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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Abbreviations

F – Fluorescent

# – size in nm

PS – Polystyrene

Si – Silica

SSC-A

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Phenotyping EVs by Flow Cytometry

Erdbrügger et al. (2017), Cytometry A

Credit: Dr. Xiaoxuan Fan, UMSOM Flow Core Facility

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# – size in nm

PS – Polystyrene

Si – Silica

SSC-A

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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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Abbreviations

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# – size in nm

PS – Polystyrene

Si – Silica

SSC-A

FIT

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ViewSizer 3000 (Inverted)

Light Scatter

ViewSizer can accurately resolve a complex, polydisperse bead mixture.

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ViewSizer 3000 (Inverted)

F110

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F110

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Fluorescence

ViewSizer 3000 Performance

1300

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Si

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Si

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Si

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Si

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F500

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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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EVs isolated from plasma require higher energy wavelengths for accurate analysis

ViewSizer 3000 Comparison with 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