Cellular Responses to Graphene Oxide Sheets: Effect of Lateral Dimension and the Oxidative Stress Paradigm
Dr. Sandra Vranic,
Nanomedicine Lab,
Faculty of Medical & Human Sciences & National Graphene Institute, University of Manchester, Manchester (UK) 19
Graphene in Nanomedicine and Nanotoxicology CLINAM 2016 - day 2 Hall Singapore 28.6.16
Introduction by Conference Hostess: ok so I think we need to wait for the
presentation of coaching so we will
start with our second speakers and
Sandra Vranic from the University of
Manchester, The National
graphene Institute, telling us about how
the lateral dimensions of graphene can
affect their oxidative stress
Sandra Vranic: Thank you very much for this
introduction
I would also like to thank
to the organizers for giving me the
opportunity to present my work so today
I will talk about cellular responses to
graphene oxide especially about the
effect of lateral dimensions and the
induction of intracellular oxidative
stress so to begin with few words about
graphene and graphene-based materials so
graphene has been isolated and
characterized by two scientists at the
University of Manchester back in 2004 so
for this discovery later on they
received Nobel Prize and since the
isolation and characterization of this
material it has been studied a lot and
many exciting applications have been
foreseen so graphene oxide belongs to
the group of graphene related materials
and it has oxygen-containing functional
groups on the surface so these
functional groups are actually giving
many also potential potentially exciting
applications to this material this is a
large surface for functionalization very
good dispersibility in aqueous solutions
and also potential for different
covalent functionalization that cannot
be interesting for biomedical
applications however the outcome of the
exposure in vitro and in vivo to this
material is unknown so why are we
interested in this first because there
can be potential adverse health effects
that we want to avoid and secondly it is
very important to understand aspects of
interactions of this material with the
cells in order to be able to exploit to
the maximum the potential that these
material kids so based on 30 previous
years of the research on nanoparticles
and also on carbon nanotubes and the
other nanomaterials there is a opinion
that actually the underlying mechanism
of the cellular response to this
material is based on oxidative stress
paradism the intracellular levels of the
duros that are induced after treatment
with the sun'll materials is increasing
and cells can actually
activate antioxidant defense in order to
bring these levels of rust to normal
values however if this is not the case
the levels of rust will continue to
increase and this can lead to activation
of inflammatory pathways or in the end
to cytotoxicity so as graphene oxide and
graphene is new material it is important
to try to correlate with the studies
that exist on nanoparticles and other
nanomaterials so our aim was actually to
determine physical chemical properties
of this material that is leading to
potential molecular and or cellular
injury so our first team was actually to
correlate the cellular responses to the
lateral dimensions of the Geo so with
this purpose we synthesized two types of
the Geo large with lateral dimensions
between five and 15 micrometers and
small with lateral dimensions below 200
nanometers furthermore we also were
interested in the effect of proteins
that can adsorb on the surface of these
sheets especially this is important in
in vitro conditions where cells are
actually cultivated in the presence of
serum so we wanted to see the difference
in the cellular response if the cells
were treated with the material
dispersing the presence or absence of
serum so with this purpose we had two
experimental designs in first of them
cells were treated with the material
dispersed in the cell culture medium in
the presence of serum for 24 hours and
in the second design we treated the
cells with the material dispersed only
in the cell culture medium for four
hours and we consider that during this
first for our cells have time to
interact with the material and the
downstream effects will already take
place so considering the endpoints we
were interested to study cytotoxicity
using several different assays oxidative
stress and activation of genes involved
in pro inflammatory response so to begin
with I will first show the results of
the toxicity study involving large and
small geo in the absence of serum so
first we performed up to
microscopy observations to have an first
idea of how the cells actually respond
to the treatment with this material and
what we could see is that first there is
a dose-dependent interaction of the
cells with the material and they
aggregate on top of the cells after 24
hours in a dose-dependent way however we
could not speculate about the optic of
this material based only on the optical
microscopy but there is a suggestion
that that the material actually is
interacting what was remarkable is
actually the treatment with the large
deal in the absence of serum where we
could see that already after the
treatment with low doses of the material
cells seem to detach and to be removed
