The need to characterize different properties of nanomaterials continues to grow rapidly. Since the commercialization of the technique in 2004, Nanoparticle tracking Analysis (NTA) has become increasingly prevalent in a wide variety of different research fields and industrial applications. In this eleventh chapter of the Nanoparticle Tracking Analysis (NTA) application and usage review, we review the reports of the use of NTA in blood and pregnancy derived-exosomes and cellular vesicles research.
Extra-cellular microvesicles and exosomes are emerging as a significant class of sub-micron structures of potentially great importance in the development and diagnosis of a wide range of disease states. Found to be generated by nearly all cells and in all organisms, they are believed to contain a wide range of signaling proteins as well as genetic material of many different types.
Their detection has, to date, only been possible by electron microscopy or by classical methods of analysis such as DLS. Flow cytometry has a lower limit in practice of some 300nm and therefore cannot see the majority of microvesicular material thought to be present.
NTA offers a means by which not only can such structures be seen and concentration measured, but variations in the technique, such as fluorescence mode NTA, have allowed exosomes to be phenotyped. This multi-parameter capability, compatible with natural structures in their native environment promises to be of significant value in the elucidation of the role these structures play in disease and the ways in which they may be exploited in a diagnostic or therapeutic application.
NTA was first assessed as a method for the analysis of exosomes and microvesicles by research groups working in the Departments of Haematology & Thrombosis and Reproductive Biology at the University of Oxford, England.
The first group (Harrison 2008 and 2009 and Harrison et al., 2009a and 2009b) were primarily interested in identifying new methods by which the then current detection limit of >500nm for the popular and widespread technique of flow cytometry could be improved on, given the proportion of microparticles below this limit was then unknown. They assessed a conventional DLS instrument and NTA and showed that while both systems gave similar results on calibration quality beads over the size range 50–650nm, measurement of either purified microparticles (MPs) or diluted normal Platelet Free Plasma (PFP) NTA gave a polydisperse MP distribution (up to 1000nm) but with a predominant population from < 50nm to above 300nm. Analysis of diluted PFP in PBS (1:40–1:160) suggested that the concentration of particles was 200–260×109/L which was 1000-fold greater than previous estimates. They concluded that while both techniques were rapid and capable of measuring over the entire size range of MP sizes to be expected in biological fluids, NTA exhibited superior resolving power in broad distributions. Gardiner et al. (2011) subsequently reported on the use of NTA in the analysis of cell-derived microvesicles and nanovesicles in plasma while Yuana et al. (2010) had compared NTA to atomic force microscopy in the detection of nanosized blood microparticles.
In further extensions of these studies, Aleman et al. (2011) investigated differential contributions of monocyte- and platelet-derived microparticles towards thrombin generation, fibrin formation and stability using a variety of techniques including transmission electron microscopy, NTA, flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability, concluding that microparticles from platelets and monocytes differentially modulate clot formation, structure and stability. Fibrinolysis was also studied by Lacroix et al. (2012) in which report they suggested leukocyte- and endothelial-derived microparticles represented a circulating source for fibrinolysis.
Siljander (2011) reviewed the subject of platelet-derived microparticles (PMV), pointing out that while the molecular properties and the functional roles of the PMV are beginning to be elucidated by the rapidly evolving research interest, novel questions are simultaneously raised regarding the methodological problems and the paradoxical role of the PMV in health and disease. Aatonen et al. (2012a) analyzed the distribution of PMV sizes by NTA and EM, confirming that size distributions by the two techniques correlated well showing that over 90% of PMVs were <500nm and over 70% were <250nm irrespective of the method of activation by various physiological stimuli in comparison to Ca2+-ionophore. These findings showed that the majority of PMVs were much smaller than previously defined by flow cytometry and that the data suggest qualitative agonist-dependent differences in the PMV-specific cargo which respectively influenced their function. They concluded that novel detection methods, such as NTA, and a broader understanding of microvesicle physiology were changing the understanding of MP/exosome sizes and properties. Aatonen subsequently reviewed the role of platelet-derived microvesicles as multitalented participants in intercellular communication (Aartonen et al. 2012b) and discussed the methodological issues of PMV detection and analysis in the light of recent advances within the field, as exemplified by NTA. Most recently, Aatonen has shown that the properties of PMV depend on the activation pathway. PMV number and size distribution were analyzed by NTA and correlated with total protein, lipids and EM, respectively. They systematically compared PMVs generated by different platelet-activating pathways and found that 64-85% of MVs were <250 nm depending on activation and 95-99% were <500 nm in diameter. Only 0.5-5% of PMVs was in the 0.5-1 mm range. Their data showed that the activation pathway significantly modulated the quantity and the molecular composition (protein/lipid) of the subsequently formed PMVs, but not so much their size distribution. They concluded that the apparent scarcity of PMVs in the 0.5-1 mm range strongly necessitated the re-evaluation of data obtained with 1st generation flow cytometers, NTA having shown that the bulk of PMVs were below the detection level of such instrumentation (Aatonen et al., 2013).
