Case Briefing Document●

Corporate Manslaughter●

Gross Criminal Manslaughter

Offences
Immediate action required:
Halt the roll-out of experimental treatments known as “COVID-19
Vaccines” immediately pending Police forensic examination

Defendants identified●

AstraZeneca●

Pfizer●Moderna●

National Health Service (NHS)●

Medicines & Healthcare products Regulatory Agency (MHRA)●

Joint Committee on Vaccination and Immunisation (JCVI)●

Her Majesty’s Government

Background Statement
The British Government began a rollout of what is now referred to as “COVID-19 vaccines” in early 2021.
The said ‘vaccines’ received a temporary Emergency Authorisation under Regulation 174 of the Human
Medicine Regulations Act (2012).
All Phase 3 COVID-19 vaccine trials are ongoing and not due to conclude until late 2022/early 2023. The
treatments are currently experimental with only 1 year of short-term data and no long-term human safety
data available. It is widely accepted now that none of the COVID-19 experimental treatments prohibit
infection of COVID-19 nor are they prohibiting transmission of any disease.
Scientists around the world have been reporting unusual findings when examining COVID-19 injection vial
contents in an effort to ascertain what is causing reported deaths, injuries and adverse reactions.
On 20th September 2021 in the pathological institute in Reutlingen, Germany, the results of autopsies ofeight people who died after COVID19 experimental treatment were presented. The fine tissue analyses
were performed by pathologists Prof. Dr. Arne Burkhardt and Prof. Dr. Walter Lang. The findings confirm
the previous report from Prof. Dr. Peter Schirmacher. Of more than 40 corpses he autopsied, who had diedwithin two weeks of COVID19 experimental treatment, approximately one-third of those deaths werecaused by the experimental treatment itself.

1

In November 2020 Dr Andreas Noack, a German chemist and one of the EU’s top graphene experts,
released a video explaining that he had discovered graphene hydroxide contained in the COVID-19
experimental treatments. He described how the graphene hydroxide nano structures injected into thehuman body act as ‘razor blades’ inside the veins of recipients and how they would not show up on an
autopsy or normal toxicology tests given their atomic size.
On 26th November 2021, just hours after publishing his latest video about graphene hydroxide, he died insuspicious circumstances.
Professor Dr Pablo Campra, University of Almeria, Spain also examined Covid-19 experimental treatments
in November 2021 using Micro-Raman Spectroscopy, the study of frequencies. He too confirmed thepresence of graphene.

Post Vaccination Death & Injury Reports
It is possible to signpost you to numerous death and injury reports made by way of statements to Policeand Lawyers around the UK.
It is incumbent upon medical staff in the UK to report any injury, adverse reaction or death following theinjection of these experimental treatments. The MHRA (Medicines & Healthcare products Regulatory
Agency) set up the Yellow Card System for this purpose and encourages the reporting of COVID-19
suspected side effects to medicines and vaccines or medical devices and diagnostic adverse incidents
used in coronavirus treatment.
As of 5th January 2022, this system shows that death has been listed as an outcome related to COVID-19
experimental treatments at least 1,932 times as well as nearly one and a half million adverse reactions to
the experimental treatments (1,414,293).
In the United States, VAERS (Vaccine Adverse Events Reporting System) reports of Deaths have been
listed as an outcome related to COVID-19 experimental treatments at least 21,745 times as of January 7,
2022 and 38,000 people are permanently disabled.
On the Euro eudraVigilance European database Death has been listed as an outcome related to COVID-19
experimental treatments at least 34,337 times as of December 18, 2021. 3.1 Million injuries have also beenreported.
It follows that the rates of increase of death and significant harm are increasing as the experimental
treatment programme is rolled out.

Investigation
Following her own experience with patients who had suffered apparent vaccine injury and adverse
reactions, a British Medical Practitioner came forward in December 2021 offering to assist in an
investigation to ascertain whether the results discovered by Dr’s Noack and Campra could be replicated in
the UK and also to examine the COVID-19 injection vials for discovery of toxins or unexpected contents.
The medical practitioner seized an injection vial from the fridge housed in the surgery in which she works
and handed it to an independent investigator assisting in investigating cases relating to injury sustained asa result of injections given as part of the roll out.
Further vials have since been obtained which cover the three main manufacturers in the UK: Pfizer,
Moderna and AstraZeneca.
2

The contents of the injection vials have been forensically examined and a laboratory report is attached tothis brief. A summary of the findings are found below.
A brief summary of a toxicology report is also found below, the full document is attached.
A full peer reviewed scientific paper is expected to be provided in due course.
As soon as we had the confirmation of the laboratory report and toxicology. Ian Clayton contacted the
Police (details) in line with duty of care to the Public to request immediate cessation of the experimental
treatment roll-out and to make an appointment to present this evidence in person to the Police.

Chain of Evidence
1. Injection vial delivered to GP surgery
2. Injection vial collected from the GP surgery refrigerator
3. Injection vial delivered to Investigation team
4. Injection vial delivered to the Laboratory
5. Injection vial forensically examined
6. Injection vial retained for disclosure purposes

Attachments
The following documents are attached in support of this submission:●

Laboratory report●

Scientific article ‘Toxicity of graphene-family nanoparticles: a general review of the origins andmechanisms’●

Layman's summary of the article ‘Toxicity of graphene-family nanoparticles: a general review of theorigins and mechanisms’Laboratory Report Summary
A summary of the findings detailed in the attached quality assured report is as follows:
RAMAN Spectroscopy discovered the following particles ●Graphene●

SP3 Carbon●

Iron Oxide●

Carbon derivatives

3

Toxicology Report Summary
A summary of the findings detailed in the attached toxicology report is as follows:●

Graphene nanomaterials (GFNs) can penetrate the body’s natural barriers and damage the central
nervous system●

Graphene oxide (GO):a. can damage internal organs
b. damages the reproduction and development systemc. destroys blood healthd. damages and destroys cells
e. can trigger cancer and accelerate ageingf.

damages mitochondria and DNA

g. triggers an inflammatory response and three different kinds of cell deathh. causes changes in gene function

Police Submission
With the information that has been provided to you today there is more than enough reasonable suspicion
that serious indictable offences have been committed.
There is also more than enough grounds for the Police to apply for a S.8 Police & Criminal Evidence Act
1984 Warrant and seize injection vials for themselves and submit these vials to a Home Office laboratory
with a view to replicating our findings.
Having replicated the examinations the Police will be in possession of their own hard evidence to support
serious indictable offences.
The first and foremost duty of the Police is the protection of life. It is therefore incumbent on the Police thatthey take action to halt the roll-out of these experimental treatments immediately and until their own
forensic investigations have been completed. Again, there are reasonable grounds for doing this.
The main potential defendants in this matter, as listed above, precludes them from conducting any
investigation themselves. It is a matter for the Police only.
Ian Clayton is a co-founder of a collective of people called UKCitizen2021, which was itself established bya small group of people who formerly worked within the legal profession. He is the
person reporting this matter to you and is your point of contact going forward.
During the course of being involved with UKCitizen2021, Ian Clayton has amassed a plethora of contacts,
information and direct access to various experts in many fields. He is more than happy to assist the Police
in signposting them.
As the Police have a duty to conduct the necessary forensic tests for themselves, it is not necessary tomention the names of the people involved in this matter. Due to the fact that Dr. Noack died in suspicious
circumstances following his video release about graphene hydroxide being present; the identities of the4

persons involved in this private forensic investigation are withheld for security reasons.

Law

Corporate Manslaughter and Corporate Homicide Act 2007
UK Public General Acts <https://www.legislation.gov.uk/ukpga>2007 c. 19<https://www.legislation.gov.uk/ukpga/2007/19/contents>
1 The offence(1)
An organisation to which this section applies is guilty of an offence if the way in which its activities aremanaged or organised—(a)causes a person's death, and(b)amounts to a gross breach of a relevant duty of care owed by the organisation to the deceased.
(2)
The organisations to which this section applies are—(a)a corporation;(b)a department or other body listed in Schedule 1;(c)a police force;(d)a partnership, or a trade union or employers' association, that is an employer.
(3)
An organisation is guilty of an offence under this section only if the way in which its activities are managed
or organised by its senior management is a substantial element in the breach referred to in subsection (1).
(4)
For the purposes of this Act—(a)“relevant duty of care” has the meaning given by section 2, read with sections 3 to 7;(b)a breach of a duty of care by an organisation is a “gross” breach if the conduct alleged to amount to abreach of that duty falls far below what can reasonably be expected of the organisation in thecircumstances;(c)“senior management”, in relation to an organisation, means the persons who play significant roles in—(i)the making of decisions about how the whole or a substantial part of its activities are to be managed ororganised, or(ii)
the actual managing or organising of the whole or a substantial part of those activities.
(5)
The offence under this section is called—(a)corporate manslaughter, in so far as it is an offence under the law of England and Wales or Northern
Ireland;(b)
corporate homicide, in so far as it is an offence under the law of Scotland.
5(6)An organisation that is guilty of corporate manslaughter or corporate homicide is liable on conviction on
indictment to a fine.
(7)
The offence of corporate homicide is indictable only in the High Court of Justiciary.

Gross Criminal Manslaughter
The Law
The ingredients of the offence were authoritatively set out in the leading case of R v Adomako [1995] 1 AC
171in which Lord Mackay of Clashfern LC at page 187 said the following:"In my opinion, the law as stated in these two authorities Bateman (1925) 19 Cr. App. R. 8 and Andrews v
DPP [1937] AC 576 is satisfactory as providing a proper basis for describing the crime of involuntarymanslaughter. Since the decision in Andrews was a decision of your Lordships' house, it remains the mostauthoritative statement of the present law which I have been able to find and it has not been departed from.
On this basis, in my opinion the ordinary principles of negligence apply to ascertain whether or not thedefendant has been in breach of a duty of care towards the victim who has died. If such breach of duty isestablished the next question is whether that breach of duty caused the death of the victim. If so, the jury
must go on to consider whether that breach of duty should be characterised as gross negligence andtherefore as a crime."
In order to prove the offence, the prosecution must therefore establish the following elements:a) The defendant owed a duty of care to the deceased;b) By a negligent act or omission the defendant was in breach of the duty which he owed to the deceased;c) The negligent act or omission was a cause of the death; andd) The negligence, which was a cause of the death, amounts to gross negligence and is therefore a crime;
More recently, the elements of manslaughter by gross negligence were stated concisely by the President of
the Queen's Bench Division in R v Rudling [2016] EWCA Crim 741at paragraph 18 as follows:
We can summarise the law shortly. The critical ingredients of gross negligence manslaughter can be takenfrom R v Prentice, Adomako and Holloway [1994] QB 302 in this court and Adomako [1995] 1 AC 171,[1994] 99 Crim App R 362 in the House of Lords as well as R v Misra [2005] 1 Cr App R 21. They can besummarised as being the breach of an existing duty of care which it is reasonably foreseeable gives rise toa serious and obvious risk of death and does, in fact, cause death in circumstances where, having regard
to the risk of death, the conduct of the defendant was so bad in all the circumstances as to amount to acriminal act or omission (see Adomako [2005] 1 Cr App Rep at 369). The elements of GNM were set out by
the House of Lords in R v Adomako [1995] 1 AC 171.

Other Offences
It follows that numerous other offences will have been committed by other individuals which I do not intend
to detail here but which the Police will have regard to in due course.
Police and Criminal Evidence Act 1984
UK Public General Acts <https://www.legislation.gov.uk/ukpga>1984 c. 60<https://www.legislation.gov.uk/ukpga/1984/60/contents> Part II<https://www.legislation.gov.uk/ukpga/1984/60/part/II>
Search warrants
86Power of justice of the peace to authorise entry and search of premises.
(1)
If on an application made by a constable a justice of the peace is satisfied that there are reasonablegrounds for believing—(a)that an indictable offence has been committed; and(b)that there is material on premises mentioned in subsection (1A) below which is likely to be of substantial
value (whether by itself or together with other material) to the investigation of the offence; and(c)that the material is likely to be relevant evidence; and(d)that it does not consist of or include items subject to legal privilege, excluded material or special procedurematerial; and(e)that any of the conditions specified in subsection (3) below applies,
he may issue a warrant authorising a constable to enter and search the premises in relation to each set ofpremises specified in the application.
(1A)
The premises referred to in subsection (1)(b) above are—(a)one or more sets of premises specified in the application (in which case the application is for a “specificpremises warrant”); or(b)
any premises occupied or controlled by a person specified in the application, including such sets ofpremises as are so specified (in which case the application is for an “all premises warrant”).
(1B)
If the application is for an all premises warrant, the justice of the peace must also be satisfied—(a)that because of the particulars of the offence referred to in paragraph (a) of subsection (1) above, there arereasonable grounds for believing that it is necessary to search premises occupied or controlled by the
person in question which are not specified in the application in order to find the material referred to inparagraph (b) of that subsection; and(b)that it is not reasonably practicable to specify in the application all the premises which he occupies orcontrols and which might need to be searched.
(1C)
The warrant may authorise entry to and search of premises on more than one occasion if, on theapplication, the justice of the peace is satisfied that it is necessary to authorise multiple entries in order toachieve the purpose for which he issues the warrant.
(1D)
If it authorises multiple entries, the number of entries authorised may be unlimited, or limited to a maximum.
(2)
A constable may seize and retain anything for which a search has been authorised under subsection (1)above.
(3)
The conditions mentioned in subsection (1)(e) above are—(a)that it is not practicable to communicate with any person entitled to grant entry to the premises;(b)that it is practicable to communicate with a person entitled to grant entry to the premises but it is notpracticable to communicate with any person entitled to grant access to the evidence;(c)that entry to the premises will not be granted unless a warrant is produced;(d)7

