Exosomes: Exploiting the Diagnostic and Therapeutic Potential of Nature’s Biological Nanoparticles
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Research on exosomes and other forms of extracellular vesicles (EVs) have rapidly expanded over the last two decades. These lipid-enclosed, nanoscale messengers are released from cells packed with diverse cargo and can travel long distances to modify the function of target cells. Found in abundant quantities in biological fluids like blood, there is great clinical interest in using EVs as diagnostic markers or altering their properties for therapeutic delivery. Tune in to find out more about what exosomes are, how researchers study them, and what challenges remain. This talk will highlight multi-laser nanoparticle tracking analysis (NTA) with the ViewSizer 3000 and what it offers in exosome research. View recorded webinars: http://bit.ly/particlewebinars
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Exosomes: Exploiting the Diagnostic and Therapeutic Potential of Nature’s Biological Nanoparticles
- 1. Exosomes: Exploiting the Diagnostic and Therapeutic Potential of Nature’s Biological Nanoparticles June 11, 2020 HORIBA Webinar Particle Characterization Series Niaz Zafar Khan MD/PhD Candidate University of Maryland School of Medicine Medical Scientist Training Program Program in Neuroscience
- 2. The Lab for the Study of Central Nervous System Injury Dr. Alan Faden Dr. Bogdan Stoica Dr. Junfang Wu Dr. Marta Lipinski Dr. David Loane
- 3. Loane and Faden (2010), Trends Pharmacol Sci Primary Injury Secondary Injury Pathophysiology of CNS Injury Secondary injury contributes to progressive cell loss after neurotrauma.
- 4. Loane & Kumar (2015), Exp. Neurol. Neuroinflammation after CNS Injury Pro-inflammatory, microglial activation persists months after neurotrauma.
- 5. Kourembanas et al. (2015), Annu Rev Physiol Extracellular Vesicles (EVs) EVs are biological messengers that can transfer proteins, lipids, and nucleic acids.
- 6. EVs were once thought to be just “dust”.
- 7. Implications: (1) Biomarker Potential (2) Long-distance communication between organ systems (3) EVs as drug delivery carriers Quinn et al. (2015), J Extracell Vesicles EVs in Biological Fluids
- 8. Shah et al. (2018), N Engl J Med EVs as Biomarkers for Disease
- 9. Holm et al. (2018), Trends Neurosci Bidirectional Cellular Crosstalk through EVs
- 10. Kumar et al. (2017), J. Neuroinflammation EVs and Neuroinflammation after TBI Blood microparticles (MPs) of microglial-origin analyzed by flow cytometry after TBI. Pro-inflammatory microglia release MPs that can promote inflammatory activation. Kumar et al. (2017), J. Neuroinflammation
- 11. • Advantages over synthetic nanoparticle systems may include: – Can be Personalized – Long circulating half-life – Reduced immunogenicity – Inherent targeting capabilities – Ability to cross biological barriers such as the blood- brain barrier Rufino-Ramos et al. (2017), J Controlled Release EVs as Therapeutics
- 12. 0 500 1000 1500 2000 2500 EV Research is Skyrocketing! 2000 2020 2010 NumberofPublications withEV-relatedtermsinTitle PubMed Filter for EVs
- 13. Need for Standardization in EV Research
- 14. Sample Collection Sample Storage EV Isolation -”Omic” analyses Transcriptomic Genomic Proteomic Lipidomic EV Characterization Quantitative and Qualitative Functional Assays Pre-analytical variables Workflow in EV Research 1 2 3
- 15. EV Nomenclature Extracellular vesicle (EV) is the umbrella term endorsed by ISEV ISEV (2014), J Extracell Vesicles
- 16. Rufino-Ramos et al. (2017), J Controlled Release CD9, CD63, CD81 <50-200nm 50-1000nm Classification of EVs Original definitions based on biogenesis >1000nm /Microparticles
- 17. Classification of EVs Original definitions based on biogenesis and physical separation Centrifugation Steps 1. 1000g, 10 min 2. 2000g, 20 min 3. 10,000g, 30 min 4. 100,000g, 2 hr Cell Debris Apoptotic Bodies Microvesicles Traditional definitions Exosomes Current ISEV recommendations • No current isolation protocol can purify based on biogenetic origin • Size is not an appropriate defining feature alone • Describe EVs based on • Physical characteristics • Size: Large EVs, medium EVs, small EVs • Biochemical characteristics • Cell origin or stimulus condition
- 18. Large EV with “cup- shaped” indentation Likely small lipoprotein structure EV Isolation Ultracentrifugation has been the gold-standard procedure but lacks purity 500 nmCredit: Dr. Ru-ching Hsia, UMSOM EM Core Facility
- 19. EV Isolation Isolation methods have differing levels of recovery and purity https://www.nanoviewbio.com/characterize
- 20. Sample Collection Sample Storage EV Isolation -”Omic” analyses Transcriptomic Genomic Proteomic Lipidomic EV Characterization Quantitative and Qualitative Functional Assays Pre-analytical variables Workflow in EV Research 1 2 3
- 21. EV Characterization Toolbox Hartjes et al. (2019), Bioengineering (Basel) Considerations in evaluating technology: • EV Size • EV Count • EV Phenotype • EV Morphology/Visualization • Single EV or Bulk analysis? • Isolation or Direct detection?