The development of standardized methods for the analysis of platelet-derived extracellular vesicles (PL-EVs) in human platelet hemapheresis products was described by Orsó et al. (2012) in which resistive pulse sensing, FFF, NTA and flow cytometry were compared and found to produce varying results though NTA showed consistency of size of exosomal preparations in different media. Schmitz et al. (2012) have discussed the differential composition of subpopulations of platelet derived extracellular vesicles(PL-Evs) related to platelet senescence.
Using differential centrifugation followed by NTA analysis, Pienimaeki-Roemer et al. (2012) have shown, for the first time, that stored platelets alter glycerophospholipid and sphingolipid species in stored platelet concentrates and which are differentially transferred to newly released extracellular vesicles with implications for the effect that storage has on the activity and viability of platelet-derived extracellular vesicles.
Coagulation has been repeatedly linked to the generation and prescence of circulating microveiscles in recent years. Owens et al. (2011) reported using NTA in demonstrating that monocyte tissue factor–dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin. de Vooght et al. (2013) have recently shown that cardiopluminary bypass procedure induces extracellular vesicle formation but these vesicles are rapidly cleared in patients. Barteneva et al. (2013) reviewed current knowledge about microparticles (MPs) and provided a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. They suggested that MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. They concluded that improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics but also for the evaluation of treatment regimens and predicting disease outcomes. Cantaluppi et al. (2013) showed that anticoagulation and enhanced permeability hemodialyzers limits sepsis-associated acute kidney injury through the increased clearance of inflammatory cytokines (IL-6) and microvesicles. Using FACS, Nanosight and RNA profiling, they showed that during extracorporeal blood purification for sepsis, regional citrate anticoagulation inhibits inflammation and decreases mortality. Enhanced permeability hemodialyzers reduce plasma levels of inflammatory mediators including IL-6. Moreover, Zecher et al. (2013) showed that erythrocyte-derived microvesicles amplify systemic inflammation by thrombin-dependent activation of complement. Given that transfusion of aged blood has been associated with increased morbidity and mortality in critically ill patients and that during storage, erythrocytes release increasing numbers of microvesicles (red blood cell–derived microvesicles [RBC-MVs]), they hypothesized that RBC-MVs mediate some of the deleterious effects of aged blood transfusions. Using NTA, their results point toward a thrombin-dependent mechanism of complement activation by RBC-MVs independent of the classical, lectin, or alternative pathway. Besides identifying RBC-MVs as potential mediators of transfusion-related morbidity, they suggested their findings may be relevant for other inflammatory disorders involving intravascular microvesicle release, for example, sickle cell disease or thrombotic microangiopathy.
Similarly, Tissot et al. (2013) discussed various detection and analytical techniques, including NTA, in their assessment of blood microvesicles and their various roles and properties from proteomics to physiology. Mullier et al. (2013) have also addressed pre-analytical issues in the measurement of circulating microparticle regarding current recommendations and pending questions while Rauova and Cines (2013) have again tried to clarify the nomenclature and definitions of microparticles in blood, empahsizing that NTA and AFM, which can resolve particle sizes 1 to 3 orders of magnitude lower than the 200-nm threshold for flow cytometry, indicate that 90% of MPs are below this detection limit.
As mentioned above, NTA was also assessed as a method for the analysis of exosomes and microvesicles by research groups working in the Department of Reproductive Biology at the University of Oxford, England.