that the purpose of a search may be frustrated or seriously prejudiced unless a constable arriving at thepremises can secure immediate entry to them.
(4)
In this Act “relevant evidence”, in relation to an offence, means anything that would be admissible inevidence at a trial for the offence.
(5)
The power to issue a warrant conferred by this section is in addition to any such power otherwise
conferred.
(6)This section applies in relation to a relevant offence as defined in section 28 of the Immigration Act 1971)
as it applies in relation to an indictable offence.
(7)
Section 4 of the Summary Jurisdiction (Process) Act 1881 (execution of process of English courts in
Scotland) shall apply to a warrant issued on the application of an officer of Revenue and Customs under
this section by virtue of section 114 below.
Home Office Guidelines on the Reporting and Recording of Crime
The Law
The Police Act 1996 Section 44 (2 and 3) states that the Home Secretary can require chief constables offorces in England and Wales to provide statistical data.
The HOCR also promote a victim-oriented approach (PDF document)<https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/387762/count-vision-decem
ber-2014.pdf> to crime recording. This means that a belief by the victim that a crime has occurred is, inmost cases, enough to justify its recording as a crime.
The crime recording process used by the police can be divided into six stages, which are set out below.
1. Incident reporting and recording
Incidents reported to the police relate to issues including public safety and welfare, crime, anti-social
behaviour <https://www.justiceinspectorates.gov.uk/hmicfrs/glossary/anti-social-behaviour/> and transport.
There are many ways that incidents can be reported to the police:•victims <https://www.justiceinspectorates.gov.uk/hmicfrs/glossary/victims/>, witnesses or other third parties
can tell a police officer, PCSO or member of staff either on the street or at the front counter of a police
station;•victims <https://www.justiceinspectorates.gov.uk/hmicfrs/glossary/victims/>, witnesses or other third parties
can telephone incidents to police control rooms;•victims <https://www.justiceinspectorates.gov.uk/hmicfrs/glossary/victims/>, witnesses or other third parties
may report an incident online;•the police might discover the crime themselves; or•other agencies such as social services may refer them. It is also possible that other agencies will refer anincident that is clearly a crime.
The HOCR require that “all reports of incidents, whether from victims<https://www.justiceinspectorates.gov.uk/hmicfrs/glossary/victims/>, witnesses or third parties and whether
crime related or not, will result in the registration of an incident report by the police”. It goes on to specify
that these must be recorded on an auditable system, which in practice means:•an incident log (sometimes referred to as a command and control log); and/or,•a record on the force crime system.
When recording an incident, staff allocate an “opening code” to the incident log. Opening codes indicate thenature of the incident, for example whether it relates to a road traffic accident or a burglary. Opening codes
are important because they allow supervisors to see immediately what type of incidents are currently open
and prioritise resources accordingly.
8

2. Deciding if a crime should be recorded
The HOCR require:“An incident will be recorded as a crime (notifiable offence)
1.For offences against an identified victim if, on the balance of probability:•The circumstances as reported amount to a crime defined by law (the police will determine this, based on
their knowledge of the law and counting rules), and•There is no credible evidence to the contrary.
2.For offences against the state the points to prove to evidence the offence must clearly be made out,
before a crime is recorded.”
Because the rules place an obligation on the police to accept what the victim says unless there is “credible
evidence to the contrary”, the following reasons are not enough to justify not recording a crime:•the victim declines to give personal details;•the victim doesn’t want to take the matter further; or•the allegation can’t be proven.
In relation to the balance of probability test, the National Crime Recording Standard (which is reproduced
within annex A of the HOCR (PDF document)<https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/534967/count-general-july-2
016.pdf_.pdf>) notes that:“In most cases, a belief by the victim (or person reasonably assumed to be acting on behalf of the victim)that a crime has occurred is sufficient to justify its recording as a crime, although this will not be the case in
all circumstances. Effectively, a more victim orientated approach is advocated.”“An allegation should be considered as made, at the first point of contact, i.e. the stage at which the victim
or a person reasonably assumed to be acting on behalf of the victim first makes contact with the police, bethat by phone, etc. or in person. If an alleged or possible victim cannot be contacted or later refuses toprovide further detail, the Crime Recording Decision Making Process (CRDMP) should be based on all
available first contact information.”

9CGC-IDS

Global Humanitarian Crisis Prevention and Response Unit

Project CUNIT-2-112Y6580

Qualitative Evaluation of Inclusions
In
Moderna, AstraZeneca and Pfizer Covid-19
vaccines.

by
UNIT

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Global Humanitarian Crisis Prevention and Response Unit

Executive Summary

UNIT was commissioned by EbMCsquared CiC under project UNITC-112980 to investigate
the contents of four injection vials (Moderna 01, Moderna 02, AstraZeneca, Pfizer) for any
undeclared ingredients that may cause bodily harm.
This report is the submission of initial findings that confirm the presence of graphene
compounds in each of the injection vials. Though a quantitative estimate has not be established
for the concentration of graphene in the samples, its occurrence is on a high frequency range
on an average 2cm transect when counts were conducted on a higher magnification (40x).

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Global Humanitarian Crisis Prevention and Response Unit

TABLE OF CONTENTS
1.

2.

Introduction ...................................................................................................................... 5
1.1.

Background ................................................................................................................ 5

1.2.

Descriptions of Vials.................................................................................................. 5

1.3.

Aims and Objectives of the study ............................................................................ 6

1.4.

Report Structure and Outline .................................................................................. 6

Methodology ..................................................................................................................... 8
2.1.

Vial Descriptions ....................................................................................................... 8

2.2.

Process of Sampling and Slide Preparation ............................................................ 8

2.3.

Raman Spectroscopy ............................................................................................... 10

3.Results .................................................................................................................................. 11
3.1. Description of Inclusions ............................................................................................ 11
3.1.1.

Graphene Composites in the form of Nano Ribbons ......................................... 11

3.1.2.

Graphene Composite form 1 .............................................................................. 12

3.1.3.

Graphene Composite Form 2 ............................................................................. 12

3.1.4.

Calcite ................................................................................................................ 13

3.1.5.

Graphene Nano Forms ....................................................................................... 13

3.1.6.

Crystalised forms of the solution ....................................................................... 14

3.2.

Moderna 01 .............................................................................................................. 15

3.2.1.

Microscopy ........................................................................................................ 15

3.2.2.

Raman Spectroscopic Investigation ................................................................... 18

3.2.3.

Counts ................................................................................................................ 20

3.3.

Moderna 02 .............................................................................................................. 21

3.3.1.

Microscopy ........................................................................................................ 21

3.3.2.

Raman Spectroscopic Investigation ................................................................... 25

3.2.3. Counts .................................................................................................................... 26
3.4.

AstraZeneca ............................................................................................................. 28

3.4.1.

Microscopy ........................................................................................................ 28

3.4.2.

Raman Spectroscopic Investigation ................................................................... 32

3.4.3.

Counts ................................................................................................................ 34

3.5.

Pfizer ......................................................................................................................... 35

3.5.1.

Microscopy ........................................................................................................ 35

3.5.2.

Raman Spectroscopic Investigation ................................................................... 38

3.5.3.

Counts ................................................................................................................ 39

4.

Interpretation, Discussions and Conclusion ................................................................ 41

5.

Bibliography ................................................................................................................... 45
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Global Humanitarian Crisis Prevention and Response Unit

THIS REPORT AND ANY OF ITS ASSOCIATED
CONTENTS MARKED BY THE IDENTITY TAG
AUC101 ARE STRICTLY CONFIDENTIAL.
ANY UNAUTHROSISED REPRODUCTION AND OR
DISTRIBUTION OF THIS REPORT IS STRICTLYPROHIBITED.

Any reproduction and distribution queries should be directed
to
UNIT
At
info@openplatform.uk

DISCLAIMER
This work is an interim report with the presentation of initial laboratory analysis results of Project UNITC-112980.
Further work is ongoing to critically analyse and process these initial results and produce a more in depth analytical andquantitative report.
UNIT takes no responsibility for interpretations drawn by readers of the report.

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Global Humanitarian Crisis Prevention and Response Unit

1. Introduction
The following report is a product of a joint cooperation between EbMCsquared CiC and UNIT
in an attempt to identify the undeclared contents of the current vaccines that are being
administered to the UK public causing high numbers of adverse reactions and deaths.

1.1. Background
UNIT was commissioned by EbMCsquared under UNITC-112980 to 91 to analyse contents offour vaccine vials and identify if any of the following components were present in these vials– graphene, graphene oxide, parasites, biological filaments.
The four vaccines that form the subject of this first investigation by UNIT belonged toModerna, Pfizer, and AstraZeneca.
The entire process of collection and delivery is reported in ANNEXE 1A.

1.2. Descriptions of Vials
1.2.1. Moderna 01 Labelling
The manufacturing label had the following information on the vial:
COVID-19 Vaccine Moderna. 020mg/mL Dispersion for injection. COVID-19 mRNA Vaccine(nucleoside modified) Multidose vial doses of .5mL.
Lot-3004737 Exp. 24/01/2022.
The liquid contained in the Moderna bottle was cloudy to naked eye against the sunlight.
1.2.2. Moderna 02 Labelling
The manufacturing label had the following information on the vial:
COVID-19 Vaccine Moderna. 020mg/mL Dispersion for injection. COVID-19 mRNA Vaccine(nucleoside modified) Multidose vial doses of .5mL.
Lot- Lot-3004737 Exp. 24/01/2022.
The bottle weight prior to breaking of the seal was 18.842gm.
The liquid contained in the Moderna bottle was cloudy to naked eye against the sunlight.
1.2.3. AstraZeneca Labelling
The manufacturing label had the following information on the vial:
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Global Humanitarian Crisis Prevention and Response Unit

Covid-19

Vaccine4ml,

Astrazeneca

Injection.

Covid-19

Vaccine:(ChAd0x1-S (recombinant)), Intramuscular use. Multidose vial (8x0.5ml doses). 108439/2 LotPW40167 Expiry- 01-22. (Figure 2.1)
The bottle weight prior to the breaking of the seal was 12.184g and after the breaking of theseal was 11.803g.
The liquid contained in the AstraZeneca bottle was transparent to naked eye.
1.2.4. Pfizer Labelling
The manufacturing label had the following information on the vial as in figure 1.1:

Figure 1.1. Pfizer vaccine vial

Lot-FC9001 Exp. 09/01/2022.
The liquid contained in the Pfizer bottle was cloudy to naked eye against the sunlight
1.2.5.

1.3. Aims and Objectives of the study
The aim of the study was to identify any solid inclusions in the vials as were undeclared by the
manufacturers.
The study was to verify the findings of graphene related compounds such as graphene oxide,
graphene hydroxide by Campara (2021) and report any other biological inclusions that may beinterpreted as toxic to human body.

1.4. Report Structure and Outline
The vials went through evaluation of contents at four different laboratory sites that are
identified in ANNEXE 1B on request and the methodology adopted is presented in Chapter
two.

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Global Humanitarian Crisis Prevention and Response Unit

The report presents an in depth analysis of the findings of this project and is divided into
five chapters. Chapter one is the introduction, the second chapter is a description andanalysis of the methods used for the evaluation. Chapter three presents the results of theanalysis, Chapter four is the interpretation, discussions and conclusions of the study. The
Bibliography forms the last chapter of the report.
The entire project involved the inputs of experts in individual fields and their names, affiliations
and expertise are identified for reference purposes in Annex 2.

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Global Humanitarian Crisis Prevention and Response Unit

2. Methodology
2.1.

Vial Descriptions

The secured vials were made of glass and the vaccines contained within the vials were in aliquid form. The different parts of the vial are shown in figure 2.1. Each of these vials werestored at 4°C in the sample storage rooms until the evaluation of their contents took place.
Each of the four vials undertook the same process for evaluation.

Figure 2-1 Different parts of the sealing process of the vial. The vial shown here is the AstraZeneca vial.
The same three seal components were present for the Moderna and Pfizer vials.

2.2.

Process of Sampling and Slide Preparation

Stage 1 - Opening of the vial
Stage 1 involved the opening of the vial while the process was recorded on the camera forposterity. Each of the vials were sealed with a plastic seal on the top (figure 2.1). This seal had
to be broken off before one could access the rubber top of the injection vial. The rubber top
was held securely in place with a metal ring.
There were two options of accessing the liquids from within the vials. One, was to use an
injection syringe and draw out the required amount of liquid. However, knowing that theobjective of the study was to identify and report any nano graphene particles within the vials,
this method of sample acquisition was not thought to be appropriate. The reasoning being, thatduring the penetration of the vial, the syringe would carry nanoscopic amounts of rubber sealmaterial, which is a carbon product, each time, it would access the liquid. Therefore, the second
option of material access was adopted, which meant, breaking off the metal ring and simply
removing the rubber seal on top each time the sample material was required.

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Global Humanitarian Crisis Prevention and Response Unit

Stage 2 – Subsampling for evaluation of experimental steps required
Stage 2 had the simple purpose of the first evaluation of the sample. As there was no priorstudy available at hand to determine the chemistry and the physical properties of the material
at hand, a small subsample was used from the vial to be evaluated under a polarising
microscope to identify the gross composition of the material of the vaccine and to have a brief
understanding of any inclusions if they were present.
Each of the vaccine’s active ingredients seemed to have been preserved in a sucrose-salt richmedium. The optical and physical properties of the preserving medium posed a challenge tothe identification of the actual inclusions. In addition to being of nearly the same refractive
index as the medium, the micro and nano crystalline nature of the expected inclusions renderedan additional impediment to the task of their isolation.
During, wet microscopic evaluation at the dry laboratories, it was noted from the onset, thatthe denser material settled at the bottom of the slide and got impregnated and encrusted withthe medium solution, while the slide was still in the process of wet examination. Additionally,
the deposition of the inclusions occurred at different depths within the medium.
Cummulatively, such characteristics, posed a grave challenge to the central objective of thestudy, in that, isolation of the objects of interest was deemed impossible. The medium masked
all signatures of the inclusions by impregnation and encrustation in a manner that the inclusions
were completely camouflaged into the background.
These challenges were attempted to be overridden by vacuum filteration. The first attempt at
vacuum filteration failed due to equipment failure. This resulted in the loss of the test material.
As, at the time of this experiment, the material at hand was quite limited, any further attempts
for both vacuum filteration processes were paused.
(It should be noted, that the ongoing continuation of this project will be using checmical
filteration to isolate the inclusions from the onset as the current project has given a clear insight
into the chemistry of the product.)Stage 3- Partitioning of sample for clean slide preparation
This stage included the partitioning of sample into two. The clean portions of the sample werestored in a pyrex glass vial which was cleaned using hydrogen per oxide, hydrochloric baths,
then rinsed with distilled water. The vials were then sterilised in the sterilisation chamber prior
to the transfer of the sample into them.

