Hi, I am Priscila Dauros-Singorenko and here to talk about importance of Zeta Potential to your EV research.
Determination of key EV physicochemical properties directly influences the outcome of investigations,
whether is to determine EV biological roles,
method development or applications into nanomedicine.
This 3D plot shows the latest innovations in advanced single particle analysis by TRPS,
where heterogeneous populations can be accurately profiled based on concentration,
size and surface charge,
vastly improving the measurement and analysis capabilities.
As you may know our technology,
Tunable Resistive Pulse Sensing (TRPS) is known to be the most precise and accurate EV analysis tool in the market.
Unlike optical technologies,
TRPS physical-based technology relies on nanoparticles passing through a nanopore,
creating a temporary increase in resistance.
The characteristics of this current blockade are used to determine particle concentration,
particle size and particle charge or zeta potential by measuring the number of pulses,
pulse magnitudes and pulse durations, respectively.
qNano, the original TRPS instrument, could be complex to use for some researchers.
Nevertheless, to respond to the researchers needs,
The Exoid was developed and launched to significantly improve user usability and advance efficiency in terms of time and effort to take measurements.
The Exoid implements semi-automation: with fully automatic Pressure System,
and resolution of sub 1 Pascal,
speeding up the process,
improving sensitivity and minimising variability,
automated Stretcher Unit which controls the stretch of the nanopore,
and Assisted nanopore unblocking mechanism to help measurement of complex samples.
What are the TRPS particle concentration and size capabilities?
Additionally, to monodisperse particle samples,
like liposomes or synthetic particles,
TRPS can resolve multimodal or polydisperse samples,
such as EV samples,
with the highest precision.
In this example, analysis of quadrimodal polystyrene standards
has shown that TRPS can resolve each one the populations by size and concentration,
unlike other single particle technologies such as Nanoparticle Tracking Analysis,
or other ensemble technologies such as Multi-Angle Dynamic Light Scattering,
which cannot fully resolve these populations.
Many of relevant biological interactions between a particle and the recipient or target cell might depend on zeta potential.
Zeta potential is a measure of effective charge of a particle in a certain medium.
It depends on factors such as medium (polar or non-polar), type of electrolyte, salt concentration, and pH.
The zeta potential is defined as the electrostatic potential at the plane of shear between the particle-associated and the stationary part of the ionic double layer.
Therefore, accurate characterisation of EVs zeta potential is important for meaningful assignations of biological roles.
In a synthetic particle mixture,
it is assumed that all particles have the same zeta potential,
however, in EVs samples this might not the case,
where a highly heterogeneous population is the norm.
For monodisperse particle mixes,
Phase Analysis Light scattering based on Laser doppler velocimetry or PALS might be the most used technique,
however this ensemble technique loses its applicability for polydisperse samples,
such as EV-based samples,
since with this technique individual particle sizes and zeta potentials are not measured.
With TRPS,
zeta potential is determined particle by particle by their electrophoretic mobility through a previously calibrated nanopore,
providing results that are unbiased from size.
In this graph we show that TRPS can resolve a particle mixture of 1:1 of 400 nm charged carboxylated polystyrene CPCs
as shown on the right of graph vs 400 nm bare polystyrene CPNs on the left of graph.
In contrast, PALS cannot resolve these two populations.
Previous and current Exoid-based zeta potential characterisations can render data as detailed histograms of concentration
vs charge or 2D plots of size vs charge.
In this histogram of counts vs charge we can see the high sensitivity of TRPS zeta potential measurements
as liposomes made with different phospholipids or different proportion of mixed phospholipids return different zeta potentials.
In this plot we can see that a pentamodal mix of particles of same sizes and different charge,
such as charged carboxylated polystyrene CPC100
vs bare polystyrene CPN100
or magnetic particles vs magnetic particles with DNA are well separated into different populations.
So far particle size and zeta potential can be determined simultaneously,
however,
one of the most recent innovations on our TRPS and Exoid technology is the possibility of analysing data of EV concentration
based on size and zeta potential simultaneously,
as shown in this figure.
TRPS measurements of zeta potential are done at very low pressures,
to keep electrophoresis as the main driving force,
resulting in small bias or inflated counts towards highly charged particles.
Nevertheless,
a new post-processing protocol of classically obtained data
can correct this distortion and convert 2D plots into 3d histograms
of particle number concentration (/ml) vs particle diameter (nm) and zeta potential (mV).
The correction is purely based on physical principles and not on arbitrary fitting models.
For example,
here is 3D plot of a trimodal 2:2:1 CPN150/CPN200/CPC200 mix,
where before data analysis correction,
charged carboxylated polystyrene CPC200 are vastly overrepresented.
However, after data analysis correction,
bare polystyrene CPN150 and CPN200 proportions
outweigh the CPC200 proportion of the mix,
as expected nominal values.
In this way,
with our TRPS-based zeta potential sensitivity and resolving power,
analysing Zeta potential in EVs as a key physicochemical parameter,
may hold significant utility in many fields of EV research.
Characterising EV composition or how certain stimuli can influence the composition of different EV subtypes.
For example, some antibiotic treatments,
used for their antibacterial properties,
has shown effects on EVs released from mammalian cells,
increasing DNA associated in surface of small EVs,
resulting in modification of their adhesion properties.
These modified or new discovered roles can be exploited for therapeutic applications,
since EV-associated DNA has shown immunomodulatory functions
(e.g. can suppress inflammatory responses or antitumor immune responses).
Zeta potential is also useful to assess clinical or pharmaceutical applicability of EV nanoformulations,
since EVs already present low immunogenicity and natural cell-targeting capabilities.
For example, in exogenous drug loading of EVs,
or loading after separation of EVs from cells,
post-loading EV characterisation can be done by checking if the drug loading process changed critical parameters of EV,
such as surface charge and size,
which can affect the biological interactions in drug delivery studies,
also, storage stability of EV preparation can be assessed due to higher zeta potential indicating higher repulsion between the particles in suspension
suggesting high dispersion stability.
EV have been shown to have critical roles in many different types of cancers,
by stimulating tumor growth, or mediating transfer of drug resistance or manipulating the tumor microenvironment.
It’s been shown that many and specific cancer EVs present a differential composition and surface charge,
for example as shown for the abundance of sialic acid in EVs, a main contributor of EV charge.
Thus, understanding the composition and roles of tumor secreted EV have in the progression of cancer,
is essential to develop effective new therapeutics.
As mentioned earlier, cancer-derived EVs,
which have a differential composition and zeta potential,
may also be exploited as targets of cancer diagnosis,
progression and monitoring treatment efficiency.
Current methods mostly rely on detection of cancer EVs by surface markers,
however coupling an extra layer of specificity by determining EV charge,
will improve the overall performance of the detection tool.
And finally, whether is labelling EVs or developing new capture system,
binding of molecules such as aptamers, proteins, antibodies, lectins, DNA, RNA,
to the surface of the EV can change zeta potential and can be precisely assessed and profiled in heterogenous EV samples.
Thank you for tuning into the IZON symposium on Zeta potential and EV research.
If you would like to know more about how TRPS and Zeta potential can impact your EV research please contact us at www.izon.com.
We can help to take your EV research to the highest potential with powerful and unparalleled precision and accurate characterisation of EVs.
From everyone here at Izon, we hope you enjoy the rest of ISEV21.
Thanks for watching.