This group was interested in the use of exosomes as potential diagnostics for the condition of pre-eclampsia - a common disorder of pregnancy characterized by hypertension, proteinuria endothelial dysfunction and systemic inflammation (Sargent 2010a and 2010b, Mincheva-Nilsson and Baranov 2010). Circulating microvesicles shed by the placenta during pregnancy include syncytiotrophoblast microvesicles (STBM) and exosomes which have the potential to interact with maternal immune and endothelial cells and may have both proinflammatory and immunoregulatory effects and it was suspected that increased shedding of STBM was associated with pre-eclampsia. NTA was used alongside flow cytometry and Western blotting to confirm that excess shedding of syncytiotrophoblast vesicles in pre-eclampsia is a cause of the maternal syndrome. Alam et al. (2012) have reported that immunomodulatory molecules are secreted from the first trimester and term placenta via microvesicles.
Historically it has been believed that in pre-eclampsia the maternal symptoms are thought to be caused by the increased shedding of STBM into the maternal circulation. However, the number of STBM observed in the peripheral blood is much lower than predicted by the rate of shedding. Gardiner et al. (2012) hypothesized that this could be due to STBM binding to platelets and tested this using fluorescent NTA to show that there was no reduction in supernatant STBM following incubation in unstimulated PRP and <5% of platelets demonstrated STBM binding and that STBM-dependent activation of the haemostatic system, and the subsequent binding of STBM to and internalization by platelets, may account for the apparent scarcity of circulating STBM.
Dragovic et al. (2011a) have most recently used both flow cytometry and NTA to rapidly size, quantitate and phenotype cellular vesicles. Their interest was in the study of cellular microvesicles (100nm-1μm) and nanovesicles (< 100nm; exosomes) isolated from the placenta as they have major potential as novel biomarkers for pre-eclampsia, such microvesicles having been previously shown to be implicated in a multitude of other pathological conditions. In common with all such studies however, developments in this area were constrained by limitations in the technology available for their measurement. Dragovic and her co-workers used a commercially available flow cytometer (BD LSRI) employing side-scatter threshold and showed that they could analyze microvesicles ≥ 290nm but nothing smaller. However, they showed that NTA could measure cellular vesicles down to approximately 50nm.
Sheldon et al. (2010), in their study on notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes, used NTA to confirm that their exosomes were only slightly larger than the suggested size of exosomes (modal size of 114nm for HUVECs and 120nm for U87 cells, compared with published sizes of 50-100nm). They stated that, while sizing of exosomes by EM was subjective and limited though underestimation of size following fixing and dehydration, NTA allowed an objective and more accurate estimation of size of exosomes in a buffer such as PBS.
Furthermore, using a human placental vesicle preparation in combination with a fluorophore labelled anti-placental alkaline phosphatase antibody (NDOG2-Qdot605), flow cytometry showed that 93.5% of the vesicles labelled positive for NDOG2 with over 90% of the vesicles being below 1000nm in diameter, the main population being between 300-400nm in diameter (Dragovic et al., 2011b). However, when the same sample was studied by fluorescence NTA, the results showed a size distribution of NDOG2-labelled vesicles ranging from 50-600nm, with peaks at 100nm and 180nm. Analysis of total cellular vesicles in ultracentrifuge pellets of platelet free plasma (n=10) revealed that ~200 fold more vesicles were detectable using NTA (mean vesicle size 251±35nm) vs. flow cytometry. They concluded that these results demonstrate that NTA is more sensitive than conventional flow cytometry and greatly extended their capabilities for the analysis of microvesicles and nanovesicles (Dragovic et al., 2011b).
In a further extension to their work, the Oxford group (Alvarez-Erviti et al., 2011) used NTA to show that exosomes played a role in the transmission of alpha-synuclein, aggregation of which is known to be important in Parkinson's disease pathology. These mechanisms they elucidated were considered to potentially provide a suitable target for therapeutic intervention.
Results generated by these groups on the use of NTA for the detection of exosomes and other circulating microvesicles has been the subject of numerous presentations in different applications (Gardiner et al., 2009, 2010, 2011 and Gardiner 2011).