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0.0125ml of sample was then transferred onto clean slides within fume cupboard and left there
to air dry at 20°C under a glass chamber.
The dried slides were mounted using glass coverslips and used for microscopic evaluation. Itshould be noted that none of the prepared clean slides dried out completely even after a few
days.
Stage 3- Optical and Petrological Microscopy
Prepped slides were examined under a reflection and transmission light microscope for organic
content and petrological microscope for mineral contents.
Microscopic stage involved preparation of several test slides to identify the typical nature of
the content.
On representative slides, counts were done along a specific transect. These were thenrepresented on psimpol to get density options on a typical 0.006ml of sample.
Stage 4 – Raman sampling and preparation
Subsamples from original vials were obtained for Raman spectroscopy. These were transferred
onto standard slides using sterilised glass pipettes. The slides were left to dry under glasschambers inside a fan heating oven before being taken for examination to the Raman
Laboratories.

2.3.

Raman Spectroscopy

As most of the observed inclusions belonged categorically to carbon compounds, Raman
Spectroscopy was the chosen method for initial identification of the inclusions. All Raman
spectra were recorded in air and at room temperature in back scattering geometry using
Renishaw in Via Raman spectrometer. A tunable Ar ion laser was used as an excitation sourceof 488 nm. The laser beam was tightly focused on the sample surface through a Leica 50x
LWD microscope objective with a numerical aperture equal to .5, leading to a laser beam
diameter of about 2µm. Spectral resolution was about 2cm-1.

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3.Results
3.1. Description of Inclusions
The analysis of all four vial contents identified objects that are similar. For ease ofnomenclature and related descriptions per vaccine, these inclusions are illustrated and defined
individually belowThe identified inclusions were1. Graphene nano ribbons coated with Polyethylene Glycol
2. Graphene Composite Form 1
3. Graphene Composite Form 2
4. Microcrystalline Calcite with Carbonaceous inclusions
5. Graphene Nano Forms with and without fluorescencea. Graphene nano objects
b. Graphene nano scrolls
3.1.1. Graphene Composites in the form of Nano Ribbons

Figure. 3.1. Carbon Composite Ribbon Forms from a sample in Moderna 01. The first picture is of a wet
sample and at a low magnification. The second picture is that of the same sample when it is dry andembedded in solution at a high magnification.

This micro inclusion form appears dark grey in wet slides, resembling filamentous ribbons. As
the material dries, the form becomes nearly transparent. On high magnifications of 40x,
lamellar structures can be identified as sheets. Raman spectrum on these sheets shows a carbon
oxygen bond with added polyethylene glycol imprints (figure 3.9). A longer range of theRaman requires to be shot for a better grasp on the distribution of defects and othercharacteristics of this form.

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3.1.2. Graphene Composite form 1
GC1 appears in a translucent folded form of about 10-15microns across. The form istransparent to translucent in transmitted light and shows light structure within it.

Figure 3.2. An assemblage of different forms with some embedded in the solution appearing translucent
and those on the top show a good relief.

GC1 is isotropic under crossed polars. Raman spectroscopic results for this form shows
dominant dual peaks of calcite at 1100 cm-1 and of some form of iron oxide nearly 500 cm-1 .
The spectrum beyond 1300 cm-1 is quite noisy due to the presence of some fluorescence.

3.1.3. Graphene Composite Form 2
These forms are visibly more intricate and give a rather complex Raman signature. The
deciphered components were graphene with iron oxide and calcite. The forms are quitedistinct in their lamellar, structure.
The signature for carbon-carbon bond is quite distinct at 1600 cm-1 , 1100 cm-1 fpeak ispicked up for calcite.

Figure 3.3. Carbon composite Form 2

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3.1.4. Calcite

Figure 3.4. Microcrystalline Calcite with graphene inclusions.

Microcrystalline Calcite is another inclusion which is present in the samples. The form can be
described as lumpy with inclusions of graphene nano forms. The form gives a very clear Raman
signal for Calcite. Calcite is also present in GC1 and GC2 as identified through Raman spectra.
3.1.5. Graphene Nano Forms
Nano forms of graphene were identified in all the samples that were evaluated (figure 3.5).
Upon shooting of the Raman when focused on some of these nano objects (in different
vaccines), the obtained signals were found to be markedly masked with fluorescence.
Identification of Graphene nano forms was therefore conducted on the basis of microscopic
morphological correlation with the forms where, the signal was clear and undebatable (figure3.24).
Nano forms of Graphene dominated the counts in all the samples. They were found to bepresent both in roundish shapes and long spiculate shapes. The rounded forms were almost
entirely found in association with nano particles.

Figure 3.5. (a) Nano forms of Graphene. (b) Nano scrolls.

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Quite conspicuously the spiculate forms were noticeably strewn in random orientations at thebottom of all the samples that were examined (figure 3.5).
These nano Graphene spicules were impossible to be evaluated by Raman, as their radius wasmeasurably smaller than the resolution of the laser.
Given, the sheer number of the Graphene scrolls that were noted to be present in the samples,
a separate project with the central focus on these scrolls is underway, where, high resolution
Raman and invasive FTIR investigations are being undertaken to establish the structures ofthese scrolls and to get a quantitative estimate of their concentrations.
3.1.6. Crystalised forms of the solution
All the four vaccines are sugar based, and on the edges of the coverslip, as the material dried it
crystallised into sugar crystals form in varied shapes. Figure 3.6. shows some varied forms ofthese crystals under polarised light.

Figure 3.6. Four different forms of sugar base crystals identified in a dried Moderna slide. The crystals
display third order interference colours under polarised light with an angular extinction in most cases.
The crystals stem from distinctive nucleii.

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3.2.

Moderna 01

3.2.1. Microscopy
Moderna 01 was the first sample that was evaluated. Under wet microscopy, the sample showed
several filamentous forms (figure 3.7). These forms seemed to shed off some of their filaments
in form of small flakes (figure 3.7).
As the slide dried, these filamentous forms became incorporated in the solution medium andwere optically difficult to distinguish from the background (figure 3.8).

Figure 3.7. Filament forms as observed under wet microscopy.

In the dried state, various forms of particulate inclusions were identified. The settlement ofthese inclusions took place at multiple levels within the solidified medium. Lighter materialcame to rest and settle on top and denser material was found at the bottom of the slide.
The crystalised solution seemed to be quite viscous, and it solidified in a thick multi-layered
pattern which is quite obvious in form of large sheets with perforations (figure 3.9). On theedges of the slide, the material crystallised into pleochroic crystals stemming from a seed
nucleus (figure 3.6). The optical properties of these structures displayed third order interference
colours along with the distinct presence of growth nucleus clearly identifying these crystals to
be formed of the host solution which includes sucrose.

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The ribbon like structures were seen in two states of deposition. One, where partial structureof the inclusion was visible above the solidified medium and part of it was almost transparent
and embedded into the medium and the other state was, where the ribbon was incorporated in
the medium and was barely distinguishable. The embedded portions seemed to create aquadrilateral geometry with a faint visual signal that seemed like a folded ribbon (figure 3.6).

Figure 3.8. Ribbon shaped forms half embedded in the medium. Square and quadrilateral crystals in the
background.

Figure 3.9. Flat, perforated several layered settlements of the medium.

Figure 3.10 exhibits various representative forms that were found in the vaccine. These forms
ranged from transparent or translucent sheet like forms, to dark almost opaque amorphous
carbon like materials of different sizes (figure 3.10j,k).

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Figure 3.10. Various inclusions found within Moderna 01.

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3.2.2. Raman Spectroscopic Investigation
Representative inclusions from Moderna 01 were examined by Raman spectroscopy. The
investigation clearly showed that all the inclusions have a strong carbon signal with confirmed
graphene compositions of some representative forms.

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Figure 3.11. Raman spectrum of representative inclusions in Moderna01.

The two clear Raman signals were obtained from two objects. The flat ribbon like inclusions
exhibited clear graphene spectra integrated with the spectrum of polyethylene glycol and otherminor compounds. The other clear signal was obtained from a calcite microcrystalline form
with a distinct strong peak at 1100cm-1.
The carbon composite forms had a highly complicated signal with clear graphene peak at
1600cm-1, but other peaks at 1100 cm-1 making the signal quite difficult to separate. Further
analysis is currently underway to isolate these signals and identify the other components of thisform of carbon. Some nano amorphous carbon forms showed a clear Graphene signal however,these forms also exhibited fluorescence which masked the Graphene peak.
These same forms were identified in other vaccine vials as well allowing for the establishmentof their composition consistency across the spectrum of various samples with confidence.
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The compositional identities derived of the inclusions through Raman spectroscopy was used
to quantify the comparative occurrence of these inclusions along a 2D track. This abundanceis presented as counts in the next section.
3.2.3. Counts

Figure 3.12. Count sheets at low and high resolutions along two tracks each for Moderna 01.

The inclusions were counted along two transects. Each transect was 2cm long. The counts were
performed at both: low and high magnifications.
The count distribution is dominated at the low resolution by GComposite 01. At low resolution,
GComposite 2 is present in very low quantities, but the abundance reverses at higher
magnifications. The counts are overall dominated by Graphene nano objects (figure 3.11).
It should be noted, that Graphene nano scrolls were omitted in the counts. This step wasnecessary because though, these nano scrolls form a significant percentage of the total counts,
a confirmation of their composition could not be attained within the limitations of this project.
As mentioned above in Section 3.1.5, thorough investigation of these forms now constitutes the
subject of enhanced second investigation project following this report.

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3.3.

Moderna 02

3.3.1. Microscopy
The sample material from Moderna 2, was translucent on slide with granular particulatematerial in suspension. On observation of the slide material under wet microscopy, several
floating bits of transparent sheet like objects were observed (figure 3.12). Heavier materialsank below as traction bedload and salted across the slide while the medium evaporated underthe microscopic lamp light.

Figure 3.13. Floating sheets of translucent material in a wet sample as observed under an optical
microscope. (b) The settled detrital material at the bottom of the slide. The shadows of the lighter floating
material can be seen as dark hazy figures.

In regions where the solution column was of a significant thickness, bubbles were observed as
flowing with the convection undercurrent towards the edge of the extent of the material. The
locus of these particles was clearly striking, even under the lowest magnification.
The graphs obtained by tracking the movement for these particles were typical of selfassemblage systems composed of particles coming together under the influence of various
intermolecular forces (figure 3.14).
An interpretation is here drawn in light of the knowledge, that these particles carry the required
m-RNA load and under the designated conditions, exhibit the self assemblage characteristics
using a combination of non-covalent intermolecular interactions such as electrostatic,hydrophobism, vander Waals and pi effects (figure 3.15).
The self assembly processes seem to be driven by a constantly changing competitiveenvironment which is driven by the kinematic and thermodynamically driven cursours
following a typical LaMer model. The seeding of the process seems to be around the nucleic
acid form of molecules and Graphene nano objects. According to Kulkarni et al. (2018), thegrowth of the particle relies on the pH neutralisation and migration of neutral, unboundPage 21 of 48

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ionisable lipid towards the LNP core regardless of the payload (mRNA, minicircular DNA or
pDNA).
These particles were observed in ubiquity across the sample preparations and each of thesestructures began with the formation of a small seed like particle to which the surrounding
particles aggregated, based on hydrophobic interactions.
What seems to be obvious through observation is that hydrophobic interactions appear to be
the dominant driving force of the LNP growth and electrostatic interactions guide the seed
formation and stability of the final assembly.

Figure 3.14. Motion graph of a characteristic self-assemblage particle. Accelerations in both X and Y
directions show typical staggered forms that typify hydrophobic/philic jumps and movements.

Figure 3.15. Self-Assembled Nano Particles with Payload of mRNA.

On drying of the sample, the solution settled into a thick layer, which retained some form ofmoisture. The relatively denser material settled to the bottom, while, the lighter material wason the top of the solidified solution with an additional detrital layer in the middle of themedium.
General overview of the dried slide at low resolution exhibited a translucent medium withforms akin to fibres and transparent sheets on the top surface (figure 3.16).
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Figure 3.16. Moderna 02 Low Magnification overview of the slide.

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On higher magnification, the slide material seemed to abound in carbon-related forms. Figure
3.17 shows different shapes and forms that were noted along various transects across the slide.
It is noteworthy, that the noted deposits are on three separate planes, with a significant
difference in the depth of focus.

Figure 3.17. Representative inclusions across Moderna 02 at high magnification.

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3.3.2. Raman Spectroscopic Investigation

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Figure 3.18. Raman spectra of various inclusions in Moderna 02.

Raman spectroscopy was used on the inclusions of Moderna 2 to identify the main
representations of the sample (figure 3.18). With the exception of calcite composite samples,
the rest showed a highly interfered spectrum. The carbon peaks at around 1600 cm-1 and 1350
cm-1 were only vaguely discernable in the Graphene nano objects. With the fluorescentbackground, it was extremely difficult to interpret the spectrum for any other component except
carbon.
A reshooting and further processing of the data is highly recommended for these Moderna
inclusions to be reasonably identified with some confidence.
In the absence of confident Raman signtures, for analysis of these inclusions, a comparative
similarity was used in the presence of the known chemistries of the other inclusions. Thesechemistries were used to do the counts which are present in figure.
3.2.3. Counts
Counting of inclusions along four tracks of two at low and two at high magnifications showed
results similar to Moderna 01 (figure 3.19). Graphene Composite 01 were prominently present

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Global Humanitarian Crisis Prevention and Response Unitat lower resolution and at higher resolutions, Graphene Nano objects are present in greatabundance.
It is clear from the counts, that the nanoscopic structures far exceed the density counts than
microscopic structures.
It should be noted, that Graphene nano scrolls were omitted in the counts. This step wasnecessary because though, these nano scrolls form a significant percentage of the total counts,
a confirmation of their composition could not be attained within the limitations of this project.
As mentioned above in Section 3.1.5, thorough investigation of these forms now constitutes the
subject of enhanced second investigation project following this report.

