Exosomes: Exploiting the Diagnostic and Therapeutic Potential of Nature’s Biological Nanoparticles
June 11, 2020HORIBA Webinar Particle Characterization Series
Niaz Zafar KhanMD/PhD Candidate
University of Maryland School of Medicine Medical Scientist Training Program
Program in Neuroscience
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
Loane and Faden (2010), Trends Pharmacol Sci
Primary Injury Secondary Injury
Pathophysiology of CNS Injury
Secondary injury contributes to progressive cell loss after neurotrauma.
Loane & Kumar (2015), Exp. Neurol.
Neuroinflammation after CNS Injury
Pro-inflammatory, microglial activation persists months after neurotrauma.
Kourembanas et al. (2015), Annu Rev Physiol
Extracellular Vesicles (EVs)
EVs are biological messengers that can transfer proteins, lipids, and nucleic acids.
EVs were once thought to be just “dust”.
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
Shah et al. (2018), N Engl J Med
EVs as Biomarkers for Disease
Holm et al. (2018), Trends Neurosci
Bidirectional Cellular Crosstalk through EVs
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
• 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
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EV Research is Skyrocketing!
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PubMed Filter for EVs
Need for Standardization in EV Research
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
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EV Nomenclature
Extracellular vesicle (EV) is the umbrella term endorsed by ISEV
ISEV (2014), J Extracell Vesicles
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
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
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
EV Isolation
Isolation methods have differing levels of recovery and purity
https://www.nanoviewbio.com/characterize
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
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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?
Nanoparticle Tracking Analysis
Single Particle Interferometric
Reflectance Imaging (SPIRI)
Flow Cytometry
EV Characterization Toolbox
Flow Cytometry provides excellent phenotyping capability but size
resolution is a limitation, especially for small EVs
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Si880
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F500
PS
Abbreviations
F – Fluorescent
# – size in nm
PS – Polystyrene
Si – Silica
SSC-A
FIT
C-A
Phenotyping EVs by Flow Cytometry
Erdbrügger et al. (2017), Cytometry A
Credit: Dr. Xiaoxuan Fan, UMSOM Flow Core Facility
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Abbreviations
F – Fluorescent
# – size in nm
PS – Polystyrene
Si – Silica
SSC-A
FIT
C-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
Flow Cytometry provides excellent phenotyping capability but size
resolution is a limitation, especially for small EVs
Phenotyping EVs by Flow Cytometry
A BA – White
B – Red
ExoView® R100 Technology
Antibody capture and imaging of tetraspanin positive EVs -- CD9, CD63, CD81
CD9 Capture Spot
ExoViewTM
CD9 AF647
CD81 AF555
Nanoparticle Tracking Analysis for EVs
NTA has been used extensively in EV research since the mid-2000s
Erdbrügger et al. (2017), Cytometry A
Nanoparticle Tracking Analysis for EVs
NTA represented an important advance over DLS for polydisperse mixtures
Filipe et al. (2010), Pharm Res
Multi-Spectral Advanced NTA (MANTA) ViewSizer 3000
ViewSizer can use three lasers simultaneously to visualize nanoparticle samples
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ViewSizer 3000 Performance
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Si880
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Si
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Si
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F500
PS
Abbreviations
F – Fluorescent
# – size in nm
PS – Polystyrene
Si – Silica
SSC-A
FIT
C-A
ViewSizer 3000 (Inverted)
Light Scatter
ViewSizer can accurately resolve a complex, polydisperse bead mixture.
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Particle Diameter (nm)
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F5
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PS
F500
PS
F110
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ViewSizer 3000 (Inverted)
F110
PS
F110
PS
F110
PS
F110
PS
Fluorescence
ViewSizer 3000 Performance
1300
Si880
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
FIT
C-A
ViewSizer can identify fluorescent particles uniquely out of a polydisperse mix.
Influence of Laser Wavelength on Particle Detection
BLUE
ViewSizer 3000
GREEN
ViewSizer 3000
RED
ViewSizer 3000
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Green Only
Red Only
All Lasers
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EVs isolated from plasma require higher energy wavelengths for accurate analysis
ViewSizer 3000 Comparison with NanoSight LM10
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ViewSizer All Lasers
NanoSight LM10
NanoSight LM10
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ViewSizer 3000
Laser wavelength can significantly affect particle count and size distribution
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
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.
• 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