from the support which could be
indication of the toxicity of the
material so in order to confirm this we
perform the cell count using slip on
blue and indeed we saw that after
treatment with the highest concentration
of the material was so significant
decrease in the cell number compared to
the untreated all the saturated with a
small geo after that we focused on the
cells that remained on the layer so not
the ones that were detached and for this
we use the Nexus 5 rupee de mayo de sa
which also showed slight but significant
decrease in a dose-dependent way in cell
viability after treatment with both
types of the material this was
furthermore confirmed using modified ldh
sa also showing those dependent slight
but still significant decrease so then
if we want to compare large versus small
geo in terms of of cytotoxicity we could
see firstly from this images that
actually there is higher toxic effect of
the large you're comparing to the small
one then we perform the same experiments
with this material in the presence of
serum from the beginning of incubation
and here we could not actually see the
same response especially for the large
deal in the presence of serum and this
was furthermore documented with the
subsequent experiment so cell count
didn't reveal any significant difference
between the treatments and here for
example performing an action 5 propidium
iodide we could see the dose-dependent
creasing percentage of a life cells only
after treatment with the Largey or
however after treatment with the small
material in the presence of steel it was
completely non-toxic for the cells this
was also confirmed with modified LD HSE
so afterwards we wanted to see how does
this actually fit with oxidative stress
pardon so is there increase in
intracellular ros production and indeed
so we performed to assess in order to
because it is very well known that after
performing experiments and using
different assets many interferences can
occur so for this reason we confronted a
few different test is to see if there
will be the same trend so first of all
we did DC FDA oxidation which is a dye
that actually enters freely the cell but
after being oxidized in the presence of
material it becomes green what we saw in
this optical microscopy images and also
we measured this fluorescence using
microplate reader then we used another
probe and another approach so it is it
was flow cytometry approach to to
confirm and see if we will see the same
trend and the principle is the same so
die enters the cells freely through
plasma membrane but then becomes
oxidated inside the cell if there is
increased dross and it's non
cell-permeable however the color is
different so using flow cytometry again
we saw the same trend that there is
increase in intracellular ros production
especially after treatment with small
and large in the absence of serum and so
if we compare the enlarge seems to
induce more intracellular as comparing
to the small and one more thing that was
also remarkable from this result is that
presence of serum seems to decrease the
intracellular loss production so the
third like tier 3 flow of the the
cellular response to the material goes
to through activation of
pro-inflammatory response and activation
of the genes that are involved in this
so as we perform 24 hours of treatment
we were interested in genes that are
involved in the expression of
a cute face of the pro inflammatory
response so we measured the activation
of genes that are for interleukin 6 1
beta and interleukin-8 and so what we
saw is that actually here there is there
was overall trend for all the cytokines
we did also tnf-alpha in parallel but I
didn't show it here so we saw that there
is a induction of the pro-inflammatory
response observed after treatment only
with the large geo in the absence of
serum for all the other conditions of
treatment there was no significant
increase so how does this fit actually
with oxy distress parenting what that
I've showed in the beginning so if we if
we classify and take a look at this
different so we had these two types of
the material and two conditions of
treatment for the small geo that was
used and dispersed in the presence of
serum we so very slight increase in
intracellular us that didn't furthermore
lead to inflammation response and
subsequent cytotoxicity however the
other extreme condition was the large
geo or in the absence of serum that had
very high increase in intracellular ros
leading to activation of pro
inflammatory response and subsequently
to through the cellular death so this is
the first big message that we got from
the study that we performed and second
one was that actually the absence of
serum is significantly having an impact
on the cellular responses to this
material so in the end I would like to
thank to the colleagues that are working
in the lab with me dr. Nelson of all
this and Mauricio Boozer and also I
would like to thank to dr. Cyril boosie
and professor kaskus trellis for
supervising thank you very much for your
attention
Audience member: Thank you. It just a technical question.
did I understand correctly when you did
the annex in five assay for live or dead
cells apoptotic cells did you look only
at the attached cells not at the
detached cells?
Sandra Vranic: Yes.