Figure 3.18. Count sheets at low and high resolutions along two tracks each for Moderna 02.

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3.4.

AstraZeneca

3.4.1. Microscopy
AstraZeneca was the third vaccine that was evaluated for its inclusions. Several fresh runs of
the wet samples were seen under the microscope. The AstraZeneca vaccine is almost
transparent when seen through the microscope, making the spotting of any inclusions with
inherent colours slightly easier.
The wetmicroscopic observation was that the fresh solution exhibited instantaneous

movement of nanoscopic particulate material (figure 3.20) which when observed closely,
evolved from being driven by the convection current, to being quite random. As the solution
dried up, the particles exhibited traction movement.

Figure 3.20. Nano Particles in motion when depth of the solution is greater than the height of the particle.
The particles coalesce to form bigger particles and the dominating influence on the direction of the
movement is through the currents within the solution.

These nanoscopic particles were quite noticeable as white specs in the beginning, as moving ina swarm like motion in the same general direction. With time, these evolved into bigger
droplets with more random vectors following the principles of self assembly.
As the solution dried, from the heat of the lamp of the microscope from the top, thesedimentation process began with sheet like forms being deposited on the top of the medium
which begins to crystallise early while the liquid below is still able to flow partially. Thisdifference in the crystallisation or solidification pace was clearly discernible from a few slide
shots off a video, as are presented in figure 3.21. In the figure, the microscopic form is clearlyvisible as it is lying on top of the solid film while the nanoparticles are still in motion in the
background as can be seen by the shifting position of the shadow. A clear output of thismechanism was that as the medium solidified, it becames more difficult for the nanoparticles
to navigate through the viscous material. The core of the nanoparticles was seeded with carbon
nanotubes and carbon nano objects: formed of some form of detrital carbon particles. These
were dragged along with the eddie currents at the bottom of the medium and as the material
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settled, the flute casts were left behind which were identified easily in the dry microscopic
sections.

Figure 3.21. Lamellar convection columns with solidified top resulting in deposition of the flaky particle.
The arrow points to the position of the nanoparticle in the deeper column of cooler material.

Even, in a wet slide under the microscope, several forms of inclusions were observed. These
are described in detail below.
Nano particle movement and self assembly process was noted in AstraZeneca, similar to what
was noted in the Moderna vaccines. Here too, the nano particles self assembled through typicalsignatory movements attributed to the inter-particle forces. While the solution dried up, theheavier carbon material that had sunk to the bottom of the slide was released from the nucleusof the nanoparticles and these now began to exhibit traction movement. Figure 3.22, shows therelative movement vectors of some of these solid materials. When these inclusions settled
down, they sank to the lower strata. A later, high resolution examination of the lower strata
shows a dump of graphene nano objects (figure 3.23) including graphene nano scrolls (figure3.24).

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Figure 3.22. Distance covered by the object through combined movement vectors

Figure 3.23. Graphene based structures settled down on drying. These structures demonstrated selfmovement against the growing viscosity of the drying solution. When the solution dried, they were foundto have settled at the bottom of the slide.

Figure 3.24. Graphene scrolls or spicules dumped at the bottom of the slide.

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Figure 3.25. Representative inclusions found within AstraZeneca vaccine.

The inclusions identified within AstraZeneca were of the same category as described in section
3.1. These included Graphene composites of two variations, Graphene ribbons impregnatedwith polyethylene glycol, Graphene Nano Objects including a vast number of nano scrolls anda high count of amorphous carbon. Calcite was also distinctly present in the vaccine in a
microcrystalline carbon composite form.
The type inclusions were targeted for Raman identification.

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3.4.2. Raman Spectroscopic Investigation

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Figure 3.26. Raman spectra of various inclusions in AstraZeneca.

Raman spectroscopy results on inclusions within AstraZeneca yielded direct confirmation of
the presence of Graphene in all the identified representative forms.
The carbon composites are of two forms as they are in the Moderna vaccines. These two forms
showed distinct graphene signatures through the characteristic Graphene peaks at 1600 cm-1
and 1350 cm-1. Besides Graphene, the spectrum is dominated by iron oxide and some otherforms of carbon associations.
Further work is ongoing to isolate the signals and help to identify individual compounds. With
the exception of one sample of graphene composite, the effect of fluorescence was found to beminimal in AstraZeneca’s selected inclusions.
Calcium composites with carbon also showed the same signature as the ones present in the twoModerna vaccines.
The inclusions in the calcite composites contained pristine graphene nano particles. These
particles were evaluated to show a clear signal for graphene. Though, the Graphene nanoobjects were present in clear association with the Graphene nano scrolls, Raman investigations
were not carried out on scrolls due to the limitation of the laser size and the microscope’s

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Global Humanitarian Crisis Prevention and Response Unitmagnification. In any future work on AstraZeneca, shooting Raman on the scrolls would be
one of the main targets in quantifying the concentration of Graphene.
The identification of the inclusions assisted in accurate counting process in the following stage.

Figure 3.25. Count sheets at low and high resolutions along two tracks each for AstraZeneca.

3.4.3. Counts
AstraZeneca counts do not pick up Graphene composite 2 in the lower magnifications howeverthese are present in significant numbers at higher magnifications. It is clear from the graph thatgraphene nano objects dominate the count at higher resolutions.
It should be noted, that Graphene nano scrolls were omitted in the counts. This step wasnecessary because though, these nano scrolls form a significant percentage of the total counts,
a confirmation of their composition could not be attained within the limitations of this project.
As mentioned above in Section 3.1.5, thorough investigation of these forms now constitutes the
subject of enhanced second investigation project following this report.

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3.5.

Pfizer

3.5.1. Microscopy
Pfizer was the fourth vaccine vial that was evaluated for its inclusions. The vaccine was noted
to be of the same offwhite colour as Moderna. 0.006 µL of the sample was transferred on theslide for wet evaluation and an equal amount was left in a slightly inclined pippette to allow
for examination in a closed environment in 3dimensions.
The pippete specimen showed some extremely interesting inclusions which were not foundwhen the slide dried. As the material was sucked into the pipette, distinct translucent totransparent sheets were seen floating about (figure 3.28). These were recognized from previous
observations in AstraZeneca and Moderna to be the Graphene Composite 1.
Active sedimentation of the denser material on the bottom curve of the cylinder was noted
immediately afterwards (figure 3.29).

Figure 3.28. Floating lighter material. In the background, the golden sparkly particles are the future selfassembly nano particles that will encapsulate the mRNA.

The two objects of interest that were clearly noted to be floating about but could not be locatedonce the slide had dried, were: (1) an extremely pointy transparent spicule like object (figure3.30) and the other was a thin translucent perforated sheet (figure 3.31).
Where, both the objects are of interest to this study, the nature of the spicule remains vital for
future work to identify.
As the solution was poured onto a slide for observation, the mixture exhibited the same
nanoparticulate self assembly mechanism as was observed in both Moderna and AstraZeneca
vaccines. As the material dried out, the inclusions settled at various depths depending upon
their relative densities.
Figure 3.32 shows an assembly of various forms of inclusion that were identified within Pfizer.
These fall in the same category as mentioned in section 3.1.

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Figure 3.29. Sedimentation of the heavier load on the curved bottom of the pipette and the floatingtransparent composite 01.

Figure 3.30. Sharp transparent spicule floating in the liquid.

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Figure 3.31. Floating perforated membrane.

Ribbon forms of nearly transparent microforms are found in fair number in the slide. These areoften half embedded in the solution with one end projecting outside the material.
The carbon composites of both form 1 and 2 also are present in great numbers. Form 1 settles
on top of the material while form 2 is found at mid levels of the solidified medium.
Graphene nano forms are present in fair numbers within the slide material along with some
scrolls.

Figure 3.32. Representative inclusions found within Pfizer vaccine.

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3.5.2. Raman Spectroscopic Investigation

Figure 3.33. Raman spectra of various inclusions in AstraZeneca.

Raman spectroscopy was conducted on four selected representative samples for Pfizer (figure3.32). Three of these samples showed carbon composite signatures with possible graphene inPage 38 of 48

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them. The signals of amorphous carbon like materials were extremely complex with carbon
along with iron oxide and several other compounds in them. The graphene complex 1 isgraphene with polyethylene glycol signal forming the bulk of the spectrum. Though, for initialassessments, this study can confirm the presence of graphene in Pfizer, however, the complex
with which it is associated still requires to be established through further work.
One of the sample that was shot, displayed a fair influence of fluorescence. Reshooting thissample at longer exposures is important to separate the signal from the article of interest withthe signal of the background.
In summary, Raman helped identify the inclusions within Pfizer and these identifications wereclassed in the counts.
3.5.3. Counts

Figure 3.34. Count sheets at low and high resolutions along two tracks each for AstraZeneca.

The counts for Pfizer were conducted along four tracks of two each for Low and Highmagnifications (figure 3.34). The relative numbers of carbon composite 2 is quite high in oneof the tracks whereas it is completely absent in the first track. At higher magnifications thecounts are dominated by the sheer number of nano graphene objects.

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It should be noted, that Graphene nano scrolls were omitted in the counts. This step wasnecessary because though, these nano scrolls form a significant percentage of the total counts,
a confirmation of their composition could not be attained within the limitations of this project.
As mentioned above in Section 3.1.5, thorough investigation of these forms now constitutes the
subject of enhanced second investigation project following this report.

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4. Interpretation, Discussions and Conclusion
Moderna

Table 1
Active Ingredients
List of declared ingredients for• mRNAthe vaccines.
Vehicles• SM102• Polyethylene Glycol• 2000 dimyristoyl glycerol (DMG)• Cholesterol• 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC)Inactive Ingredients• Tromethmine• Tromethamine hydrochloride• Acetic Acid
• Sodium Acetate• Sucrose

AstraZeneca
Active Ingredients• Adenovirus
Vehicles• L-histidine• L-histidine hydrochloride monohydrate
Inactive Ingredients• Magnesium Chloride Hexahydrate• Polysorbate 80• Ethanol• Sucrose• Sodium Chloride• Disodium edetate dihydrate (EDTA)• Water

PfizerActive Ingredients• mRNA
Vehicles• 4-hydroxybutyl azanediyl bis hexane -6.1-diyl bis 2 hexyldecanoate• Polyethylene Glycol• N,N di tetra decylacetamide• 1,2 di stearoyl sn glycerol 3phosphocholine• Cholesterol
Inactive Ingredients• Potassium Chloride• Monobasic Potassium Phosphate• Sodium Chloride• Dibasic sodium, phosphate dehydrate• Sucrose.
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The study subjects were four vaccine samples (2 of Moderna, 1 of AstraZeneca and 1 of Pfizer).
The objective of this study was to examine these four vaccine vials and document any
undeclared ingredients in the composition of the vaccines, with special focus on graphene andrelated products as well as any biological forms.
It should be noted, that all three vaccines have very different chemical ingredients as declaredby the manufacturers (Table 1). Despite, this genetic difference between them, upon
examination, the results reveal common undeclared inclusions in the four vials.
These inclusions are primarily with a focus on graphene and carbon related nano structures inform of carbon or graphene composites, graphene in association with polyethylene glycol,
graphene oxide, iron oxide compounds, and calcite. The variation in shapes of the logged
inclusions, points to their varied purposes in the wider arena of drug delivery and biosensory
fields.
The shapes identified during the course of this project can be classified into five different
categories1. Ribbon Forms (G-PEG)
2. Sheet forms (GC-1)
3. Tubular forms (GC-2)
4. Nano Dots
5. Nano Scrolls
Where, the roles of the Ribbons and sheets remain unclear due to their microscopic sizes, thetubular forms along with nano dots and scrolls seem to be aimed at enhancing cell acceptancefor the drug.
All three vaccines commonly employ the self-assembling lipid nano particles as drug delivery
mechanisms. Where, the central find of this project has been the confirmation of the presenceof graphene in all four samples, it is important to evaluate this find in the context of the subject
itself.
As mentioned above, self-assembly processes are the core delivery mechanisms for the three
types of vaccines. As has been reported under observations in the Chapter 3, the process initself, is primarily dependent on weak intermolecular interactions (Mendes et al. 2013) which,in the case of lipid nanoparticles is driven and determined by the kinetics and thethermodynamic environment. However, based on the observations within this study, tokickstart the nucleation or seeding of these particles in the first state, Graphene nanoparticles
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seem to play an important role. This interpretation is drawn from the observation that each of
the nanoparticles seem to have a graphene core made of nano particles that float in the
beginning and then saltate prior to settling down. This process creates a defect in the solution
medium thereby, facilitating the formation of the nano particle seed. Given, the highconcentration of graphene nano objects in all four vaccines, as the nanoparticles move throughthe medium, they must be incorporating not only graphene nanodots, but also nano scrolls,
thereby increasing their binding efficiency and their ability to carry their pay load.
As is already well known, the structure of the lipid nano particle plays a crucial role indetermining its efficiency to carry the nucleic acid payload. However, it is the payload’s
structure that defines the geometry of the lipid nanoparticles (Hajj et al. 2019, Kulkarni et al.
2018).
Though, these structures can be varied, depending upon the payload itself, the core
responsibilities of the nano lipid particles are the same; viz (1) protection of nucleic acids from
nucleases, (2) controlled release of nucleic acid, (3) cell and tissue selectivity, (4) high delivery
yield, (5) minimal toxicity, and (6) stability especially in long-term storage. These required
characteristics for a vector can been enhanced significantly by use of carbon nanotubes,
graphene and graphene oxide. This process was observed in action, when the experiments werebeing conducted during the course of this study and as reported in detail within in Chapter 3.
Nano tubes, both single and multiwalled, along with graphene forms are increasingly, paving
their way into targeted drug delivery (Sattari et al. 2021, Wu et al. 2018, Wierzbicki 2017,
Eatemadi et al. 2014). The samples evaluated during the course of this project have identified
graphene and other carbon composites as forming a striking percent portion of the ingredientsof these vaccines. Given the context of the increasing use of graphene in drug delivery, thefindings of this project seem to fit in the reference frame of attempts towards enhanced andtailored drug delivery.
The work carried out so far has been essentially a qualitative assessment of the contents. There
are several forms within these vaccines that require quantitative assessment and determination.
A major hurdle in achieving a good quantitative result was the failure to isolate the solid
faction. The method used in this project was the traditional method of slide preparation. It is

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Global Humanitarian Crisis Prevention and Response Unit

hoped, that if similar work were to be performed on additional samples, vacuum filtering would
be adopted as a mechanism of attaining cleaner samples for both Raman and SEM imaging.
Further, it is quite important to mention, that the source of fluorescence within the samples was
unknown while Raman investigations were underway. Due to extremely tight timescales, it
was not possible to complete the Raman re-runs where the effects of fluorescence would havebeen removed through targeted emittance and processing of the data. Despite this, the signals
were interpreted using similar data from various catalogues. It is hoped that further work willcontinue on the Raman spectroscopic investigations to attain cleaner spectra and to isolate theindividual spectrums of the current specimens.
In conclusion, it can be stated that the four samples of vaccines (Moderna 1, Modern 2,
AstraZeneca, Pfizer) all contain significant amount of carbon composites, graphene
compounds and iron oxide. These ingredients were undeclared by the manufacturers and areabsent from the list of ingredients for the vaccines.

