Small extracellular vesicles – A host for advanced bioengineering and “Trojan Horse” of non-coding RNAs

The physiological origins and functions of extracellular vesicles (EVs) and lipoproteins (LPs) propel advancements in precision medicine by offering non-invasive diagnostic and therapeutic prospects for cancers, cardiovascular, and neurodegenerative diseases. However, EV/LP diagnostics (ExoLP-Dx) face considerable challenges. Their intrinsic heterogeneity, spanning biogenesis pathways, surface protein composition, and concentration metrics complicate traditional diagnostic approaches. Commonly used methods such as nanoparticle tracking analysis, enzyme-linked immunosorbent assay, and nuclear magnetic resonance do not provide any information about their proteomic subfractions, including active proteins/enzymes involved in essential pathways/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. In this perspective, we propose a charge-based electrokinetic membrane sensor, with silica nanoparticle reporters providing salient features, that can overcome the interference, long incubation time, sensitivity, and normalization issues of ExoLP-Dx from raw plasma without needing sample pretreatment/isolation. A universal EV/LP standard curve is obtained despite their heterogeneities.

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Immunojanus Particles for low-volume and isolation-free unlabeled characterization of small Extracellular Vesicle in biofluids: Characterization of disease type by surface marker profiling

Kumar,

Sinclair,

Shi

et al. 2024

Preprint

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Small extracellular vesicles (sEVs) are vital for cellular communication and serve as critical biomarker carriers for diseases such as cancer. However, quantifying and profiling sEV surface markers presents significant challenges due to the low concentration of specific sEV-bound proteins and interference by more abundant dispersed proteins. This paper presents Immunojanus Particles (IJPs), a new method that enables the direct detection of sEVs in less than an hour without isolation. The design of IJPs incorporates fluorescent and non-fluorescent halves, utilizing rotational Brownian motion to detect captured sEVs through the change in the blinking rate, without interference from the 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 small 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 high 0.90-0.99 AUC in a blind setting. Compared with an 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.

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Small extracellular vesicles – A host for advanced bioengineering and “Trojan Horse” of non-coding RNAs

Cited by 4 publications

References 145 publications

Emerging biologic and clinical implications of miR-182-5p in gynecologic cancers

Emerging biologic and clinical implications of miR-182-5p in gynecologic cancers

Extracellular vesicle and lipoprotein diagnostics (ExoLP-Dx) with membrane sensor: A robust microfluidic platform to overcome heterogeneity

Immunojanus Particles for low-volume and isolation-free unlabeled characterization of small Extracellular Vesicle in biofluids: Characterization of disease type by surface marker profiling

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