Knowing that flow cytometry detects only a fraction of cell-derived microvesicles and nanovesicles in plasma (PMV), Gardiner et al. (2011) recently exploited the sensitivity of NTA and showed NTA sizing is not dependent on the refractive index of the exosomes, whereas sizing of exosomes by flow cytometry requires suitable calibration. Furthermore, fluorescence NTA of PMV, achieved by labelling with a quantum dot-conjugated cell-tracker peptide, produced vesicle concentration measurements of 1.49x107/μL for PFP and 1.20x107/μL for the reconstituted pellet with >95% of all pelleted particles being labelled with the cell tracker, compared to <0.1x107/μL (<0.02%) of the vesicles in the supernatant. The latter were stained with a lipophilic dye, indicating that these were probably lipoprotein vesicles which have a similar size profile to PMV and low density. This suggested that PFP comprises a large population of low density vesicles that are not cellular derived. The presence of lipoproteins will become problematical for flow-cytometry as particle size detection limits continue to fall. The mean PMV (pelleted) concentration measurement was 1.82x107/μL (SD 0.78), with a mean modal size of 92.7nm (SD 6.9nm) and a mean median size of 107.3nm (SD 9.8). The size distribution showed that the 75% of PMV were <150nm, while <2% were greater than 300nm; the minimum size detection limit of conventional flow-cytometers. Pointing out that even the new ultra-sensitive flow-cytometers only detect between 10,000 and 40,000 PMV/μL, Gardiner concluded that NTA detects approximately 100 times more PMV than the most sensitive flow-cytometers.
More recently, Redman and his co-workers have established that there is a large ‘hidden’ population of microvesicles and nanovesicles (including exosomes) which are hard to investigate because of their size, despite being of significant importance in signaling in the maternal syndrome of pre-eclampsia. Using NTA to measure the size and concentration of syncytiotrophoblast vesicles prepared by placental perfusion, they found that the vesicles range in size from 50nm to 1μm with the majority being <500nm (which includes both exosomes and microvesicles). They speculated whether changes not only in the numbers, but also in the size (beneficial syncytiotrophoblast exosomes and harmful microvesicles) might be important in pre-eclampsia (Redman et al., 2011).
To enable the identification of the cellular origin of plasma microvesicles and exosomes, specific markers are required and in vitro derived vesicles provide the ideal platform to determine whether surface antigens specific for a particular cell type are also present on vesicles derived from them. Dragovic et al. (2012) used flow cytometry and NTA in parallel to rapidly size, quantitate and phenotype in vitro derived vesicles from platelets, red blood cells (RBCs), endothelial cells, lymphocytes, monocytes and granulocytes. They found that, while using a side-scatter threshold to determine that their standard BD LSRII flow cytometer could analyze vesicles ≥ 290nm but nothing smaller, NTA could measure cellular vesicles down to approximately 50nm in size and that NTA of platelets, RBC and endothelial-derived vesicles revealed that their size distribution differed, ranging from 50-900nm, 50-400nm and 50-650nm respectively. They showed that vesicle concentration measurements, as determined by NTA vs. flow cytometry were elevated by 75-fold for platelet vesicles, 2855-fold for RBC vesicles and >10,000 fold for endothelial vesicles. From differences in the expression of cell surface antigens on these populations (as determined by NTA vs. flow cytometry) they concluded that vesicles do not necessarily have the same antigenic repertoire as their parent cells and brings into question the use of several standard cellular markers for quantifying plasma vesicles. Dragovic et al. (2013) have most recently employed multicolor flow cytometry and NTA of extracellular vesicles in the plasma of normal pregnant and pre-eclamptic women. They developed a multi-color flow cytometry antibody panel to enumerate and phenotype STBM in relation to and other EVs in plasma from non-pregnant (NonP), normal pregnant (NormP) and women with late-onset PE using NTA to determine EV size and concentration. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller. In general EVs were elevated in pregnancy plasma apart from platelet EVs which were reduced. They also suggested there is scope to develop NTA using a fluorescence capability and could therefore be used to phenotype EVs using fluorescent antibodies.
In an attempt to standardize the characterization and enumeration of exosomes, El-Andaloussi et al. (2012) have published a standardized (3 week) protocol for the exosome-mediated delivery of siRNA in vitro and in vivo. Their protocol covers i) the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand, ii) how to purify and characterize exosomes from transfected cell supernatant (crucial steps for loading siRNA into exosomes) and finally, iii) how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. As part of the crucial characterization step, they describe a 30minute protocol for NTA analysis of exosome preparations comprising verification using NIST-traceable polystyrene microspheres, dilution to appropriate concentrations, repeat measurements for adequate statistical reproducibility and, finally, data analysis.
Hypothesizing that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation, Ng et al. (2013) have shown that exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.
