Exosome and Microvesicle Characterization
Exosomes and microvesicles are small vesicles that are shed by cells and play an integral role in intercellular communication. An exosome can be broadly defined as a 40–100 nm diameter membrane vesicle of endocytic origin released by most cells upon fusion of the multivesicular bodies with the plasma membrane—presumably as a vehicle for intercellular communication.Microvesicles are generally classed as 100 nm -1 µm vesicles that directly bud from the plasma membrane. It is their role as potential biomarkers that makes this field of research so exciting. If one can understand the message being transported by these vesicles and understand where that message is originating from, one can potentially develop a diagnostic test that can detect and perhaps predict the onset of cardiac disease, for example.
Furthermore, their ubiquitous presence in a broad range of biological and physiological processes, as well their elevated levels in blood, open avenues for the development of exosome/microvesicle-based diagnostics across a broad range of conditions.
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Figure 1. Schematic of technology
It is becoming increasingly apparent that the techniques traditionally used to both isolate and characterize biological materials are either unsuitable or have not been developed and refined for work with exosomes and microvesicles. As a result, the rate of development and discovery is hampered. Nanoparticle tracking analysis (NTA) and perhaps more importantly fluorescent nanoparticle tracking analysis (FNTA) have recently been identified by NanoSight as potentially well-suited technologies for the measurement of these particles.
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Figure 2. Light scattering from particles
What Does NTA Measure?
Nanoparticle tracking analysis is a technique that visualizes the light that is scattered from an exosome or microvesicle in liquid suspension (Figure 1). The technique can measure the size of individual exosomes as small as 30 nm particles that are visualized by the technology.
The fact that particle size is derived on a particle-by-particle basis overcomes the inherent intensity bias associated with ensemble techniques such as dynamic light scattering (DLS), which produces average particle sizes biased toward the larger brighter microvesicles within a sample.
Figure 2 shows a typical image generated by the technology, and provides a qualitative view into the sample (one can clearly see large and small vesicles within this sample), the images form the basis of analysis. Each particle is tracked, and the Brownian motion of each particle measured and size calculated.
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Figure 3. Tracks of individual particles
Figure 3 shows the tracking of each particle with the red trace denoting the Brownian walk that each particle plotted over the course of the analysis.
Possibly more important than its ability to measure particle size, the technique can also measure the concentration of particles within a preparation and can plot the number of particles within a given size class—expressed in terms of particles per mL. Figure 4shows a number versus size distribution generated by the technique.
Finally the technique can image the fluorescent signal from an appropriately labelled vesicle (range of excitation wavelength possible); particle size and concentration can be measured simultaneously while operating in fluorescence mode.
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Figure 4. Size distribution plot
Changes in exosome/microvesicle concentrations associated with the onset of disease can also be assessed. In addition, the technology offers the ability to discriminate target exosomes/microvesicles associated with a specific disease and cellular origin from the host of naturally occurring exosomes/microvesicles that are present due to the naturally occurring physiological processes within the body.
The ability to work in fluorescence mode potentially bypasses the requirement to purify clinical samples (by ultracentrifugation/size exclusion chromatography/chromatography, etc.) and avoids the potential difficulties and variables presented by the method of purification itself.
The suitability of FNTA to study syncytiotrophoblast microvesicles (STBMs) associated with preeclampsia was recently evaluated. STBMs are present in normal pregnancy but found in significantly elevated levels in preeclampsia. Researchers were able to prove the accuracy of FNTA first through particles of a known and controlled size, i.e., reference particles.
Accurate measurement of a simple mono-dispersed 400 nm bead (sized at 401 nm) was demonstrated, moving on to prove that in a bimodal population of 100 and 300 nm particles, accurate sizing could still be demonstrated in a sample that more closely mimics the natural spread of sizes that might be found in a microvesicle preparation.
Accuracy of particle counting was demonstrated (again using reference beads) in the range of 2x108 to 2x1010particles per mL, with only a slight deviation in the measured concentration from the calculated concentration. The study also measured preparations of STBMs generated by a placental perfusion showing a bimodal distribution (peaks at 100 nm and 160 nm with a tail out to 500 nm) postulated to be a mixed population of exosomes and microvesicles.
Researchers were able to fluorescently label this population using quantum dots antibody labelled to placental alkaline phosphatise (NDOG2), showing an almost identical match between the fluorescently labeled sample and the sample measured under light scatter. The distributions were subsequently confirmed by electron microscopy.
This research confirmed the ability of FNTA to both measure the size and size distribution of exosomes and microvesicles, measure their concentration, and when fluorescently labeled confirm the phenotype of the microvesicles measured, with the goal of linking the observations to a clinically relevant observation, in this case preeclampsia.
Fonte: GEN