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Ou et al. Particle and Fibre Toxicology (2016) 13:57
DOI 10.1186/s12989-016-0168-y

REVIEW

Open AccessToxicity of graphene-family nanoparticles:
a general review of the origins andmechanisms
Lingling Ou2, Bin Song1, Huimin Liang1, Jia Liu1, Xiaoli Feng1, Bin Deng3, Ting Sun2 and Longquan Shao1*
Abstract
Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many
fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility andtoxicity of GFNs in vivo and in intro. Generally, GFNs may exert different degrees of toxicity in animals or cell
models by following with different administration routes and penetrating through physiological barriers, subsequently
being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies
on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine thetoxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, andcorona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance,
physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In
these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha(TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial rolein these pathways. In this review, we summarize the available information on regulating factors and the mechanisms
of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim ofcompleting the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs andfacilitate their wide application.
Keywords: Graphene-family nanomaterials, Toxicity, Toxicokinetics, Mechanisms, Physicochemical properties, Future
prospects

Background
Graphene, which is isolated from crystalline graphite,
is a flat monolayer composed of single-atom-thick,
two-dimensional sheets of a hexagonally arranged
honeycomb lattice [1]. Because of its unique structural,
specific surface area and mechanical characteristics, thefunctions and applications of graphene have gained
considerable attention since the discovery of the material
in 2004 [2, 3]. Graphene and its derivatives include monolayer graphene, few-layer graphene (FLG), graphene oxide(GO), reduced graphene oxide (rGO), graphene nanosheets (GNS), and graphene nanoribbons, etc. [4–7].
GO is one of the most vital chemical graphene derivativesof the graphene-family nanomaterials (GFNs), which* Correspondence: shaolongquan@smu.edu.cn
1
Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
Full list of author information is available at the end of the article

attracts increasing attention for its potential biomedical
applications. Graphene-based materials usually have sizes
ranging from several to hundreds of nanometer andare 1-10 nm thick [8, 9], which is also the definitionof ‘nanoparticles’ or ‘nanomaterials’. Due to their exceptional physical and chemical properties, graphenematerials have been widely used in various fields, including energy storage; nanoelectronic devices; batteries[10–12]; and biomedical applications, such as antibacterials [13, 14], biosensors [15–18], cell imaging [19, 20],
drug delivery [8, 21, 22], and tissue engineering [23–25].
Along with the application and production of GFNs
increasing, the risk of unintentional occupational orenvironmental exposure to GFNs is increasing [26]. And
recently, there are some investigation on GFNs exposurein occupational settings and published data showed that

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Ou et al. Particle and Fibre Toxicology (2016) 13:57

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the occupational exposure of GFNs had potential toxicity to the workers and researchers [27–29]. GFNs canbe delivered into bodies by intratracheal instillation [30],oral administration [31], intravenous injection [32], intraperitoneal injection [33] and subcutaneous injection[34]. GFNs can induce acute and chronic injuries intissues by penetrating through the blood-air barrier,
blood-testis barrier, blood-brain barrier, and bloodplacenta barrier etc. and accumulating in the lung, liver,and spleen etc. For example, some graphene nanomaterials aerosols can be inhaled and substantial depositionin the respiratory tract, and they can easily penetrate
through the tracheobronchial airways and then transit
down to the lower lung airways, resulting in the subsequent formation of granulomas, lung fibrosis and adverse
health effects to exposed persons [2, 29]. Several reviews
have outlined the unique properties [35, 36] and summarized the latest potential biological applications of GFNs
for drug delivery, gene delivery, biosensors, tissue engineering, and neurosurgery [37–39]; assessed the biocompatibility of GFNs in cells (bacterial, mammalian andplant) [7, 40, 41] and animals (mice and zebrafish) [42];collected information on the influence of GFNs in the soil
and water environments [43]. Although these reviews
discussed the related safety profiles and nanotoxicology of
GFNs, the specific conclusions and detailed mechanismsof toxicity were insufficient, and the mechanisms of toxicity were not summarized completely. The toxicologicalmechanisms of GFNs demonstrated in recent studies
mainly contain inflammatory response, DNA damage,apoptosis, autophagy and necrosis etc., and those mechanisms can be collected to further explore the complex
signalling pathways network regulating the toxicity of
GFNs. It needs to point out that there are several factors which largely influence the toxicity of GFNs, suchas the concentration, lateral dimension, surface structure and functionalization etc. Herein, this review presents
a comprehensive summary of the available information on
the mechanisms and regulating factors of GFNs toxicity invitro and in vivo via different experimental methods, withthe goals of providing suggestions for further studies of
GFNs and completing the toxicology mechanisms to
improve the biological safety of GFNs and facilitate
their wide application.

toxicity of GFNs [44–46]. So to make them clear may be
helpful to better understand the laws of the occurrence
and development of GFNs toxicity.

Toxicity of GFNs (in vivo and in vitro)
GFNs penetrate through the physiological barriers orcellular structures by different exposure ways or administration routes and entry the body or cells, eventually
resulting in toxicity in vivo and in vitro. The varying
administration routes and entry paths, different tissue
distribution and excretion, even the various cell uptake
patterns and locations, may determine the degree of the

GFNs entry paths

Administration route

The common administration routes in animal models
include airway exposure (intranasal insufflation, intratracheal instillation, and inhalation), oral administration,
intravenous injection, intraperitoneal injection and subcutaneous injection. The major exposure route for GFNs
in the working environment is airway exposure, thus
inhalation and intratracheal instillation are used mostly
in mice to simulate human exposure to GFNs. Though
the inhalation method provides the most realistic simulation to real life exposure, instillation is more effective
and time-saving method, and GFNs was found that causing longer inflammation period using instillation (intratracheal instillation, intrapleural installation and pharyngeal
aspiration) than inhalation [24, 30, 47, 48]. GFNs were investigated to deposit in the lungs and accumulate to ahigh level, which retained for more than 3 months in thelungs with slow clearing after intratracheal instillation[49]. Intravenous injection is also widely used to assess the
toxicity of graphene nanomaterials, and graphene
circulates through the body of mice in 30 min, accumulating at a working concentration in the liver and bladder[32, 50–52]. However, GO derivatives had rather finite
intestinal adsorption and were rapidly excreted in adultmice via oral administration [31, 53]. Nano-sized GO(350 nm) caused less mononuclear cells to infiltrate subcutaneous adipose tissue after subcutaneous injection in
the neck region compared to micron-sized GO (2 μm)[34]. GO agglomerated near the injection site after intraperitoneal injection, and numerous smaller aggregates
settled in the proximity of the liver and spleen serosa[31, 33]. Experiments on skin contact with or skin permeation of GFNs were not found in the papers reviewed
here, and there is insufficient evidence available to conclude that graphene can penetrate intact skin or skinlesions. The route of nasal drops, which has been widelyused to test the neurotoxicity or brain injury potential ofother nanomaterials, was not mentioned in the papers
reviewed here.

GFNs reach various locations through blood circulationor biological barriers after entering the body, which
results in varying degrees of retention in different
organs. Due to their nanosize, GFNs can reach deeperorgans by passing through the normal physiologicalbarriers, such as the blood-air barrier, blood-testis
barrier, blood-brain barrier and blood-placental barrier.

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Blood-air barrier

Blood-placenta barrier

The lungs are a potential entrance for graphene nanoparticles into the human body through airway. The
inhaled GO nanosheets can destroy the ultrastructure
and biophysical properties of pulmonary surfactant (PS)film, which is the first line of host defense, and emerge
their potential toxicity [54]. The agglomerated or dispersed particles deposit on the inner alveolar surface
within the alveoli and then be engulfed by alveolar
macrophages (AMs) [55]. Clearance in the lungs is facilitated by the mucociliary escalator, AMs, or epithelial
layer [56–58]. However, some small, inhaled nanoparticles infiltrate the intact lung epithelial barrier and canthen transiently enter the alveolar epithelium or theinterstitium [59, 60]. Intratracheally instilled graphene
can redistribute to the liver and spleen by passing
through the air-blood barrier [61]. The study of bloodair barrier may draw an intensive attention, since theresearchers and workers occupational exposure of GFNs
usually through inhalation. To make clear how the
blood-air barrier plays a role in the toxicity of GFNs
may become a research hot topic.

The placental barrier is indispensable in maintaining
pregnancy, as it mediates the exchange of nutrients andmetabolic waste products, exerts vital metabolic functions and secretes hormones [67]. A recent review
suggested that the placenta does not provide a tightbarrier against the transfer of nanoparticles to foetuses,
specifically against the distribution of carbonaceous
nanoparticles to and in the foetus [42]. It was suggested
that rGO and gold particles (diameter of 13 nm) arebarely present or are absent in the placenta and foetus
in late gestation after intravenous injection [44, 68].
However, other reports showed that transplacental transfer does occur in late gestational stages [69, 70]. Muchattention had been paid to the developmental toxicity ofnanomaterials, and reports showed that many nanoparticles did cross the placental barrier and strongly influenced the development of embryos [71–75]. But studiesof the exposure to graphene materials through theplacenta barrier are deficient, and how these particles
transfer to embryos should be evaluated in detail in the
future.
These four barriers were the most frequently mentioned barriers in the literature, and other barriers havenot been evaluated in recent studies, such as skinbarriers, which have not been mentioned in any of thehundreds of GFNs toxicity studies searched. Moreover,the mechanism by which GFNs pass through thesebarriers is not well understood, and more systematicinvestigations are urgently needed.

Blood-brain barrier

The intricate arrangement of the blood-brain barrier,consisting of numbers of membrane receptors andhighly selective carriers, only exerts subtle influence on
blood circulation and the brain microenvironment compared to the peripheral vascular endothelium [62]. The
research on the mechanism of blood-brain barrier had
made some progress involved in diseases and nanotoxicity. Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry imaging (MSI) revealed
that rGO, with an average diameter of 342 ± 23.5 nm,
permeated through the paracellular pathway into theinter-endothelial cleft in a time-dependent manner bydecreasing the blood-brain barrier paracellular tightness [63]. In addition, graphene quantum dots (GQDs),
with a small size of less than 100 nm, can cross throughthe blood-brain barrier [64]. Studies on how graphenematerials pass through the blood-brain barrier and cause
neurotoxicity are very rare, and more data are neededto draw a conclusion.
Blood-testis barrier

The blood-testis and blood-epididymis barriers arewell known for being some of the tightest bloodtissue barriers in the mammalian body [65]. GO particles with diameters of 54.9 ± 23.1 nm had difficulty
penetrating the blood-testis and blood-epididymis
barriers after intra-abdominal injection, and the sperm
quality of the mice was not obviously affected even at
300 mg/kg dosage [66].