Audience member: may be the reason for
asking is that when cells undergo
apoptosis they detach so aren't you
missing an apoptotic population
potentially then
Sandra Vranic: okay so there are two
reasons why we did this experiment in
this way but so first is that actually
the material that remains in the
supernatant and that you cannot actually
remove so you collect supernatant but at
the same time with yourselves you also
have the material which we saw that can
quench the fluorescence from propidium
iodide so I in the beginning I did this
set up collecting supernatant collecting
the layer and then I saw that actually
does not reflect what we see using
optical microscopy so then I separated
and I saw doing cell count which is
complementary to the experiment with the
propidium iodide in the next in five so
I did analyze cells from the
supernatant for propidium iodide and an
axe in five and I have the percentage of
dead cells with different cell types
which shows that there is fifty percent
of dead cells but this result is only
indicative because actually we know that
the material did quench the fluorescence
of the next in five
so there is
indication but actually I cannot say the
percentage like accurate percentage of
the cell death
Audience Member: sure no I understand that
there may be interferences with the
essay but I think then you you may still
want to consider an alternative method
where you have less interference but
where you take into account both
detached and and non detach cells
I if I
may follow up with another question I
mean apoptosis is not necessarily the
only type of cell death and
our papers in the literature suggesting
that there is regulated necrosis or
necroptosis following exposure to
graphene oxide so you could test for
this if you add apoptosis inhibitors or
necroptosis inhibitors and then just
measure cell death this could give you
an indication of the mode of cell deaths
have you consider this?
Sandra Vranic: well yes maybe
for the future study we will take this
into consideration
Melanie Cookie: hello my name is
Melanie cookie and I would like to ask
you what is a modified ldh essay is it
an absorbent space or is aggressive one
is it for full lysis assay?
Sandra Vranic: okay so I
have the this is the method that has
been developed in the in the lab before
I joined so this is also especially to
to avoid interference with the material
okay
so this is the original one that
has been used with non carbon-based
materials and it works fine but when it
comes to the carbon-based materials such
as carbon nanotubes and then we also can
say the same for graphene there some
interferences were observed so it's
actually indirect in this direct method
you are measuring what the cells that
are dead actually released in the
supernatant
however in the modified one
you remove the supernatant and then you
have the cells that are on the support
that remained on the support then you
lyse these cells and actually inverse so
you measure the cells that remains as
alive and with indirect to measure that
sells but this is also because there is
this step of centrifugation in the
so yeah so there is a step of the
centrifugation when you lyse the cells
and this is because you want to
obviously to remove the material from
the supernatant wash and lyse the
cells and be sure that actually you
excluded your material from the
measurement
Melanie Cookie: ok.thank we're talking about
the same I just want to know if it's
absorbing space the essence one because
the fluorescence one is highly capable
for the quenching
Sandra Vranic: so yeah but this is
with this purpose so to eliminate the
material in order to remove the
quenching of the that comes from the
material becomes
Conference Hostess: more questions?
Audience member: I have
one so you showed us at the end that the
large sheets are bad yeah so they're
about 1 micrometer
Sandra Vranic: so there between five
and 15 originally but in the cell
culture medium this is how they look
like so this is in the cell culture
medium is actually much more so while we
didn't really measure that this until
the distribution of the size in the cell
culture medium but already from the
picture we see that only in the case of
small do in the presence of serum it
looks homogeneous well in all other
conditions we see the especially without
serum we see these clumps of the
material indicating that the size is
actually much bigger
Audience member: so your cells are
not fat aesthetics you have a epithelial
cells so do you have any explanation why
actually the large and one larger ones
could be more toxic despite having less
than bincy to enter into the cells
Sandra Vranic: yes
so we there are studies that show that
colosseo is actually but in the
macrophages and it goes through the
physical contact with tlr receptors that
are further done with the stream effect
of the seller that so beast officials
are having this dlr but we haven't
explored what is actually going on so is
it this physical physical contact with
this receptor or is it maybe the stars
are covered
fully with the material and then this is
what induces them to for the toxicity so
we don't know but we see that it's the
the size-dependent definitely okay this
effect so
Conference Hostess: one more question yeah
Audience member: why are
you adding under Fe essence or less
toxic and making a corona and the worse
mechanisms
Sandra Vranic: yeah so there are three
possibilities that serum can actually
have so first it can be corona effects
as you said with the hiding this surface
of the material and then having
influence on surface reactivity of the
material which like second explanation
but third is also modulating optic and
interactions with the cells so so for
future experiments will show does it
actually have impact on the uptake or
does it have influenced only from this
surface reactivity it can be also
extracellular so it doesn't have to go
inside the cell but we have so far we
haven't we did some studies for the
uptake but it is still ongoing so it can
be different reasons
Conference Hostess: ok so if no more
questions we thank sandra