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

Xian Wu1, Xiangxiang Hu1, Yiqin Li1

  • 1Department of Pharmaceutics, School of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.

Journal of Pharmaceutical Sciences
|December 5, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed a new high-throughput assay to study how cells communicate using extracellular vesicles (EVs). This enhanced method uses horseradish peroxidase (HRP) for sensitive detection of nanoparticle transfer between cells.

Keywords:
Drug deliveryExtracellular vesicleHigh throughput screeningHorseradish peroxidaseIntercellular exchangeNanoparticles

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Area of Science:

  • Cell Biology
  • Nanotechnology
  • Biochemistry

Background:

  • Extracellular vesicles (EVs) are crucial for intercellular communication and regulating cellular processes.
  • Previous 3D assays showed EVs mediate nanoparticle (NP) transfer, aiding in vitro and in vivo delivery.
  • Limitations in fluorescence-based detection hindered adaptation of previous assays for high-throughput screening (HTS).

Purpose of the Study:

  • To develop an enhanced, HTS-compatible assay for studying intercellular exchange.
  • To identify small molecules that modulate EV-mediated intercellular transfer of nanoparticles.
  • To improve the sensitivity and adaptability of existing intercellular exchange assays.

Main Methods:

  • Developed a novel assay using horseradish peroxidase (HRP) labeling for signal amplification.
  • Utilized cell-penetrating peptide-conjugated silver nanoparticles (CPP-AgNPs) labeled with HRP.
  • Optimized gel composition for HTS compatibility and achieved an improved signal-to-noise ratio (approx. 6:1).

Main Results:

  • The HRP-based assay demonstrated significantly improved signal-to-noise ratio compared to previous methods.
  • Successfully validated LDN-214117 as an agonist that promotes CPP-AgNP transfer between cell pairs.
  • The assay proved effective in identifying modulators of intercellular exchange in various cell types.

Conclusions:

  • Presents a novel, rapid, and versatile HRP-based platform for HTS of intercellular exchange modulators.
  • This enhanced assay overcomes limitations of previous methods, enabling efficient screening.
  • The developed platform facilitates the discovery of compounds that regulate EV-mediated cellular communication and NP delivery.