- 22. Nanoparticle Tracking Analysis Single Particle Interferometric Reflectance Imaging (SPIRI) Flow Cytometry EV Characterization Toolbox
- 23. Flow Cytometry provides excellent phenotyping capability but size resolution is a limitation, especially for small EVs 1300 Si 880 Si 590 Si 180 Si 240 Si 300 Si F110 PS F500 PS Abbreviations F – Fluorescent # – size in nm PS – Polystyrene Si – Silica SSC-A FITC-A Phenotyping EVs by Flow Cytometry Erdbrügger et al. (2017), Cytometry A Credit: Dr. Xiaoxuan Fan, UMSOM Flow Core Facility
- 24. 1300 Si 880 Si 590 Si 180 Si 240 Si 300 Si F110 PS F500 PS Abbreviations F – Fluorescent # – size in nm PS – Polystyrene Si – Silica SSC-A FITC-A Phenotyping EVs by Flow Cytometry Sizing with beads to estimate EV size is inaccurate due to biophysical differences van der Pol et al. (2018), Nanomedicine
- 25. Flow Cytometry provides excellent phenotyping capability but size resolution is a limitation, especially for small EVs Phenotyping EVs by Flow Cytometry A B A – White B – Red
- 26. ExoView® R100 Technology Antibody capture and imaging of tetraspanin positive EVs -- CD9, CD63, CD81
- 27. CD9 Capture Spot ExoViewTM CD9 AF647 CD81 AF555
- 28. Nanoparticle Tracking Analysis for EVs NTA has been used extensively in EV research since the mid-2000s Erdbrügger et al. (2017), Cytometry A
- 29. Nanoparticle Tracking Analysis for EVs NTA represented an important advance over DLS for polydisperse mixtures Filipe et al. (2010), Pharm Res
- 30. Multi-Spectral Advanced NTA (MANTA) ViewSizer 3000 ViewSizer can use three lasers simultaneously to visualize nanoparticle samples
- 31. 180Si F110PS 240Si 300Si F500PS 590Si 880Si 1300Si 0 500 1000 1500 0 100 200 300 400 Particle Diameter (nm) Countsperbin ViewSizer 3000 Performance 1300 Si 880 Si 590 Si 180 Si 240 Si 300 Si F110 PS F500 PS Abbreviations F – Fluorescent # – size in nm PS – Polystyrene Si – Silica SSC-A FITC-A ViewSizer 3000 (Inverted) Light Scatter ViewSizer can accurately resolve a complex, polydisperse bead mixture.
- 32. 0 500 1000 1500 0 50 100 150 Particle Diameter (nm) Countsperbin F110PS F500PS F500 PS F110 PS ViewSizer 3000 (Inverted) F110 PS F110 PS F110 PS F110 PS Fluorescence ViewSizer 3000 Performance 1300 Si 880 Si 590 Si 180 Si 240 Si 300 Si F110 PS F500 PS Abbreviations F – Fluorescent # – size in nm PS – Polystyrene Si – Silica SSC-A FITC-A ViewSizer can identify fluorescent particles uniquely out of a polydisperse mix.
- 33. Influence of Laser Wavelength on Particle Detection BLUE ViewSizer 3000 GREEN ViewSizer 3000 RED ViewSizer 3000 0 50 100 150 200 250 300 0 1×107 2×107 3×107 4×107 Particle Diameter (nm) DensityofPSD (Particles/mL/nm) Blue Only Green Only Red Only All Lasers 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 80 90 100 Particle Diameter (nm) CumulativeFrequency Percent EVs isolated from plasma require higher energy wavelengths for accurate analysis
- 34. ViewSizer 3000 Comparison with NanoSight LM10 0 50 100 150 200 250 300 0.000 0.005 0.010 0.015 Particle Diameter (nm) DensityofPSD (NormalizedUnits) ViewSizer Red Only ViewSizer All Lasers NanoSight LM10 NanoSight LM10 RED 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 80 90 100 Particle Diameter (nm) CumulativeFrequency Percent RED ViewSizer 3000 Laser wavelength can significantly affect particle count and size distribution
- 35. Biggest Advantages Accurate counting and sizing of individual nanoparticles Fluorescence NTA may help distinguish real EVs from contaminants Current Limitations Conventional NTA requires a clean EV isolation procedure Minimum size detected for biologics – 50nm? Future Directions What design features can be added to improve lower detection limit? Can instruments be designed for multiplex phenotyping like flow cytometry? Future Potential of NTA in EV Research
- 36. Bill Travers, Ph.D. Sean Travers, Ph.D. Jeff Bodycomb, Ph.D. Julie Chen Nguyen, Ph.D. Alan Faden, M.D. Junfang Wu, M.D., Ph.D. Steven Jay, Ph.D. Mentor Team …and the rest of the lab! Acknowledgements Funding Sources RF1 NS110637-01 (JW/SJ) R01 NS094527-03 (JW) R01 2NR013601-07 (JW/AIF) R01 NS110635-01 (JW/AIF) R01 NS110567-01 (JW) Kuba Tatarkiewicz, Ph.D.
- 37. • Latest MISEV guidelines (2018) https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750 • Original ISEV position statement (MISEV 2014) https://www.tandfonline.com/doi/full/10.3402/jev.v3.26913 • Coursera Course “Basics on Extracellular Vesicles” https://www.coursera.org/learn/extracellular-vesicles#about • Extracellular Vesicle Club for latest advances in research https://www.youtube.com/channel/UC0nhdTaTEUqpO8anXZqRdkQ Resources to learn more about EVs/exosomes