To determine the particle size distribution of samples in liquid suspension, NTA makes use of the properties of Brownian motion and light scattering. A laser is sent through the sample chamber and the particles in suspension in the path of this beam scatter light such that they can be visualized easily through a 20x magnification microscope having a camera. The camera operates at 30fps and captures a video file of the particles under Brownian motion within the field of view of approximately 100x80x10µm (Figure 1).
Figure 1. Schematic of the optical configuration used in NTA.
Particle movement is captured on a frame-by-frame basis. The proprietary NTA software identifies and tracks the center of each of the observed particles simultaneously, and determines the average distance moved by each particle in the x and y planes. This value allows the particle diffusion coefficient (Dt) to be determined from which, if the sample temperature T and solvent viscosity η are known, the sphere-equivalent hydrodynamic diameter, d, of the particles can be identified using the Stokes-Einstein equation (Equation 1).
where KB is Boltzmann’s constant.
Figure 2 shows an example of the size distribution profile generated by NTA.
Figure 2. An example of the size distribution profile generated by NTA. The modal size for this sample is found to be approximately 70 nm, with larger sized particles also present.
Since microvesicles and exosomes are present in a wide range of eukaryotic and prokaryotic organisms and play a significant role in both pathological and physiological processes, they are under intense investigation.
Based on reviews done by Gyorgy (2011), exosomes can be defined as 50 to 100nm diameter and microvesicles 100-1000nm diameter and the techniques used most frequently in their purification, isolation, detection and analysis have been detailed (Gyorgy et al, 2011).
There are different definitions available. Simpson et al (2009) has defined exosomes as 40 -100nm diameter membrane vesicles of endocytic origin released by large number of cell types on fusion of multivesicular bodies with the plasma membrane, mostly as a vehicle for cell-free intercellular communication.
Most frequently encountered descriptors are microparticles, MVs, exosomes, ectosomes, exosome-like vesicles, shed vesicles and oncosomes. Other names used include argosomes, promininosomes, P4 particles, prostasomes, and several others (Lee et al, 2011).
Platelet-derived microparticles (PMP) can be defined as heterogeneous populations of vesicles (<1µm) generated from the plasma membrane on platelet activation by a number of stimulii. Exosomes, however originate from intracellular multivesicular bodies.
PMP is also different from microparticles originating from megakaryocytes even though microparticles and PMP have identical surface markers.
It can be said that the different definitions of microvesicular bodies, be they microvesicles or exosomes, is because they have varied cellular origins through several causes and serve various functions, all of which need to be clarified still.
There are three distinct mechanisms through which microvesicles originate:
- Breakdown of dying cells into apoptotic bodies
- Blebbing of the cellular plasma membrane (ectosomes) and
- The endosomal processing and emission of plasma membrane material in the form of exosomes
Pathways involved in oncogenic transformation, microenvironmental stimulation, cellular activation, stress, or death may trigger their generation. Vesiculation events take place either at the plasma membrane (ectosomes, shed vesicles) or within endosomal structures (exosomes) (Gyorgy et al, 2011; Lee et al, 2011). Microvesicles, as confirmed by Lee et al (2011) are considered as mediators of intercellular communication since they can merge with, and transfer a repertoire of bioactive molecular content (cargo) to, recipient cells.
The key features are:
- Exosomes are found in body fluids such as amniotic fluid, urine, synovial fluid, malignant ascites, bronchoalveolar lavage fluid, breast milk, saliva and blood (Simpson et al, 2009) and many other roles have been given to exosomes as they have different molecular structures related to their construction
- Exosomes derived from breast milk may be essential for developing the infant’s immune system (Admyre et al, 2007)
- Exosomes also play an important role in cell signaling and as such exhibit a strong relationship to disease progression
- Physiological function of exosomes is still under debate
Methods of isolation and preparing microvesicles and exosomes differ greatly and such differences can have a very strong impact on any investigative results obtained. Researchers compared dynamic light scattering (DLS) and NTA and concluded that DLS sensitivity was lower in polydisperse sample types as represented by cell-derived MPs. NTA is capable of precisely sizing particles in a sample.
Ludwig and Giebel (2011) utilized both NTA and EM to size their exosome-enriched solutions, implying they mostly contained particles ranging from 80 to 160nm, whereas the same sample when documented and prepared for with EM-based technologies, appears considerably smaller.
Huang et al (2012) compared ultra-filtration, a technique that can potentially separate exosomes rapidly based on the characteristics of the physical size with more traditional ultra-centrifugation methods. The method was proved quite effective as the particle size was defined as 30 to 150nm.
Additional researches on using myristoylated alanine-rich C-kinase substrate (MARCKS) peptide as a probe to target microvesicles (Morton et al, 2012) used NTA.
Since both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is important to perform high-quality exosome purification. Several techniques for exosome purification were defined by Thery et al (2006) and discussed methods to evaluate the purity and homogeneity of the purified exosome preparations.
Present isolation protocols use a two-step differential centrifugation process. Since exosomes have a low density, they are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high-speed (200,000 x g).
Techniques for the isolation and analysis of exosomes and microvesicles has been the subject of much recent patent activity, in which NTA is used as proof of the exosomal nature of the isolates (Vlassov et al, 2013; Antes and Kwei, 2013; Jones and Knox, 2013).
In order to determine whether spin filtration with size exclusion chromatography (SEC) fractioning might represent a more scalable and reliable technique than conventional ultracentrifugation (UC), Nordin et al (2013) compared UC, spin filtration and spin filtration with sequential LC fractioning for isolation of exosomes from cell culture media. Uaing RNA and protein content analysis, Western blotting (WB), NTA and electron microscopy, it was shown that by simple spin-filtration and sequential LC fractionation, high yields of exosomes can be purified from large media volumes but more development was needed to make it the gold standard for exosome purification.
Another method, involving a new peptide having affinity for canonical heat shock proteins (HSPs) as a tool for capture and enrichment of extracellular microvesicles (eMV), was proposed by Chute et al (2013). They proved that eMVs can be purified and evaluated by protein content, and NTA was as good as other established methods of eMV isolation.
Finally, Stensballe et al (2013) reported the proteomic analysis of exosomes enriched using exosome microarray.
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