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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

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