Using STBM prepared by placental lobe dual perfusion and mechanical disruption and analyzing them by four color flow cytometry, NTA and Western blotting to determine vesicle size, purity and Flt-1 and endoglin (Eng) expression, Tannetta et al. (2013) have recently found differences in physical and antigenic characteristics of normal and PE placenta STBM preparations produced by placental perfusion or mechanical disruption.
Ferreira et al. (2013) have shown that human embryos release extracellular vesicles (EV) which may act as indicators of embryo quality. Using NTA to provide real-time visualization and characterization of EV size and concentration, they confirmed that EV characterization may predict recovery of slow developing embryos, which might be associated with miscarriages though the relationship between EV, embryo quality and oocyte quality requires further investigation. Nevertheless, EV may be a key way in which the embryo signals to the endometrium which may have important implications for both IVF protocols and determining the implantation potential of an embryo. Ouyang et al. (2013) have recently reviewed placenta-specific microRNA in exosomes centering on extracellular miRNAs that originate in trophoblasts and that could mediate crosstalk between the feto-placental unit and the mother during pregnancy. They specifically detailed the function of miRNAs from the primate-specific chromosome 19 miRNA clusters which are highly expressed in human placentas and in the serum of pregnant women. They also showed, using NTA, that such miRNAs are also packaged into extracellular vesicles of diverse sizes, including exosomes, and endowed non-trophoblastic cells with resistance to a variety of viruses.
Kilpinen et al. (2013) have recently shown extracellular membrane vesicles (MV) from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning. NTA-analyzed MVs resuspended with PBS following ultracentrifugation, they demonstrated by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.
Sargent (2013a and 2013b) has recently reviewed the role of MVs in pregnancy and the use of NTA in their detection.
The potential of microvesicles and exosomes as diagnostic agents, based in the presence of multiple biomarkers on and in such structures acting as early diagnostics for the onset of a wide range of disease conditions, has been described extensively.
As well as the work carried out by the University of Oxford described previously, several other groups have been studying the use of exosomes in diagnostics. Schorey (2012) proposed that exosomes can be used as diagnostic and prognostic markers in detection and treatment of prostate cancer. Thamilarasan et al. (2012) investigated the presence and differential expression of microRNA (miRNA) located in peripheral blood microvesicles of multiple sclerosis patients under treatment of interferon-beta-1b, in which they confirmed the presence of microvesicles in their preparation using two laser-based detection systems: Fluorescence-activated cell sorting (FACS) analysis and NTA.
While Gercel-Taylor et al. (2012) confirmed that cell-derived vesicles are recognized as essential components of intercellular communication, and that many disease processes are associated with their aberrant composition and release and, as such, circulating tumor-derived vesicles have major potential as biomarkers, they pointed out that the diagnostic use of exosomes is limited by the technology available for their objective characterization and measurement. In their study, they compared NTA with submicron particle analysis (SPA), DLS and EM to objectively define size distribution, number and phenotype of circulating cell-derived vesicles from ovarian cancer patients. Using vesicles isolated from ovarian cancer patients, they demonstrated that NTA could measure the size distributions of cell-derived vesicles, comparable with other analysis instrumentation. Size determinations by NTA, SPA, and DLS were more objective and complete than that obtained with the commonly used electron microscopic approach. They confirmed that NTA could also define the total vesicle concentration. Further, the use of fluorescently-labelled antibodies against specific markers with NTA allowed the determination of the phenotype of the cell-derived vesicles. Recently, using NTA to determine particle size distribution profile and concentration estimation, Marcus and Leonard (2012) have modified exosomes to interrogate cargo incorporation and Witwer (2012) has studied the influence of food intake on circulating extracellular vesicles and microRNA profiles based on the fact that circulating miRNAs have provoked intense interest as potential diagnostic or prognostic biomarkers for a wide variety of diseases, from cancers to sepsis. Dietary influence on circulating miRNA profiles - including the potential direct contribution of dietary miRNAs - has received comparatively less attention but could profoundly influence our understanding of proposed biomarkers, since qualitative and quantitative diet alterations have been reported in association with, for instance, cancers and infectious disease. The influence of food intake and fasting on circulating biological nanoparticle carriers of miRNAs was assessed by NTA), which was used to quantitate and characterize small (<500 nm) particles in serial pre- and post-prandial (1, 4, and 12 hour) plasma samples from an animal model. MicroRNAs were isolated from the same samples and profiled using low-density qPCR arrays.