Distribution and excretion of GFNs in tissue

The absorption, distribution, and excretion of graphene
nanoparticles may be affected by various factors including the administration routes, physicochemical properties, particle agglomeration and surface coating of GFNs.
The different administration routes influence the distribution of GFNs, for example, intratracheally instilled
FLG passing through the air-blood barrier mainly accumulated and was retained in the lungs, with 47 %remaining after 4 weeks [61]. Intravenously administered
GO entered the body through blood circulation and washighly retained in the lung, liver, spleen and bonemarrow, and inflammatory cell infiltration, granuloma
formation and pulmonary edema were observed in thelungs of mice after intravenous injection of 10 mg kg/
body weight GO [49]. Similarly, high accumulation of
PEGylated GO derivatives was observed in the reticuloendothelial (RES) system including liver and spleen after
intraperitoneal injection. In contrast, GO-PEG and FLG
did not show detectable gastrointestinal tract absorption
or tissue uptake via oral administration [31].
The different properties of GFNs, such as their size,
dose and functional groups, always lead to inconsistentOu et al. Particle and Fibre Toxicology (2016) 13:57

results in the distribution profiles of graphene. For instance, Zhang et al. found that GO was mainly entrappedin mouse lungs [49]; however, Li et al. observed that GO
accumulated in mouse liver [76]. Notably, small GO
sheets, with diameters of 10–30 nm, were mainly distributed in the liver and spleen, whereas larger GO sheets(10–800 nm) mainly accumulated in the lungs [49, 52,77]. If the size of GO is larger than the size of the vessels,
GO usually becomes stuck in the arteries and capillaries
in the proximity of the injection site. The accumulation of
GO in the lungs was shown to increase with an increasein the injected dose and size, but that in the liver significantly decreased [78]. Coating biocompatible polymers
onto GO also affects the biodistribution, for instance, theintravenous injection of GO-PEG and GO-dextran (GODEX) accumulate in the reticuloendothelial system (RES),
including the liver and spleen, without short-term toxicity[31, 79]. Moreover, the charge of plasma proteins and adsorption of GO by plasma proteins also affects the biodistribution [34].
The excretion and clearance of GFNs vary in different
organs. In the lungs, observations indicated that NGO isdrawn into and cleared by AMs, which might be eliminated from the sputum through mucociliary clearance
or other ways [57], and 46.2 % of the intratracheallyinstilled FLG was excreted through the faeces 28 d after
exposure [61]. In the liver, nanoparticles can be eliminated thorough the hepato-biliary pathway following thebiliary duct into the duodenum [80]. In addition, PEGylated GNS that mainly accumulates in the liver andspleen can be gradually cleared, likely by both renal andfaecal excretion. As recently reviewed, GO sheets larger
than 200 nm are trapped by splenic physical filtration,
but small sizes (approximately 8 nm) can penetrate therenal tubules into the urine and be rapidly removed
without obvious toxicity [81]. The excretion paths of
GFNs have not yet been clearly explained, but renal andfaecal routes appear to be the main elimination routes
for graphene.
Recently, the distribution and excretion/toxicity strategy has become an important part of nano-toxicologicalstudies. To date, several controversial results regarding
the distribution and excretion of graphene in vivo havebeen reported in several papers, and a systematic evaluation of the toxicokinetics of GFNs is still needed. The
metabolism and excretion of nanomaterials are longperiod processes, however, the recent studies of GFNs
had been limited to short-term toxicological assessments, and the long-term accumulation and toxicity of
GFNs on different tissues remain unknown. Therefore,
long-term studies on the deposition and excretion of
GFNs need to be performed using different cells andanimals to ensure the materials’ biosafety before utilizationin human biomedical applications.

Page 4 of 24

Uptake and location of GFNs in cells

The uptake and location of GFNs have also been
observed to exert different effects in different cell lines.
Graphene is taken up into cells via various routes [82, 83].
Basically, the physicochemical parameters such as thesize, shape, coating, charge, hydrodynamic diameter,
isoelectric point, and pH gradient are important to allow
GO to pass through the cell membrane [84]. As statedpreviously, nanoparticles with diameters <100 nm canenter cells, and those with diameters <40 nm can enter
the nucleus [85]. For example, GQDs possibly penetratecell membranes directly, rather than through energydependent pathways [86, 87]. Larger protein-coatedgraphene oxide nanoparticles (PCGO) (~1 μm) enter cells
mainly through phagocytosis, and smaller PCGO nanoparticles (~500 nm) enter cells primarily through clathrinmediated endocytosis [88]. GO sheets could adhere andwrap around the cell membrane, insert in the lipid bilayeror be internalized into the cell as a consequence of interactions with cells [89]. Similarly, PEGylated reduced
graphene oxide (PrGO) and rGO were shown to adhere
onto the lipid bilayer cell membrane prominently due tothe interaction of hydrophobic, unmodified graphitic
domains with the cell membrane [90, 91]. Consequently,
it was suggested that prolonged exposure to or a highconcentration of graphene induces physical or biologicaldamage to the cell membrane, along with destabilizationof actin filaments and the cytoskeleton [92].
Current data demonstrates that GO sheets interact
with the plasma membrane and are phagocytosed bymacrophages. Three major receptors on macrophages
take part in the phagocytosis of GNS: the Fcg receptor(FcgR), mannose receptor (MR), and complement receptor (CR). Furthermore, FcgR is a key receptor in the
mediated phagocytic pathway [90, 93, 94]. The protein
corona of GO promotes the recognition by macrophage
receptors, especially the IgG contained within the protein corona. Macrophages were observed to undergo
prodigious morphological changes upon contact withGO [34]. After internalization, graphene accumulated in
the cell cytoplasm, perinuclear space, and nucleus,
which induced cytotoxicity in murine macrophages by
increasing intracellular ROS through depletion of themitochondrial membrane potential and by triggering
apoptosis through activation of the mitochondrial
pathway [83]. The possible interactions and accumulation sites of GFNs are summarized in Fig. 1.
Toxicity of GFNs in organs

The toxicity and biocompatibility of GFNs has been
observed and assessed through theoretical and animalmodel studies. At present, there are a mass of data demonstrating the toxicity of GFNs in different organs or
systems in animals, so that it is hard to list all the dataOu et al. Particle and Fibre Toxicology (2016) 13:57

Page 5 of 24

Fig. 1 Graphene materials and their biological interactions. (A) A parameter space for the most widely used graphene materials can be
described by the dimensions and surface functionalization of the material, the latter defined as the percentage of the carbon atoms in
sp3 hybridization. Green squares represent epitaxially grown graphene; yellow, mechanically exfoliated graphene; red, chemically exfoliatedgraphene; blue, graphene oxide. Note that a number of other graphene-related materials (such as graphene quantum dots and graphene
nanoribbons) are also being used in experiments. (B) Possible interactions between graphene-related materials with cells (the graphene
flakes are not to scale). (a) Adhesion onto the outer surface of the cell membrane. (b) Incorporation in between the monolayers of theplasma membrane lipid bilayer. (c) Translocation of membrane. (d) Cytoplasmic internalization. (e) Clathrin-mediated endocytosis. (f) Endosomal or
phagosomal internalization. (g) Lysosomal or other perinuclear compartment localization. (h) Exosomal localization. The biological outcomes fromsuch interactions can be considered to be either adverse or beneficial, depending on the context of the particular biomedical application. Different
graphene-related materials will have different preferential mechanisms of interaction with cells and tissues that largely await discovery. [90]Copyright (2014), with permission from American Association for Advancement of Sciencein this review. Thus we summarized a certain number
literature and chose some in vivo toxicological studies of
GFNs listed in Table 1.
Toxicity in internal organs

GO can result in acute inflammation response andchronic injury by interfering with the normal physiological functions of important organs [32, 81]. Oralgavage experiments did not show detectable absorptionof GO through the gastrointestinal tract [95]. Interesting, a low dose of GO caused serious damage to thegastrointestinal tract after maternal mice drank a GO
suspension rather than a high-dose of GO because a low
dose of GO without agglomeration can easily attach to
the gastrointestinal surface and cause destruction
through its abundant sharp edges [53]. GFNs caused
inflammation and remained in the lung on day 90 after asingle intratracheal instillation, and even translocated tolung lymph nodes by a nose-only inhalation [96, 97]. A
high dose of GO that forms aggregations can block pulmonary blood vessels and result in dyspnea [50, 98], andplatelet thrombi were observed at high concentrationsof 1 and 2 mg/kg body weight via intravenous injection [89]. GO reportedly disrupted the alveolarcapillary barrier, allowing inflammatory cells to infiltrate into the lungs and stimulate the release of proinflammatory cytokines [99]. Fibrosis and inflammation could be verified by the increased levels of theprotein markers collagen1, Gr1, CD68 and CD11b in
the lungs. The use of Tween 80 to disperse FLG or apluronic surfactant to disperse graphene wassuggested to reduce the likelihood of lung fibrosis formation in cells or mice, whereas lung fibrosis was observed when graphene was suspended with bovine
serum albumin (BSA) [100]. In addition, radioactive
isotopes can be delivered into the lungs, accompanied
by a depth distribution of 125I-NGO in the lungs, andthe isotopes might deposit there and result in mutations and cancers [30]. However, recent publications
claimed no obvious pathological changes in mice exposed to low dosages of GO and functionalized graphene by intravenous injection, including aminated
GO (GO-NH2), poly(acrylamide)-functionalized GO (GOPAM), poly(acrylic acid)-functionalized GO (GO-PAA) and
GO-PEG; only GO-PEG and GO-PAA induced less toxicity
than pristine GO in vivo [31, 79, 89]. So the functionalgroups of GFNs and the working concentration or aggregate state largely influence the toxicity of GFNs.
Recently, the ways to modify the functional group of
GFNs, decrease the working concentration or change
the aggregate condition are usually used to decrease
the toxicity of GFNs.
Toxicity in the central nervous system

Graphene has largely benefited neurosurgery with theapplication of drug/gene delivery for brain tumour treatment, intracranial and spinal biocompatible devices,
biosensing and bioimaging techniques. Studies regarding
the potentialities or risks of graphene in the brain haveemerged. In the chicken embryo model, pristine graphene flakes decreased the ribonucleic acid level and therate of deoxyribonucleic acid synthesis, leading toGraphene family
nanomaterials

Physiochemial properties
and functionalization

Dose and time incubation

Effects

Reference

Nanoscale graphene oxide No information(NGO)

C57BL/6 mice0, 1, 5, 10 mg/kg, intratracheal instillation
0 h, 24 h, 48 h, 72 h and 1 week

Result in acute lung injury (ALI) and chronic
pulmonary fibrosis[30]

Few layer graphene (FLG)

No information

ICR mice

0.1, or 1 mg/mL, oral gavage or intratracheal Intratracheally instilled FLG resulted in acuteinstillation 3 or 28 days
lung injury and pulmonary edema, FLG didn’t
show detectable absorption through thegastrointestinal tract by oral gavage.

[61]

Graphene platelets (GPs)

No information

Mice

inhalation exposure, 1 day-6 weeks

GP caused acute inflammation in lung at
1 day, and alleviated inflammation in lungafter 6 weeks[48]

Graphene nanoplatelets(GPs)

Thickness of 10 nm
Size of 5–30 μm

Female C57BL/6
strain mice

50 μg per mouse, pharyngeal aspiration or
intrapleural installation, 24 h- 7 days

Large GP were inflammogenic in both thelung and the pleural space

[24]

GO

Thickness of 0.93 nm
Size of 150–250 nm

Sprague-Dawley rats

0.5 or 4 mg/m3, inhalation exposure,single 6 h

The single inhalation exposure to GO induce
minimal toxic responses in rat lungs

[235]

GO

Thickness of 0.9 nm
size of l-GO: 1–5 μm
size of s-GO:100–500 nm

Male ICR mice

1.0 mg/kg, intravenous injected, 24 h

Accumulated mainly in the liver and lungs

[78]

GO

Thickness of < 4 nm
size of l-GO:237.9 ± 79.3 nm; size ofs-GO: 54.9 ± 23.1 nm

Male and female
ICR-strain mice

24 mg/kg, tail vein injected, 5 days

Didn’t effect pup numbers, sex ratio, weights,
pup survival rates or pup growth, low toxicity
for male reproduction

[66]

GO

Thickness of ~1.0 nm
sizes of 10–800 nm

Kun Ming mice

1,10 mg/ kg, intravenous injection 14 days

Led to high accumulation, long-time retention,
pulmonary edema and granuloma formation[49]

NGO-PEG

Thickness of 1 nm
size of 10–800 nm

Male Kunming mice

5 mg/kg, tail intravenous injection
10 min-24 h

NGO-PEG alleviated acute tissue injuries,
decreased the early weight loss

[81]

GO
GO-PEG
RGO-PEGnRGO-PEG

Thickness of 0.94,1.22, 4.43 and5.66 nm,size of 450, 25, 50 and 27 nm

Balb/c mice

4 mg/kg, intraperitoneal injection
1, 7 and 30 days

Accumulated in the reticuloendothelial (RES)
system including liver and spleen over along time[31]

GO
Graphene quantum dots
(GQD)

Thickness of GO, GQD: 0.5–1 nm
sizes of GO, GQD: 3–5 nm

Balb/c mice

20 mg/kg intravenous injection or
intraperitoneal injection 14 days

GO appeared toxic and caused death
GQD revealed no accumulation in organs andcaused low cytotoxicity[176]

Purified graphene oxide(pGO)

Thickness of 1–2 nm,
lateral dimension of
100–500 nm

Female C57Bl/6 mice 50 μg/animal, intraperitoneal injection
24 h, 7 days,

Induced moderate inflammation and granuloma
formation following

[99]

GO

Thickness of 3.9 and 4.05 nm,
size of 350 nm and 2 μm

C57BL/6 male mice

Series concentrations, subcutaneous
injection21 days

The micro-size of GO induced much stronger
inflammation responses than the nanosized GO

[34]

GO

Size of 1110 to 16 200 nm

C57BL/6 J mice

2 or 20 mg/kg, subcutaneous andintraperitoneal injection

Both GO and a reduction of GO result in immune
cell infiltration, uptake, and clearance.

[84]

RGO-iron oxide
nanoparticles (rGO-IONP)Thickness of ˂10 nm
Size of 15.0 ± 2.0 nm

Female Balb/c mice

400 μg, subcutaneous injection,

RGO–IONP can effectively inactivate multiple-drug- [236]resistant bacteria in subcutaneous abscesses

Page 6 of 24

Animals

Ou et al. Particle and Fibre Toxicology (2016) 13:57

Table 1 Toxicity of GFNs in organs

GO
GO-PEG

Thickness of 0.94, 1.22, 4.43
and 5.66 nm,
size of 450, 25, 50 and 27 nm

Female balb/c mice

100 mg/kg, Oral administration; 50 mg/kg,
intraperitoneal injection, 1, 7 and 30 days

No obvious tissue uptake via oral administration,
indicating the rather limited intestinaladsorption of those nanomaterials

[237]

RGO

sizes of small rGO: 87.97 ± 30.83,
sizes of large rGO:472.08 ± 249.17 nm

Male C57black/6
mice

60 mg/kg, oral gavage, 5 days

RGO affected general locomotor activity, balance,and neuromuscular coordination, but showed
little change in exploratory, anxiety-like, or
learning and memory behaviors.

