{
  "abstract": "Background Extracellular vesicles (EVs) have emerged as important mediators of both health and disease. Given their intrinsic capacity to carry biological activity, they are also emerging as clinical biomarkers and a mechanism to deliver treatments. However, the variability in EV characterization and analysis across nanotechnology platforms poses a significant barrier for their use in clinical and research advancements. To address this, we have developed a cross-platform EV standard derived from human red blood cells (RBC) engineered with a membrane-anchored Programmable Cellular Interface (sbPCI) system (EV-CMS) which enables multiplexed detection and standardization across different analytical modalities.Methods EV-CMS is constructed as a multi-component membrane anchor that supports multi-analyte detection. To enable detection by fluorescence, nucleotide sequence and protein-based methods, EV-CMS was engineered to contain three distinct analytic features: 1) fluorescent beacon, 2) a unique nucleotide barcode, and 3) a synthetic epitope tag. The analytes within the construct can be configured to meet the requirements of a wide range of nanotechnologies. The construct is incorporated into human RBC isolated from whole blood under 42 days old. Following incorporation, EVs are generated through PKC-mediated induction, sonication, or membrane extrusion. RBC-derived vesicles are separated from free protein, lipoproteins, and debris using size exclusion chromatography. Each batch is characterized by size, label incorporation, and concentration. Long-term stability is achieved through -80°C storage and lyophilization in the presence of Trehalose. Cross-modality calibration was successfully performed using flow cytometry, quantitative PCR (qPCR), and fluorescent enzyme linked immunosorbent assay (ELISA).Results EV-CMS demonstrated stable membrane association and consistent detectability with stoichiometry between the three analytic features that enabled calibration across technologies. Moreover, dose-dependent incorporation enabled the development of an effective quantitative standard. Specific capabilities include a) enhanced specificity through detection of highly sensitive fluorophores, b) quantitative detection of the DNA barcode by qPCR, and c) paired detection of EV-CMS and endogenous markers using ELISA.Conclusions The PCI platform offers a modular and versatile solution for EV standardization by enabling multiplexed detection, quantitative yield quantification, and cross-assay validation. Its compatibility with multiple analytical technologies makes it a valuable tool for improving reproducibility, sensitivity, and comparability in EV research and diagnostics. Use cases include technology sensitivity assessment, yield tracking during EV isolation, and cross-platform benchmarking against industry standards.",
  "authors": [
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Ayomide Oloyede"
    },
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Jinchun Wang"
    },
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Andres Campos"
    },
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Siddharth Subham"
    },
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Aurora Fabry-Wood"
    },
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Colleen M OConnor"
    },
    {
      "affiliations": [
        "Stimulus Bio, Houston, TX, USA"
      ],
      "name": "Andries Zijlstra"
    }
  ],
  "title": "55 A configurable, cross-modality extracellular vesicle standard (EV-CMS) enables quantitative evaluation across the nanotechnology landscape",
  "uid": "a49a5d23-fff5-57c8-9a1f-584710b0dd44"
}