Kasper Stephen H, Otten Stephanie, Squadroni Brian, Orr-Terry Cionna, Kuang Yi, Mussallem Lily, Ge Lan, Yan Lin, Kannan Srinivasaraghavan, Verma Chandra S, Brown Christopher J, Johannes Charles W, Lane David P, Chandramohan Arun, Partridge Anthony W, Roberts Lee R, Josien Hubert, Therien Alex G, Hett Erik C, Howell Bonnie J, Peier Andrea, Ai Xi, Cassaday Jason
Merck & Co., Inc. Cambridge Massachusetts USA.
Merck & Co., Inc. West Point Pennsylvania USA.
Bioeng Transl Med. 2023 May 13;8(5):e10542. doi: 10.1002/btm2.10542. eCollection 2023 Sep.
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 ( = 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.
环肽有望靶向历史上难以成药的细胞内蛋白质-蛋白质相互作用，然而，它们普遍难以穿透细胞膜，这对表征其功能构成了挑战。微流控技术的最新进展使得通过物理细胞变形（即机械穿孔）实现细胞质膜的通透化成为可能，从而在无载体和无电泳的方法中实现了不可渗透的大分子的细胞内递送。然而，通过微流控机械穿孔递送的有效载荷（如肽）数量和/或浓度受到必须预先混合细胞和有效载荷这一人工密集过程的限制。在这项工作中，我们表明细胞在微流控涡旋脱落（μVS）后会瞬间通透（持续时间为1.1 - 2.8分钟），并且在细胞经过处理/通透化后立即暴露时，较低分子量的大分子可以递送至细胞质中。为了提高筛选肽的能力，我们构建了一个系统，即分配-微流控涡旋脱落（DμVS）系统，该系统将μVS芯片与基于微孔板的在线分配相结合。为此，我们在软件驱动的反馈回路中同步了电子压力调节器、流量传感器、开关分配阀和xy运动平台。使用该系统，我们能够在几分钟内（例如，在不到2.5分钟内填满96孔板）将低微升规模的瞬时机械穿孔细胞递送至微孔板上的数百个孔中。我们使用基于点击化学和纳米BRET的细胞通透性测定法，在96孔板中验证了针对肿瘤抑制因子p53的负调节因子MDM2的不可渗透肽的递送，整个板上的递送效果良好。此外，我们证明了DμVS可用于使用96孔和384孔格式的p53报告细胞测定法，鉴定原本无细胞活性的MDM2结合肽的功能性、低微摩尔细胞活性。总体而言，DμVS可与下游细胞测定法相结合，以高通量方式研究细胞内靶点的相互作用，既用于改进构效关系研究，也用于非优化肽（或潜在的其他大分子）工具的早期生物学验证。
