DOI: 10.7274/27934785.v1
title: Characterization of Extracellular Nanocarriers Using Novel Biosensors for Diagnosis of Cancer, Cardiovascular and Alzheimer’s disease

description: The physiological origins and functions of Extracellular Nanocarriers can propel advancements in precision medicine by offering non-invasive diagnostic and therapeutic prospects for cancers, cardiovascular, and neurodegenerative diseases. However, utilizing Extracellular Nanocarriers for diagnostics face considerable challenges due to their intrinsic heterogeneity, spanning biogenesis pathways, surface protein composition, and concentration metrics. Commonly used methods such as Nanoparticle Tracking Analysis (NTA) and Nuclear Magnetic Resonance (NMR) do not provide information about their proteomic subfractions, including active proteins and enzymes involved in essential pathways and functions. Size constraints limit the efficacy of flow cytometry for small EVs and LPs, while ultracentrifugation isolation is hampered by co-elution with non-target entities.

To address these challenges, we developed two novel platforms: Ion Exchange Membrane Sensors (IEMS) and Immunojanus Particles (IJP). These platforms provide comprehensive solutions for the characterization and quantification of extracellular nanocarriers without the need for sample pretreatment or isolation.

IEMS is a charge-based electrokinetic membrane sensor that utilizes highly negative nanoparticles to produce a signal, and is also able to overcome the issues of interference, long incubation times, sensitivity, and normalization in using Extracellular Nanocarriers for diagnostics from raw plasma. This approach achieves a universal standard normalization curve despite their heterogeneities. Utilizing IEMS, we developed an enzyme-free IEM platform for the quantification of PON1-HDL in plasma within 60 minutes, with a sub-picomolar limit of detection and a 3–4 log dynamic range. Using IEMs, we reported a study on human plasma PON1-HDL as a cardiovascular risk marker, achieving an AUC of ~0.99, significantly outperforming current cholesterol and triglyceride tests. Validation for a larger cohort can establish PON1-HDL as a biomarker with the potential to reshape the cardiovascular landscape.

Furthermore, we present a quantitative sandwich immunoassay for CD63 EVs and a constituent surface cargo, EGFR and its activity state. This assay offers a sensitive, selective, fluorophore-free, and rapid alternative to current EV-based diagnostic methods. The sensing design employs a charge-gating strategy with a hydrophilic anion exchange membrane functionalized with capture antibodies and charged silica nanoparticle reporters. This design minimizes interference from dispersed proteins, enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a limit of detection of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay allows accurate quantification of the EV marker, CD63, with colocalized EGFR. Analysis of untreated clinical samples of Glioblastoma demonstrated this platform's efficacy, with an AUC of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Moreover, we explore supermeres, recently identified as lipid membrane-free nanoparticles, focusing on their distinct surface charge and protein composition. We developed an IEM platform to characterize supermeres efficiently without isolation steps, achieving a detection limit of 10^6—10^7 supermeres/mL. Validation with ultracentrifugation and surface plasmon resonance (UC+SPR) confirmed the specificity and robustness of IEMs. Supermeres demonstrated superior diagnostic potential for colorectal cancer compared to sEVs and exomeres, with significant reductions in supermere concentration post-resection surgery. These findings underscore the critical need for detailed supermere characterization and highlight their superiority over sEVs in diagnostic applications.

We also developed Immunojanus Particles (IJPs), a novel method for the direct detection of sEVs in less than an hour without isolation. The design incorporates fluorescent and non-fluorescent halves, utilizing rotational Brownian motion to detect captured sEVs through changes in the blinking rate, without interference from smaller dispersed proteins. We demonstrate a detection limit of 2E5 sEVs/mL with low sample volumes and the capability to characterize sEVs directly from plasma, serum, cell culture media, and urine. In a pilot study involving 87 subjects, including individuals with colorectal cancer, pancreatic ductal adenocarcinoma, glioblastoma, Alzheimer's disease, and healthy controls, our method accurately identified the type of disease with a high AUC of 0.90-0.99 in a blind setting. Compared with the orthogonal ultracentrifugation plus surface plasmon resonance (UC+SPR) method that requires about 24 hours, the sensitivity and dynamic range of IJP are better by 2 logs.