The fact that exosomes and related microvesicular structures frequently contain proteins on the surface that can act as biomarkers for a wide range of disease conditions has attracted the attention of many researchers and it is widely recognized that NTA, in combination with other techniques, can be used to detect these structures at unprecedentedly early stages in disease progression. Burger et al. (2012) undertook one of the earlier reviews of this subject and in which NTA was explicitly proposed as a suitable detection methodology and Marcus and Leonard (2012) also discussed the concept of engineering exosomes to interrogate cargo incorporation. As discussed earlier, Morton et al. (2012) have discussed the potential of biomarkers on microvesicles as indicators of cancer progression.
As a platform for glioblastoma biomarker development, Akers et al. (2013) investigated the use of miR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF). While NTA was used to determine EV number and size, reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. Accordingly, in a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21 and that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development. The differential expression of miR-145 in children with Kawasaki disease was similarly studied by Shimizu et al. (2013) again using NTA for exosome quantification and sequencing of small RNA species allowed the discovery of microRNAs that may participate in Kawasaki disease pathogenesis. miR-145 may participate, along with other differentially expressed microRNAs, in regulating expression of genes in the TGF-b pathway during the acute illness suggesting a model of Kawasaki disease pathogenesis whereby miR-145 modulates TGF-b signaling in the arterial wall. Hajj et al. (2013) have also shown that astrocytes secrete a heterogeneous population of microvesicles that share many exosomal markers and contain the co-chaperone STI1 while Kowal et al. (2013) have re-assessed the distribution of exosome markers within subpopulations of extracellular vesicles.
In an extension of his earlier work, Cantaluppi and his colleagues have discussed the potential protective role of blood purification techniques in their work on the use of plasma extracellular vesicles as biomarkers and mediators of sepsis associated acute kidney injury (AKI). EV analysis was carried out by NTA, FACS, proteomic and RNA profiling during different blood purification techniques. They were able to conclude that in sepsis, plasma EVs are potential biomarkers of outcome and are involved in the pathogenetic mechanisms of AKI through the triggering of apoptosis in kidney cells. Blood purification using citrate may limit the release of EVs from activated leukocytes and platelets protecting from AKI and multiple organ failures (Cantaluppi et al., 2013).
Nieuwland (2013) also comprehensively discussed the metrological characterization of microvesicles from body fluids as non-invasive diagnostic biomarkers given the recognition that while measurement results need to be quantitative and traceable, the detection of EV is a challenge due to their small size (average diameter less than 100 nm) and heterogeneity and the complexity of body fluids. He included NTA among the state-of-the-art EV detection techniques considered. As part of a major European study (Euramet, 2011) into a multidisciplinary approach to improve the traceable characterization of MV in body fluids, he discussed methods to (a) explore, compare and develop methodologies for dimensional characterization and to measure concentration, morphology and (bio) chemical composition using techniques such as small-angle X-ray scattering, anomalous SAXS and AFM, (b) develop methods for standardized collection and handling of human body fluids and (c) select and test synthetic and biological reference materials to allow traceable calibrations of EV measurements.
Zarovni et al. (2013) have described improved immunoassays for detection and quantitative analysis of selected protein biomarkers in human plasma exosomes in order to overcome the problem of unspecific immunoreactivity that interferes with assays outcome and results in false positives or blockade of a specific signal. NTA and IR spectrometry were used to validate FACS and ELISA analysis of plasma exosomes which revealed unspecific binding of secondary Ab that hampered detection and comparison of specific target proteins in different samples. This issue was complicated by interindividual variability in immunoreactivity and exosome amount in healthy individuals and affected by exosome purification method, while unaltered by sample storage.
The potential for exosomes to be employed as drug and gene delivery vehicles has been discussed earlier (Lakhal and Wood, 2011; Maguire et al., 2012; Morton et al., 2012 and 2013; Fonsato et al., 2012; Biancone et al., 2012 and O'Loughlin et al., 2012; Yuana et al., 2012).
van Dommelen has reviewed the potential for microvesicles and exosomes to be used in drug delivery given they would appear to be capable of delivering lipids, proteins, mRNA and microRNA to change the phenotype of the receiving cells. He concludes that although a number of limiting factors in the clinical translation of the exciting research findings so far exist, it is promising for the development of a potentially novel generation of drug carriers (van Dommelen et al.,2011).
