دورية أكاديمية
أعرض في EDS

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

التفاصيل البيبلوغرافية
العنوان: A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.
المؤلفون: Kasper SH; Merck & Co., Inc. Cambridge Massachusetts USA., Otten S; Merck & Co., Inc. Cambridge Massachusetts USA., Squadroni B; Merck & Co., Inc. West Point Pennsylvania USA., Orr-Terry C; Merck & Co., Inc. Cambridge Massachusetts USA., Kuang Y; Merck & Co., Inc. Cambridge Massachusetts USA., Mussallem L; Merck & Co., Inc. West Point Pennsylvania USA., Ge L; Merck & Co., Inc. Kenilworth New Jersey USA., Yan L; Merck & Co., Inc. Kenilworth New Jersey USA., Kannan S; Agency for Science, Technology and Research (A*STAR) Singapore Singapore., Verma CS; Agency for Science, Technology and Research (A*STAR) Singapore Singapore., Brown CJ; Agency for Science, Technology and Research (A*STAR) Singapore Singapore., Johannes CW; Agency for Science, Technology and Research (A*STAR) Singapore Singapore., Lane DP; Agency for Science, Technology and Research (A*STAR) Singapore Singapore., Chandramohan A; MSD International Singapore Singapore., Partridge AW; MSD International Singapore Singapore., Roberts LR; Merck & Co., Inc. Cambridge Massachusetts USA., Josien H; Merck & Co., Inc. Kenilworth New Jersey USA., Therien AG; Merck & Co., Inc. Cambridge Massachusetts USA., Hett EC; Merck & Co., Inc. Cambridge Massachusetts USA., Howell BJ; Merck & Co., Inc. West Point Pennsylvania USA., Peier A; Merck & Co., Inc. Kenilworth New Jersey USA., Ai X; Merck & Co., Inc. Kenilworth New Jersey USA., Cassaday J; Merck & Co., Inc. West Point Pennsylvania USA.
المصدر: Bioengineering & translational medicine [Bioeng Transl Med] 2023 May 13; Vol. 8 (5), pp. e10542. Date of Electronic Publication: 2023 May 13 (Print Publication: 2023).
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: John Wiley & Sons Inc Country of Publication: United States NLM ID: 101689146 Publication Model: eCollection Cited Medium: Print ISSN: 2380-6761 (Print) Linking ISSN: 23806761 NLM ISO Abbreviation: Bioeng Transl Med Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: Hoboken, NJ : John Wiley & Sons Inc., [2016]-
مستخلص: 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.
Competing Interests: All authors that are present or former employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ or MSD International, Singapore may hold stocks and/or stock options in Merck & Co., Inc., Rahway, NJ. Srinivasaraghavan Kannan and Chandra S. Verma are co‐founders of Sinopsee Therapeutics and Aplomex; the current work does not have a conflict.
(© 2023 Merck Sharp & Dohme LLC and The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)
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فهرسة مساهمة: Keywords: automation; cell‐based assays; cyclic peptides; intracellular delivery; microfluidics; protein–protein interactions
تواريخ الأحداث: Date Created: 20230911 Latest Revision: 20230913
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC10487316
DOI: 10.1002/btm2.10542
PMID: 37693049
قاعدة البيانات: MEDLINE
الوصف
تدمد:2380-6761
DOI:10.1002/btm2.10542