https://openalex.org/W2399229449, https://openalex.org/W2072435450, https://openalex.org/W2120620959, https://openalex.org/W2094130001, https://openalex.org/W2109871942, https://openalex.org/W3126197379, https://openalex.org/W4242372416, https://openalex.org/W2937172014, https://openalex.org/W4230212633, https://openalex.org/W1981013004, https://openalex.org/W2898894410, https://openalex.org/W2112424756, https://openalex.org/W1999318360, https://openalex.org/W2766528098, https://openalex.org/W2912930263, https://openalex.org/W2790196520, https://openalex.org/W2226584831, https://openalex.org/W2743372157, https://openalex.org/W2051516831, https://openalex.org/W2095799297, https://openalex.org/W3083070838, https://openalex.org/W4220780925, https://openalex.org/W2148558870, https://openalex.org/W2889279246, https://openalex.org/W2896287573, https://openalex.org/W3155361251, https://openalex.org/W2135732933, https://openalex.org/W2949803925, https://openalex.org/W3165296617, https://openalex.org/W4289847513, https://openalex.org/W4376503689, https://openalex.org/W3128713567, https://openalex.org/W4225364558, https://openalex.org/W2018910009, https://openalex.org/W2100589388, https://openalex.org/W4205379795, https://openalex.org/W3166277383 |
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