[31]

Ou et al. Particle and Fibre Toxicology (2016) 13:57

Table 1 Toxicity of GFNs in organs (Continued)

Page 7 of 24

Ou et al. Particle and Fibre Toxicology (2016) 13:57

harmful effects on brain tissue development and theatypical ultrastructure was observed in the brain [101].
The recent researches of GFNs in the central nervous
system are mostly involved in the application rather than
the toxicity. The data of the toxic study on GFNs isunderway.
Toxicity in reproduction and development system

Pristine graphene reduced the vascularization of theheart and the density of branched vessels after injection
into fertilized chicken eggs followed by incubation for 19
d [101]. GO and rGO damage zebrafish embryos byinfluencing the embryo hatching rate and body length ina concentration-dependent manner. Although no obvious malformation or mortality was observed in exposedzebrafish embryos [102], GO adhered to and was wrappedin the chorion of the zebrafish embryos, causing remarkable hypoxia and hatching delay. GO aggregates wereretained in many organelles, such as the eyes, heart, yolksac, and tail of the embryos, and apoptosis and reactive
oxygen species (ROS) generation were observed in theseregions [103].
The GFNs exert different toxicological effects on maleor female reproductive system. Data showed that GOexerted very low or nearly no toxic effects on male
reproduction even at a high dose via intra-abdominal
injection [66]. Additionally, rGO did not change theserum estrogen levels of non-pregnant female mice. The
condition is different in the female mouse: mouse dams
could give birth to healthy offspring after rGO injection
before mating or during early gestation, and only a few
abnormal foetuses were present among the rGO-injected
dam litters. However, the pregnant mice had abortions
at all dose, and most pregnant mice died when the highdose of rGO was injected during late gestation [44]. Notably, the development of offspring in the high dosage
group was delayed during the lactation period. The highdose of GO decreased the maternal mice’s water consumption by oral exposure, which reduced milk production and thus postponed the growth of offspring [53].
Though the findings indicate that GFNs are potentiallyharmful to development, but data on reproductive anddevelopmental toxicity are still deficient. Studies of theinfluence of GFNs on male and female reproduction
and development are still required to elucidate theunderlying toxicity mechanism.
Influence of haemocompatibility

GO release into the blood is ineluctable. The haemocompatibility of GO was found to be dependent on thefunctional coating and the exposure conditions. GOwith submicron size resulted in the greatest haemolytic activity, while aggregated graphene induced thelowest haemolytic reaction. Pristine graphene and GO

Page 8 of 24

demonstrated haemolytic effect up to 75 μg/mL [104].
GO-polyethylenimine (GO-PEI) exhibited notable toxicity
by binding to HSA, even at 1.6 μg/mL [105]. Carboxylated
graphene oxide (GO-COOH) showed significant cytotoxicity toward T lymphocytes at concentrations above
50 μg/mL and had good biocompatibility below 25 μg/
mL, whereas GO-chitosan nearly inhibited haemolytic
activity [106]. Until now, the corresponding risk of haemocompatibility has remained largely unknown.
In conclusion, the lung injury induced by GFNs has
been studied in several studies, the results of which havedemonstrated inflammatory cell infiltration, pulmonary
edema and granuloma formation in the lungs. However,
only a few specific studies have evaluated in other organs, such as the liver, spleen, and kidney, and the injury
symptoms, damage index and level of damage to theseinternal organs were not fully investigated. Moreover,studies on the neurotoxicity of GFNs are quite rare; nodata has revealed which nerves or brain areas experiencedamage, nor have the related behavioural manifestations
been studied. The developmental toxicity of GFNs may
induce structural abnormalities, growth retardation, behavioural and functional abnormalities, and even death.
A study on the reproductive and developmental toxicity
of GFNs will be extremely significant and gain extensive
attention in the future. Almost all the GFNs toxicity
studies were short-period experiments, and no studies
have investigated long-term chronic toxic injury. However, based on studies of other nanomaterials toxicity,
long-term GFNs exposure may be an important factor
harming health [107–109]. Therefore, the long-term
study of GFNs is necessary.

Toxicity of GFNs in cell models

The cytotoxicity of GFNs in vitro has been verified invarious cells to change the cell viability and morphology,
destroy the membrane integrity, and induce DNA
damage [110–112]. GO or rGO decrease cell adhesion;induce cell apoptosis; and enter lysosomes, mitochondria, cell nuclei, and endoplasm [113]. GQDs enteredcells and induced DNA damage by the increased expression of p53, Rad 51, and OGG1 proteins in NIH-3 T3cells [87]. However, GQDs did not pose significant toxicity to human breast cancer cell lines (at a dose of
50 μg/mL) or human neural stem cells (at a dose of
250 μg/mL) [114, 115]. GO derivatives dramaticallydecreased the expression of differential genes that are
responsible for the structure and function of the cell
membrane, such as regulation of the actin cytoskeleton,
focal adhesion and endocytosis [89]. In rat pheochromocytoma cells (PC12 cells), graphene and rGO caused
cytotoxic effects and mitochondrial injury, such as therelease of lactate dehydrogenase (LDH), an increase inOu et al. Particle and Fibre Toxicology (2016) 13:57

the activation of caspase-3, and the generation of ROS[82, 116].
Graphene can increase cell viability [117] or cause cell
death [118] depending on the cell line, type of graphenematerial and the doseage. GO cytotoxicity was observedin human fibroblasts and lung epithelial cells at concentrations above 20 μg/mL after 24 h, but minimal toxicity
was found in A549 cells at concentrations higher than
50 μg/mL [119]. The biological responses induced by
GO such as ROS, malondialdehyde (MDA), and LDH increased, whereas superoxide dismutase (SOD) decreased
dose-dependently in HeLa cells [120]. However, GOmolecular beacon (GO-MB) showed low cytotoxicity
even at 20 μg/mL in HeLa cells [121]. GO decreased theviability of A549 cells, while the same concentration andtime of exposure increased the cell viability of CaCo2
colorectal carcinoma cells [122]. Another study reported
that GO dramatically enhanced the differentiation of
SH-SY5Y, accompanied by increasing neurite length andthe expression of neuronal marker MAP2 at low concentrations but that GO suppressed the viability of SHSY5Y cells at high doses (≥80 mg/mL) [123]. Functionalized coatings on GO, such as GO-PEG [124] and GOchitosan [125], can profoundly attenuate the particles’
cytotoxicity by inhibiting the interactions between cells.
The toxicity of GFNs in vitro is summarized in Table 2.
Data on the cytotoxicity of graphene nanomaterials arecontrasting, and varying characteristics influence the results. The mechanisms and influencing factors of
toxicity need to be elucidated in detail.

Origins of GFNs toxicity
Reportedly, the characteristics of graphene, including its
concentration, lateral dimension, surface structure, functional groups, purity and protein corona, strongly influence its toxicity in biological systems [2, 7, 104, 126–129].
Concentration

Numerous results have shown that graphene materials
cause dose-dependent toxicity in animals and cells, suchas liver and kidney injury, lung granuloma formation,
decreased cell viability and cell apoptosis [130–134]. In
vivo studies, GO did not exhibit obvious toxicity in mice
exposed to a low dose (0.1 mg) and middle dose
(0.25 mg) but induced chronic toxicity at a high dose
(0.4 mg). The high content of GO mainly deposited in
the lungs, liver, spleen, and kidneys and was difficult to
be cleaned by the kidneys via a single tail vein injection[135]. Intriguingly, increasing the dose resulted in a dramatic decrease in the hepatic uptake but an increase in
the pulmonary uptake of s-GO by intravenous injection[31], because the high dose of GO potentially surpassed
the uptake saturation or depleted the mass of plasma
opsonins, which consequently suppressed the hepatic

Page 9 of 24

uptake. Moreover, an in vitro study reported that 20 μg/mL
GO nanosheets exhibited no cytotoxicity in A549 within
2 h of incubation, but a higher concentration (85 μg/mL)
decreased the cell viability to 50 % within 24 h [136, 137].
Lü et al. also demonstrated that GO had no obvious
cytotoxicity at low concentrations for 96 h in a humanneuroblastoma SH-SY5Y cell line, but the viability of cells
sharply decreased to 20 % after treatment with 100 mg/mL
GO for 96 h of incubation [123]. The results in HeLa cells,
NIH-3 T3 cells, and breast cancer cells (SKBR3, MCF7)
treated with graphene nanoribbons also showed a dose(10–400 mg/ml) and time-dependent (12–48 h) decrease incell viability [138]. Increasing concentrations of GO entered
the lysosomes, mitochondria, endoplasm, and cell nucleus[119]. Several data indicated that rGO caused apoptosismediated cell death at a lower dose and early time point
but that necrosis was prevalent with the increase in time/dose [110, 135].
Lateral dimension

Nanoparticles with sizes <100 nm can enter the cell,<40 nm can enter nucleus, and smaller than <35 nm cancross the blood brain barrier [85]. One study showedthat GO (588, 556, 148 nm) did not enter A549 cells
and had no obvious cytotoxicity [112]. When the diameter of graphene is between 100 ~ 500 nm, the smallest
size may cause the most severe toxicity, and when the
diameter is below 40 nm, the smallest sizes may be thesafest. For instance, rGO with a diameter of 11 ± 4 nm
could enter into the nucleus of the hMSCs and cause
chromosomal aberrations and DNA fragmentation atvery low concentrations of 0.1 and 1.0 mg/mL in 1 h.
However, rGO sheets with diameters of 3.8 ± 0.4 nm
exhibited no notable genotoxicity in hMSCs even at ahigh dose of 100 mg/mL after 24 h [118].
In an in vivo study, s-GO (100–500 nm) preferentiallyaccumulated in the liver, whereas l-GO (1–5 μm) was
mainly located in the lungs because l-GO formed larger
GO-protein complexes that were filtered out by thepulmonary capillary vessels after intravenously injection[31]. Given the relative lateral sizes (205.8 nm, 146.8 nm
and 33.78 nm) of the three GO nanosheets at the same
concentration, smaller GO experiences much greater uptake than larger GO in Hela cells [139]. The high uptakeof s-GO changed in the microenvironment of cells andconsequently induced the greatest viability loss and mostserious oxidative stress among three sizes of GO samples[119]. As a result, one study delineated that GO sizedependently induced the M1 polarization of macrophages and pro-inflammatory responses in vitro and invivo. Larger GO showed stronger adsorption onto theplasma membrane with less phagocytosis, eliciting
robust interactions with TLRs and activating NF-κB
pathways, compared to smaller GO sheets, which werePhysiochemial properties andFunctionalization

Cells

Dose and time incubation

Effects

Reference

Pristine graphene

Thickness of 2–3 nm, size of
500–1000 nm

Murine RAW 264.7 macrophages 5, 10, 20, 40, 80 and 100 mg/
mL, 48 h

Pristine graphene

Thickness of 3–5 nm, size of
100–110 nm

Rat pheochromocytoma cells
PC12 cells

10–100 μg/mL 1–48 h

Increased LDH release, ROS levels andcaspase3 activation, induced apoptosis[82]

Graphene oxide(GO)Four different diameters(342–765 nm)

Human Erythrocytes
Human skinfibroblasts CRL-2522

3.125-200 μg/mL 24 h

Hemolytic activity, ROS generation, LDH
release, decreased cell viability

[106]

GO

Thickness of 0.9 nm
lateral size: s-GO, 160 ± 90 nm;
m-GO, 430 ± 300 nm;
l-GO, 780 ± 410 nm

Human lung epithelial A549
cells

10, 25, 50, 100 and 200 μg/mL
24 h

Dose-dependent oxidative stress, cell viability [119]decreased at high concentration

GO

Thickness of 1 nm, lateraldimension of 200–500 nm

Human lung fibroblast cells HLF
cells

10–500 μg/mL 2–24 h

Oxidative stress induced, concentrationdependent cytotoxicity and genotoxicity

[148]

GO

Size distribution: 592 ± 10.9 nm
in PBS, 1272 ± 56.2 nm in FBS

HeLa cells

0–80 μg/mL 24 h

Released LDH, increased MDA and ROS
generation, decreased SOD, reduction ofcell viability,

[120]

GO

smaller-sized GO: 50–350 nm
Macrophage cell J774A.1
intermediate-sized GO: 350–750 nm THP-1 cells
HEK293 cells
larger-sized GO: 750–1,300 nm
MEL cells
HUT102 cells

20 μg/mL 1-24 h

Size-dependent M1 induction ofmacrophages,
pro-inflammatory responses

[94]

GO

thickness: < 2 nm,
lateral size: 450 nm

Mouse CT26 colon carcinoma
cell

50–100 μg/mL 18 h

Triggered autophagy, enhances cell death[206]

Reduced graphene oxide (rGO)

Thickness of 11 ± 4 nm
lateral size of 3.8 ± 0.4 μm

Human mesenchymal stem
cells (hMSCs)

0.01–100 μg/mL 1–24 h

Induced DNA fragmentations andchromosomal aberrations

[118]

RGO

Thickness of 7 nm
lateral size of 40 nm

human liver carcinoma
cells (HepG2 cells)

1–200 mg/L 4–72 h

Dose-dependent DNA damage, oxidative
stress, cytotoxicity

[31]

RGO

Lateral size of 100–1500 nm

U87 and U118 glioma
cell lines

0–100 μg/mL 24 h

Reduction of cell proliferation and cell
viability, induced apoptosis[238]

Bacterially reduced graphene oxide Thickness of 4.23 nm(B-rGO)
average size of 3833 nm

MCF-7 cells

20–100 μg/mL 24–72 h

Increased ROS generation, released LDH,
dose-dependent toxicity[181]

Reduced graphene oxide
Nanoribbons(rGONR)

Thickness of 1 nm,
length of 10 μm,
width of 50–200 nm,

hMSCs

0.01, 0.1, 1.0, 10, 100 μg/mL
96 h

Caused DNA fragmentations andchromosomal aberrations[239]

Reduced graphene oxide sheets(rGOSs)Thicknesses of ~1.2 nm,
lateral sizes of ~2 μm

hMSCs

0.01, 0.1, 1.0, 10, 100 μg/mL
96 h

Caused slight cell membrane damage andcytotoxicity[239]

