Development of HRP-based 3D intercellular exchange assay for high throughput screening

Extracellular vesicles (EVs) play a vital role in mediating intercellular communication and regulatory processes. Using a three-dimensional (3D) intercellular exchange assay, we previously demonstrated that EVs facilitate the intercellular transfer of nanoparticles (NPs) among cells in vitro and are essential for the efficient delivery of NPs in vivo. This assay was further employed to identify small molecules capable of regulating the intercellular exchange process. However, the original assay relied on fluorescence labeling and flow cytometry for detection, which posed limitations for adaptation to high-throughput screening (HTS) platforms. In this study, we developed an enhanced intercellular exchange assay based on horseradish peroxidase (HRP) labeling. Specifically, cell-penetrating peptide-conjugated silver nanoparticles (CPP-AgNPs) were labeled with HRP, allowing signal amplification through the enzymatic reaction between HRP and its substrate. This modification significantly improved the signal-to-noise ratio (S/N) to approximately 6:1. Additionally, we optimized the gel composition within the assay system to ensure compatibility with HTS workflows. Using this HRP-based assay, we validated that LDN-214117, a previously identified agonist of intercellular exchange, significantly promoted the transfer of CPP-AgNPs between various cell pairs. Collectively, our work presents a novel, rapid, and versatile platform for identifying modulators of intercellular exchange in a high-throughput format.