Tumor microvesicles isolated from a variety of cell lines were analyzed for exoRNA content as a function of exosome particle size distribution profile as determined by NTA (Balaj et al., 2011), from which they proposed that tumor microvesicles also carry DNA in addition to a selected set of proteins and RNAs, thus expanding the nucleic acid content of tumor microvesicles to include: elevated levels of specific coding and non-coding RNA and DNA; mutated and amplified oncogene sequences; and transposable elements. Thus, tumor microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer. In a related paper, van der Vos et al. (2011) used NTA to identify microvesicles shed by brain tumor cells in their study of the novel intercellular communication route they represent the potential physiological role of microvesicles in brain tumorigenesis.
Powis et al. (2011) suggested the capabilities of NTA may represent a significant step forward in the characterization of exosomes, allowing them to monitor the release of exosomes in the range 30-150nm after activation with a variety of immune stimuli, relevant to both normal and aberrant immune responses in a way not previously visible with flow cytometry. Most recently, Montecalvo et al. (2011) have used NTA to size exosomes during their investigation into the mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Similarly, Weisshaar et al. (2012) used TEM to visualize the exosomes isolated from autologous conditioned cell free serum (ACS ) and NTA to quantify them but both techniques matched size and concentration of 5.2 x 108/ml with a mean size of 180nm which proved to be mainly aggregates. In their study of cellular stress conditions being reflected in the protein and RNA content of endothelial cell-derived exosomes, de Jong et al. (2012) used NTA to help quantify exosome concentration, from which they could show that several proteins and mRNAs displayed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis.
Cantaluppi and his co-workers have shown that microvesicles derived from endothelial progenitor cells protect the kidney from ischemia–reperfusion injury by microRNA-dependent reprogramming of resident renal cells, indicating the potential of microvesicles to reverse acute kidney injury by paracrine mechanisms and that microvesicles released from these progenitor cells activate an angiogenic program in endothelial cells by horizontal mRNA transfer. The mean size and particle concentration values were calculated by NTA (Cantaluppi et al., 2012).
In an interestingly orthogonal study, Maguire et al. (2012) have recently shown that Adeno-associated virus (AAV) vectors, known to exhibit remarkable efficiency for gene delivery to cultured cells and in animal models of human disease, show limitations after intravenous transfer, including off-target gene delivery (e.g. liver) and low transduction of target tissue. They have, however, shown that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). These were visualized by EM and their size and concentration routinely determined by NTA allowing their purification for future use as a unique entity which offers a promising strategy to improve gene delivery.
In describing a systematic approach to exosome-based translational nanomedicine, Hood and Wickline (2012) compared DLS with NTA, concluding that NTA has an advantage over DLS in that it is multimodal. Furthermore, they confirmed that if fluorescent antibody labelling of exosomes is combined with NTA, the result is a highly effective means to identify exosome subpopulations and pursue exosome biomarker studies, but the use of fluorescent antibody-based NTA is not appropriate for the production of exosome-based semi-synthetic nanovesicles (EBSSNs) because of its inability to discern single vesicles from vesicle clumps, whose formation is exacerbated by antibody-mediated vesicle cross linking. They suggested it is important to size exosomes prior to pelleting as described above or develop new methods to carefully disaggregate exosomes prior to sizing.
Recently, Vojtech et al. (2012) have studied the effect of exosomes in semen on mucosal immunity to viral pathogens in which they used NTA to measure concentration of seminal exosomes and found them to number between 4.7x1011 and 1.2x1012/ml (equivalent to 2-34 trillion per ejaculate). Weisshaar (2012) has also studied the anti-inflammatory and anti-microbial activity of exosomes isolated from autologous conditioned cell free serum.
Yeo et al. (2013) have recently reviewed the development of mesenchymal stem cell (MSC) exosomes as a potential first-in-class therapeutic given the rationale for MSC therapeutic efficacy remains tenuous and is increasingly rationalized on a secretion rather than differentiation mechanism and in which recent studies have identified exosomes as the secreted agent.
