Measuring Extracellular Vesicle Stability: A New Frontier in Analytical Technology
The study of extracellular vesicles is an area that has recently become the subject of intense interest. These vesicles are apparently ubiquitous in prokaryotic and eukaryotic organisms and it is believed they have a wide role to play in many physiological and pathological processes. They are typically described either as exosomes, which are produced from the cell endosome, or microvesicles, produced by cell membrane budding. Despite increased academic and commercial interest, much of the understanding of their cellular origin, structure, functions and size is still the subject of debate, as are the preferred methods of isolation and characterization.
Fundamental to the research efforts taking place is having tools that help understand the stability of sample under study. This is essential to ensure that any differences observed during the course of experimentation are truly attributable to the test conditions and not to any inherent instability of the sample itself. Nanoparticle Tracking Analysis (NTA) has a unique application in measuring the size and concentration of exosomes. This report illustrates its use in characterizing exosome samples stored at 4oC and at room temperature for up to five days.
Analysis on an extracellular level
Extracellular vesicles play important roles in a variety of clinical conditions including cancer, infectious diseases and neurodegenerative disorders. Consequently they have been identified as potential novel targets for therapeutic intervention. Both natural and engineered exosomes also have applications in macromolecular drug delivery, particularly where there is a need to deliver drugs that have low solubility or are easily hydrolyzed. Knowing the size, concentration and behavior of extracellular vesicles in their natural environment elucidates the role these structures play in disease mechanisms and the ways in which they may be exploited for diagnostic and therapeutic applications.
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Quantification and characterization of exosomes has traditionally been a challenging task, not least because of the need to measure species that are 100 nm in diameter or smaller. Techniques such as Dynamic Light Scattering (DLS) or Scanning Electron Microscopy (SEM) provide a combination of size, concentration and electrostatic charge measurements, properties that help define functionality. However, the sensitivity of extracellular vesicles to minor changes in the sample medium or preparation makes it difficult to rationalize these results with actual behavior, and many researchers struggle to validate measurements made with these techniques. Recent developments in nanoparticle visualization technology offer greater insight into exosome behavior by combining size and concentration measurements with real-time particle visualization.
Introducing Nanoparticle Tracking Analysis
NTA uses the properties of both light scattering and Brownian motion to obtain the particle size distribution of samples in liquid suspension. As a laser beam is passed through the sample chamber 
the suspended particles scatter light in such a way that they can be visualized using a 20x magnification microscope, onto which a camera is mounted. The camera captures a video file of the particles moving under Brownian motion on a frame-by-frame basis. NTA software then simultaneously identifies and tracks the center of each observed particle, and determines the average distance it moves in the x and y planes (Figure 1). This value allows determination of the particle diffusion coefficient (Dt), from which the sphere-equivalent hydrodynamic diameter of the particles is inferred using the Stokes-Einstein equation.
Movement of the particles is also measured within a fixed field of view, enabling estimation of the scattering volume of the sample. By measuring the concentration of particles within this field of view and extrapolating to a larger volume it is possible to achieve a concentration estimation of particles per mL for any given size class, or for an overall total. The following case study illustrates how this real time data provides users withunprecedented insight into exosome stability.
Case Study: Monitoring the stability of naturally produced exosomes
Two samples of lypophilized exosomes extracted from human urine and plasma were suspended in a phosphate buffered saline (PBS) solution and prepared for NTA analysis (NanoSight LM10, Malvern Instruments, fitted with a scientific CMOS camera and 405 nm laser). Samples were stored at both room temperature and 4oC, conditions commonly used in exosome assay studies. Measurements were then taken across incubation times of 0, 60, 120, 180 and 300 minutes, as well as after 24 hours and five days.
Figure 2a and 2b show the particle size distributions taken at time zero for the plasma and urine exosomes. The exosome suspensions were found to be moderately polydisperse, as expected for samples that are biological in nature. The modal peaks were around 90 nm, however smaller peaks around 140 nm, 200 nm and 300 nm were also seen.
Figure 3 shows how the concentration and modal size of the exosomes changed over the five-day period. The urine-derived exosomes demonstrated very little variation in the size at both storage temperatures; however, the plasma-derived exosomes have decreased in size slightly at day five. Both sources of exosomes showed a decrease in concentration over the measurement period. After two hours, the plasma exosomes had only 60 percent of the initial concentration remaining, and after five days just 30 percent of the initial concentration remained. The concentration of the urinary exosomes was fairly stable for up to three hours, but fell to 50 percent of the starting concentration at 24 hours. Since the size distribution profiles for both samples do not show an increase in particle size, the loss in concentration may result from exosomes degrading into small fragments of less than 25 nm, below the detectable size for the instrument.
Overall, the exosomes from urine had a more stable size and concentration profile than those from plasma. However, the loss of integrity in both samples has potential consequences because even moderate storage times may have a detrimental effect on measurable concentration. This means that any post-formulation modification must be carried out within a relatively brief three-hour window. It also suggests that 4oC storage offers no additional benefit over maintaining at room temperature. A comprehensive review of this experimental work and the findings is presented in the Malvern Instruments application note ‘Measuring Exosome Stability with Nanoparticle Tracking Analysis’.
Furthering understanding
Understanding the behavior of extracellular vesicles in solution is essential to developing therapeutic products that meet specific performance targets. Real-time data capture provides high resolution size distributions and concentration measurements, revealing both qualitative and quantitative information with the added benefit of direct visual confirmation. The ability of the NTA to offer this capability has made it an indispensable technology for furthering understanding of extracellular vesicles.
