Exosome RNA Exosome RNA Research & Industry News
Extracellular vesicles (EVs) are tiny membrane-bound particles released by cells into body fluids like blood and urine. They carry proteins, lipids, and RNA from their parent cells, making them powerful messengers, and promising tools for disease diagnosis and therapy. But EVs are incredibly diverse. Different cells release different types of EVs, and even the same cell can produce vesicles with different sizes and surface markers. This complexity makes it hard to analyze EVs accurately.
Researchers from University of Technology Sydney, Australia describe an improved way to measure and characterize EVs one particle at a time using a method called fluorescent nanoparticle tracking analysis (F-NTA).
Schematic diagram of the whole F-NTA workflow
A) Plasma EVs are isolated using the SEC column. The elute volume of 4.7–7.5 mL is collected to maximise the EV purity. B) EVs and other investigated particles, like lipoproteins and liposomes, are labelled in solution with fluorescent antibodies or proteins. Different fluorescent dyes, including AF488 (green) and AF647 (pink), are used for multiplexed detection. To remove excess unbound labels, the labelled samples were further processed through the same SEC column but collected at a different elution volume of 4.0–6.8 mL to maximise the EV recovery. C) F-NTA is then used to measure fluorescently labelled particles.
Why traditional methods fall short
Standard nanoparticle tracking analysis (NTA) is widely used to measure the size and number of nanoparticles like EVs by tracking how they scatter light as they move in solution. While useful, this approach cannot tell EVs apart from other particles of similar size, such as lipoproteins, or distinguish between different EV subtypes.
That’s a big limitation if you want to use EVs as biomarkers or monitor engineered vesicles for therapy.
Adding fluorescence for specificity
The team enhanced NTA by adding fluorescent labels that bind to specific proteins on the EV surface. These fluorescent antibodies allow the instrument to “see” only the EVs carrying certain markers, making it possible to identify and count specific EV subpopulations rather than just all particles.
However, free fluorescent labels floating in solution can create background noise and false signals. To solve this, the researchers combined F-NTA with size-exclusion chromatography (SEC), a simple separation method that removes unbound labels while keeping the EVs intact.
Testing the workflow
The researchers first tested their workflow using artificial liposomes and EVs from cultured human cells. This showed that SEC efficiently removed excess labels and that the fluorescent tagging worked reliably.
They then demonstrated that F-NTA could distinguish EV subtypes based on surface markers. For example, they identified EpCAM-positive EVs, which are often associated with epithelial and cancer cells, from different cell lines such as HT29 and HEK293.
Finally, they applied dual fluorescent labeling to EVs isolated from human plasma. This allowed them to measure EVs and lipoproteins at the same time, giving a clearer picture of EV purity, an important issue when working with blood samples.
To strengthen confidence in their approach, the team compared their results with total internal reflection fluorescence microscopy, another high-resolution imaging method, and found strong agreement.
Significance
This optimized F-NTA workflow provides a practical way to profile EVs at the single-particle level, measuring:
- how many EVs are present,
- their size distribution, and
- which surface markers they carry.
- Such detailed profiling is essential for:
- discovering EV-based biomarkers,
- monitoring responses to therapy, and
- ensuring quality control for engineered or therapeutic vesicles.
For researchers and developers working with EVs or exosomes, this method offers a more accurate and accessible tool to unlock their diagnostic and therapeutic potential.