According to Biancone et al. (2012), several studies have demonstrated that mesenchymal stem cells have the capacity to reverse acute and chronic kidney injury in different experimental models by paracrine mechanisms. They discussed whether MVs released from mesenchymal stem cells have the potential to be exploited in novel therapeutic approaches in regenerative medicine to repair damaged tissues, as an alternative to stem cell-based therapy. Biancone et al. (2012) subsequently pointed out that this paracrine action may be accounted for, at least in part, by microvesicles (MVs) released from mesenchymal stem cells, resulting in a horizontal transfer of mRNA, microRNA and proteins. MVs, released as exosomes from the endosomal compartment, or as shedding vesicles from the cell surface, are now recognized as being an integral component of the intercellular microenvironment. By acting as vehicles for information transfer, MVs play a pivotal role in cell-to-cell communication. Both these studies have used NTA as the means by which MVs can be detected and enumerated in support of such studies. Iglesias et al. (2012) have also reported, using NTA-based supported data, that stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.
Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and, because the Protein Kinase C (PKC) family controls the sub-cellular distribution of EAATs, Gosselin et al. (2013) have explored whether PKCs drive EAATs into eMV. While Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment, NTA revealed that PKC activation did not change particle concentration which raises the possibility that microvesicular EAAT-1 may exert extracellular function.
Tetraspanin molecules are commonly used as protein markers of extracellular vesicles, although their role in the unexplored mechanisms of cargo selection into exosomes has not been addressed. For that purpose, Perez-Hernandez et al. (2013) have characterized the intracellular tetraspanin-enriched microdomains (TEM) interactome by high-throughput mass-spectrometry, in both human lymphoblasts and their derived exosomes, revealing a clear pattern of interaction networks. Using NTA to measure concentration and size of their extracellular vesicles, their data provided evidence that insertion into TEMs may be necessary for protein inclusion into the exosome structure.
The loading of siRNA into extracellular vesicles by electroporation has been studied by Kooijmans et al. (2013). Using NTA to determine aggregate formation (while loading efficiency was determined by fluorescence spectroscopy (FS) and quantitative reverse transcription PCR), they showed that electroporation of EVs with siRNA is accompanied with extensive siRNA aggregate formation, which may cause severe overestimation of the amount of siRNA actually loaded into EVs. The low loading efficiency under precipitate-reducing conditions highlights the necessity for more efficient loading methods.
Verhage et al. (2013) have looked at cardiomyocyte progenitor cells (CMPCs) type stem cells for the regeneration of myocardium and the role that secretion of paracrine signals by CMPCs may play in enhancing endogenous repair. Vesicles derived from CMPCs showed presence of several exosomal characteristics, including an average NTA-determined size of 85 nm and expression of exosomal markers flottilin-1 and CD9 around a density of 1.12 to 1.16 g/ml. They concluded that the constitutive secretion of exosomes by CMPCs was shown to be a distinct component of the paracrine signaling pathway by inducing angiogenesis both in vitro as in vivo, and implicates the use of exosomes as potential therapeutic for cardiac regeneration. Yellon et al. (2013) have also reported that rat plasma exosomes are cardioprotective.
In order to better understand the relationship between exosomes and infection, Mantel et al. (2013) have studied the production of microvesicles derived from the red blood cells (RBCs) from humans and mice infected with different Plasmodium strains. NTA ensured correct identification of microvesicles in their study which allowed them to show that malaria-infected erythrocyte-derived microvesicles mediated cellular communication within the parasite population and with the host immune system. NTA sizing of extracellular vesicles (EV) also allowed Kang et al. (2013) to show that EV derived from gut microbiota, especially Akkermansia muciniphila, protect the progression of dextran sulphate sodium-induced colitis.
Given that a small subset of unconventional human T cells, gdT cells, play complex roles in host immunity against pathogens and cancers and play a key function in antigen presentation, comparable to that of dendritic cells, Welton et al. (2013) used cryo-EM and NTA to reveal the majority of extracellular vesicles secreted by these cells to be <100 nm, with very small electron dense (30 nm) vesicles being the most prevalent. They used flow cytometry to show the expression of gdT cell receptor, MHC Class I and II and tetraspanins while multivesicular body and lysosomal markers, TSG101 and LAMP2, were observed by Western blot. They accordingly were able to describe a previously unstudied exosome source, showing that gdT cells produce a heterogeneous population of mainly small and dense vesicles phenotypically similar to exosomes. These vesicles exhibited peptide-presenting function, activating chemokine secretion by antigen-specific abCD8 T cells with a potential to be therapeutically valuable.
Finally, NTA was discussed in a recent review of the current knowledge of exosome composition, biological functions and diagnostic and therapeutic potential (Vlassov et al., 2013).
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