Unlocking the Secrets of Intracellular Communication: NanoSight Pro Masters Extracellular Vesicle Analysis

Extracellular vesicles (EVs) have emerged as crucial biomarkers in various biological and medical research fields due to their ability to transport molecular signals between cells. This article highlights the significance of EVs and how the NanoSight Pro can be employed to explore various labeling approaches for unraveling different characteristics of EVs.

Understanding Extracellular Vesicles

Extracellular vesicles are small, membrane-bound particles released by cells into the extracellular environment and include exosomes and microvesicles each differing in size, origin, and function. Initially thought to be cellular waste, research has unveiled their pivotal role in influencing processes such as immune responses and tumor progression. Due to their presence in bodily fluids, EVs make promising biomarkers for diseases, ranging from cancer to neurodegenerative disorders and inflammatory conditions.

Importance of Characterising EVs

Characterising EVs involves determining their concentration, size distribution, and biochemical composition. The change in concentration of released EVs can indicate an increase in communication, which has diagnostic potential. The composition of EVs, including their cargo molecules such as proteins, lipids, and nucleic acids (including RNA and DNA), can reflect the physiological or pathological state of the cell they originated from. The ability to detect and quantify RNA within EVs serves as a valuable tool for QC to measure success of encapsulation for therapeutic applications.

NanoSight Pro: An Advanced Tool for EV Analysis

The NanoSight Pro is an essential tool that is increasingly utilised in EV characterisation, owing to its ability to track individual particles for size, size distribution and concentration measurements, as well as its fluorescent mode, which facilitates exosome detection through fluorescent labeling. It employs Nanoparticle tracking analysis (NTA) technology, which combines laser light scattering and Brownian motion to measure particle size and concentration simultaneously.

Quantify single EVs with confidence

NanoSight Pro provides high sensitivity detection and analysis of nanoparticles as small as 10 nm. This allows researchers to quickly gain comprehensive insights into the heterogeneity of EV populations. This is particularly important for studying smaller EVs, which are often challenging to characterise with traditional methods. Using real-time analysis, NanoSight Pro enables dynamic processes such as EV release and uptake to be monitored, which is valuable for time-sensitive studies and kinetic analyses.

In the NanoSight Pro, the laser is intermittently deactivated, and fresh sample is introduced between measurements to mitigate the effects of photobleaching. These novel features, combined with the trained neural network for particle identification and the continuous and controlled pumping of samples during the measurement, are essential for enhancing and ensuring the quality of fluorescence detection. The machine learning algorithms and smart tools built into the software fuel productivity and increase confidence, while eliminating any concerns about subjectivity or human error.

A standout feature of the NanoSight Pro is its fluorescence detection capability. By using specific dyes such as CellMask™ Orange (CMO) and ExoGlow™, researchers can label and track EVs with high specificity. This enables the differentiation of various EV populations within a sample. In addition, simultaneous detection of multiple fluorescence signals is particularly beneficial for studying EVs derived from different cell types or carrying distinct biomarkers.

Using NanoSight Pro, biomarker detection methods developed using two different dyes, Alexa Fluor^® 488 (488 nm excitation wavelength) and Pe/Cyanine (either 488 or 532 nm excitation wavelength), have confirmed the presence of CD9, CD63, and CD81 biomarkers. In addition, a novel cargo detection and quantification method has been established, using the Quant-iT™ RiboGreen dye, revealing RNA labeling inside EVs.

Figure 1: Particle concentrations in scatter and fluorescence measurements for exosomes labeled with Alexa Fluro 488 and PE/Cyanine7 anti-CD9, anti-CD63 and anti-CD81 antibody dyes measured using fluorescence NTA on NanoSight Pro.

The NanoSight Pro, with its specialised fluorescence mode, holds limitless capacity in advancing R&D efforts concerning EVs for both diagnostic and treatment applications. As the field of EV research continues to grow, NanoSight Pro will be instrumental in unlocking new insights and advancing the development of new vaccines and drug delivery systems.

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_{Malvern Panalytical. “Extracellular Vesicles and Nanoparticle Tracking Analysis.” https://www.malvernpanalytical.com/en/learn/knowledge-center/whitepapers/wp240326-extracellular-vesicles-nta [Accessed 25 July 2024].}

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