A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays
Abstract 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 membran...
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-09-01
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| Series: | Bioengineering & Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/btm2.10542 |
| _version_ | 1827821902217871360 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Stephen H. Kasper |
| collection | DOAJ |
| description | Abstract 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 (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. |
| first_indexed | 2024-03-12T01:51:11Z |
| format | Article |
| id | doaj.art-4ede5586001347c2a4d447e4b2a014ec |
| institution | Directory Open Access Journal |
| issn | 2380-6761 |
| language | English |
| last_indexed | 2024-03-12T01:51:11Z |
| publishDate | 2023-09-01 |
| publisher | Wiley |
| record_format | Article |
| series | Bioengineering & Translational Medicine |
| spelling | doaj.art-4ede5586001347c2a4d447e4b2a014ec2023-09-08T13:29:53ZengWileyBioengineering & Translational Medicine2380-67612023-09-0185n/an/a10.1002/btm2.10542A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assaysStephen H. Kasper0Stephanie Otten1Brian Squadroni2Cionna Orr‐Terry3Yi Kuang4Lily Mussallem5Lan Ge6Lin Yan7Srinivasaraghavan Kannan8Chandra S. Verma9Christopher J. Brown10Charles W. Johannes11David P. Lane12Arun Chandramohan13Anthony W. Partridge14Lee R. Roberts15Hubert Josien16Alex G. Therien17Erik C. Hett18Bonnie J. Howell19Andrea Peier20Xi Ai21Jason Cassaday22Merck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. West Point Pennsylvania USAMerck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. West Point Pennsylvania USAMerck & Co., Inc. Kenilworth New Jersey USAMerck & Co., Inc. Kenilworth New Jersey USAAgency for Science, Technology and Research (A*STAR) Singapore SingaporeAgency for Science, Technology and Research (A*STAR) Singapore SingaporeAgency for Science, Technology and Research (A*STAR) Singapore SingaporeAgency for Science, Technology and Research (A*STAR) Singapore SingaporeAgency for Science, Technology and Research (A*STAR) Singapore SingaporeMSD International Singapore SingaporeMSD International Singapore SingaporeMerck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. Kenilworth New Jersey USAMerck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. Cambridge Massachusetts USAMerck & Co., Inc. West Point Pennsylvania USAMerck & Co., Inc. Kenilworth New Jersey USAMerck & Co., Inc. Kenilworth New Jersey USAMerck & Co., Inc. West Point Pennsylvania USAAbstract 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 (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.https://doi.org/10.1002/btm2.10542automationcell‐based assayscyclic peptidesintracellular deliverymicrofluidicsprotein–protein interactions |
| spellingShingle | 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 A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays Bioengineering & Translational Medicine automation cell‐based assays cyclic peptides intracellular delivery microfluidics protein–protein interactions |
| title | A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays |
| title_full | A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays |
| title_fullStr | A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays |
| title_full_unstemmed | A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays |
| title_short | A high‐throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell‐based assays |
| title_sort | high throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell based assays |
| topic | automation cell‐based assays cyclic peptides intracellular delivery microfluidics protein–protein interactions |
| url | https://doi.org/10.1002/btm2.10542 |
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