Graphene-dextran(GO-DEX)

Thickness of 2.8 nm
size of 50–100 nm

HeLa cells

10, 50,200 mg/L 24, 48, 72 h

GO-DEX remarkably reduced cell toxicity[91]

GNP-COOH
GNP-NH2

Thickness of GNP-COOH: 735.9 nm
thickness of GNP-NH2: 945.5 nm

Human bronchial epithelial
cells (BEAS-2B cells)

10, 50 mg/L 24 h

Caused single stranded DNA damage,genotoxicity and hypomethylation[240]

Depleted of the mitochondrial membrane[83]
potential, increased ROS, triggered apoptosis

Page 10 of 24

Graphene family
nanomaterias

Ou et al. Particle and Fibre Toxicology (2016) 13:57

Table 2 Toxicity of GFNs in cell models

10–400 μg/mL 24–48 h

Dose-dependent and time-dependent
decrease in cell viability[138]

Thickness of 1–2 nm lateral width of Human lung fibroblast cells
100–500 nm

1, 10, 50, 100 μg/ml 24 h

Caused concentration-dependent
cytotoxicity and genotoxicity[15]

PEG-GQD

Sizes of 3–5 nm

HeLa cells and A549 cells

10–160 μg/mL 24 h

No noticeable cytotoxicity[176]

FBS-GO

Thickness of 4.0–18.0 nm

A549 cells

0–200 μg/mL 24 h

Cytotoxicity of GO was greatly mitigatedat 10 % FBS[166]

PEG-DSPE (O-GNR-PEG-DSPE)

Width of 125–220 nm, lengths
between of 500–2500 nm

PEI-GO,PEG-GO,
LA-PEG-GO

HeLa cells
NIH-3 T3 cells
SKBR3 cells
MCF7 cells

Ou et al. Particle and Fibre Toxicology (2016) 13:57

Table 2 Toxicity of GFNs in cell models (Continued)

Page 11 of 24

Ou et al. Particle and Fibre Toxicology (2016) 13:57

more likely taken up by cells [94]. To further uncover
the detailed mechanism underlying these effects, morestudies are needed to illustrate the vital mechanism of
the lateral size of graphene materials.
Surface structure

GFNs possess widely varying surface chemistries. For
example, the pristine graphene surface is hydrophobic,
GO surface is partially hydrophobic with carboxylategroups [140–142], and rGO has intermediate hydrophilicity [143]. GFNs were observed to disrupt the function
and structure of cell membranes and proteins probably
by exceptionally strong molecular interactions with cells[2, 91]. For instance, rGO bonded to cell membranes,
stimulated receptors and activated mitochondrial pathways, inducing apoptosis [110, 111, 144]. Limited evidence showed that GO is smaller and less toxic thanrGO because of the high oxygen content, smootheredges, and hydrophilic properties of the former species[104, 145, 146]. Because of the different surface oxidation states of GO and rGO, GO possessing distincthydrophilicity might be internalized and taken up by
HepG2 cells easily. On the contrary, rGO with evident
hydrophobicity, could be adsorbed and aggregated at cellsurfaces without (or with lower) uptake [110]. Due tostrong π-π stacking interactions, graphene is highly
capability of breaking many residues of the protein,
particularly the aromatic ones, such as the villin headpiece

Page 12 of 24(HP), F10, W23, and F35. The protein’s secondary and tertiary structures are largely lying on the graphene surface,disrupting the structure and function of the protein [41](Fig. 2). In addition, GO can insert between the basepairs of double-stranded DNA and disturb the flow ofgenetic information at the molecular level, whichmight be one of the main causes of the mutagenic
effect of GO [7, 112, 146, 147].
ChargeA number of studies have highlighted the importance of
the GO surface charge because of its ability to affect theinternalization and uptake mechanism of cells [148–150].
GO internalization was negligible in non-phagocytes,
which was likely due to the strong electrostatic repulsion
between the negatively charged GO and the cell surface[34]. However, others have suggested that negatively
charged nanoparticles can be internalized into nonphagocytic cells by binding to available cationic sites
on the cell surface and be taken up by scavenger receptors [110, 146, 150]. GO/GS particles reportedly
cause morphological changes and significant lysis,
leading to high haemolysis in red blood cells (RBCs).
RBC membrane disruption is probably attributed tothe strong electrostatic interactions between the negatively charged oxygen groups on the GO/GS surface
and positively charged phosphatidylcholine lipids on
the RBC outer membrane [106].

Fig. 2 A representative trajectory of HP35 adsorbing onto the graphene. (a) Representative snapshots at various time points. The proteins
are shown in cartoons with red helix and green loop, and the graphene is shown in wheat. The aromatic residues that form the π-πstacking interactions are shown in blue, others are shown in green. (b) The contacting surface area of HP35 with the graphene. (c)The RMSD of HP35 from its native structure and the number of residues in the α-helix structure. Here, the secondary structures are
determined by the DSSP program. (d) The distance between the graphene and the aromatic residues, including F35, W23, F10, F17,
and F06. To show the adsorbing process clearer, the χ-axis had been truncated and rescaled. [41] Copyright (2011), with permission
from Journal of Physical of ChemistryOu et al. Particle and Fibre Toxicology (2016) 13:57

Functionalization

Studies confirmed that functionalization with PEG [52],PEGylated poly-L-lysine (PLL) [151], poly(ε-caprolactone)[152], polyvinyl alcohol [3], Pluronic [153], amine [98],carboxyl, and dextran [79] groups largely decreases the
toxicity and improves the biocompatibility of graphene. In
vivo results revealed that only mild chronic inflammation
emerged after the subcutaneous injection of GO-Pluronichydrogel and no noticeable short-term toxicity was tested
after the intravenous injection of GO-DEX [79, 154].
PEGylated GS did not induce appreciable toxicity in mice
exposed to 20 mg/kg for 3 months, as evaluated by bloodbiochemistry and histological examinations, and showed
relatively low retention in the RES [52, 155]. Coating GOwith chitosan almost eliminated the haemolytic activity inblood [39]. Moreover, the PEG coating effectively alleviated GO-induced acute tissue injuries; decreased GO
aggregation and retention in the liver, lungs, and spleen;and promoted the clearance of GO [81], GO-DEX [79],and fluorinated graphene oxide (FGO) [156].
In vitro, several cell function assays showed clear
evidence that the surface functionalization of pristinegraphene or GO was critical for reducing the strong
toxicity effects [91]. PEG-GO, PEI-GO and LA-PEG-GO
damaged human lung fibroblast cells less than GO[148]. PEG-GO exhibited no cytotoxicity toward several
cell cultures, such as glioblastoma cells (U87MG), breast
cancer cells (MCF-7), human ovarian carcinoma cells(OVCAR-3), colon cancer cells (HCT-116), and lymphoblastoid cells (RAJI), at concentrations up to 100 μg/mL[119, 157, 158]. GQDs-PEG exhibited very low or no
toxicity against lung and cervical cancer cells even atvery high concentrations (200 μg/mL) [159]. However, asa non-biodegradable material with great potential forcellular internalization, further investigation is needed toassess the possible long-term adverse effects of functionalized graphene.
Aggregations and sedimentation

Reportedly, nanomaterials have a propensity to form
aggregates rather than individual units, particularly underphysiological conditions. GS surfaces allowed fewer RBCs
attach comparing to GO, and GS had the lower haemolytic activity for more aqueous aggregations formation. In
contrast, the fast sedimentation and aggregate formationof GS greatly inhibited the nutrient availability of humanskin fibroblast cells that were grown on the bottom ofwells [106]. Therefore, the aggregations and sedimentationof graphene particles exert varying effects on different
cells.
Impurities

Nanomaterial purity is an important consideration because residual, contaminating metals may be responsible

Page 13 of 24

for the observed toxicity, rather than the nanomaterial
itself, which has resulted in conflicting data on GFNs
cytotoxicity [35, 160]. Traditionally prepared GO often
contains high levels of Mn2+ and Fe2+, which are highly
mutagenic to cells. The nonspecific release of these ionsfrom traditionally prepared GO might lead to unusuallyhigh levels of cytotoxicity and DNA fracturing [39]. In
particular, Peng et al. [161] produced high-purity GO
containing only 0.025 ppm Mn2+ and 0.13 ppm Fe2+,
and Hanene et al. [162] invented a new method toprepare high-purity, single-layer GO sheets with goodaqueous dispersibility and colloidal stability. GO produced by these new methods did not induce significant
cytotoxic responses (at exposure doses up to 100 μg/mL)
in vitro, and no obvious inflammatory response or
granuloma formation (exposure doses up to 50 μg/animal)
were observed in vivo. Therefore, the purity of GFNs
deserves attention and is a vital step towards the determination of GFNs involved in bioapplications.
Protein corona effectBecause of the high free surface charge, nanomaterials
can easily form “coronas” with proteins in biologicalsystems [163, 164]. The protein corona is suggested toaffect the circulation, distribution, clearance and toxicity
of nanoparticles. Several papers reported that GO forms
GO-protein coronas with adsorbed plasma proteins inserum and these GO-protein coronas play an importantrole in deciding the fate of the GO biokinetic behaviourin vivo. Such GO-protein coronas can regulate the adhesion of GO to endothelial and immune cells throughboth specific and nonspecific interactions [165]. Basically, immunoglobulin G and complement proteins in
the protein corona help to reorganize nanoparticles in
immune cells, causing the particles to be engulfed by
the RES, and IgG-coated GO was taken up by either
specific or nonspecific interactions with cell membrane
receptors [31, 165]. However, another study found that
GO could not adhere to mucosal epithelial cells directly in
the intestinal tract after the filial mice drank an aqueous
GO solution because abundant proteins in the milk had
adsorbed on the surface of the GO and thus inhibited
their direct interaction with the mucosal epithelial cells[53]. Protein corona mitigated the cytotoxicity of GO bylimiting its physical interaction with the cell membraneand reducing the cellular morphological damage in HeLa,
THP-1 and A549 cells [166–168]. The cytotoxic effectwas largely reduced when GO was pre-coated with FBS
and incubated with cells; nearly ∼ 90 % survival wasobserved with 100 μg/mL FBS-coated GO and 100 %survival with 20 μg/mL FBS-coated GO. Similar trends
were observed for GO covered by BSA [166, 169]. Consistently, additional serum could neutralize the toxicity ofpristine GO in J774.A1 cells at a dose of 4 μg/mL, whichOu et al. Particle and Fibre Toxicology (2016) 13:57

lead to a decrease in cell number of 52.5 % compared tountreated cells [89].
After reviewing many studies, it can be concluded thatthe toxicity of graphene is influenced by multiple factors.
Those factors combined to largely change the toxicity of
GFNs in many cases. Scientific studies often need theclear identification of cause and effect, which should
keep only one factor different at a time, so that the effectof that single factor can be determined. But in some
papers, several factors influencing GFNs toxicity werestudied at the same time, which led to confused results.

Possible toxicity mechanisms of GFNs
Although some physicochemical properties and the
toxicity of GFNs have been well studied by manyscholars, the exact mechanisms underlying the toxicity
of GFNs remain obscure. A schematic of the main mechanisms of GFNs cytotoxicity is illustrated in Fig. 3.

Page 14 of 24

Physical destruction

Graphene is a unique nanomaterial compared with otherspherical or one-dimensional nanoparticles due to its
two-dimensional structure with sp2-carbons. The physical interaction of graphene nanoparticles with cell
membranes is one of the major causes of graphene cytotoxicity [7, 170, 171]. Graphene has high capability tobind with the α-helical structures of peptides because of
its favourable surface curvature [172]. At concentration
above 75 μg/mL, pristine graphene largely adhered tothe surfaces of RAW 264.7 cells and resulted in abnormal stretching of the cell membrane [104]. The stronghydrophobic interactions of GFNs with the cell membrane lead to the morphological extension of F-actin
filopodial and cytoskeletal dysfunction. Furthermore, thesharpened edges of GNS may act as ‘blades’, insertingand cutting through bacterial cell membranes [173].
Moreover, GO also damaged the outer membrane ofFig. 3 Schematic diagram showed the possible mechanisms of GFNs cytotoxicity. GFNs get into cells through different ways, which induce in
ROS generation, LDH and MDA increase, and Ca2+ release. Subsequently, GFNs cause kinds of cell injury, for instance, cell membrane damage,inflammation, DNA damage, mitochondrial disorders, apoptosis or necrosisOu et al. Particle and Fibre Toxicology (2016) 13:57

E. coli bacteria directly, resulting in the release of intracellular components [173]. However, TEM imaging revealed
that pre-coating GO with FBS eliminated the destructionof cell membranes [166].
ROS production leading to oxidative stressOxidative stress arises when increasing levels of ROS
overwhelm the activity of antioxidant enzymes, including
catalase, SOD, or glutathione peroxidase (GSH-PX)[174]. ROS act as second messengers in many intracellular signalling cascades and lead to cellular macromolecular damage, such as membrane lipid breakdown, DNA
fragmentation, protein denaturation and mitochondrial
dysfunction, which greatly influence cell metabolism andsignalling [175–177]. The interactions of GO with cells
can lead to excessive ROS generation, which is the firststep in the mechanisms of carcinogenesis, ageing, andmutagenesis [83, 122]. Oxidative stress had a significant
role in GO-induced acute lung injury [30], and theinflammatory responses caused by oxidative stress often
emerged upon exposure to GFNs [133, 177, 178]. The
activity of SOD and GSH-PX decreased after exposed to
GO in a time- and dosage-dependent manner [82, 106,119]. Similarly, oxidative stress was the key cause of
apoptosis and DNA damage after HLF cells were exposed to GO [148]. Both the mitogen-activated protein
kinase (MAPK) (JNK, ERK and p38) and TGF-betarelated signalling pathways were triggered by ROS generation in pristine graphene-treated cells, accompaniedby the activation of Bim and Bax, which are two proapoptotic members of the Bcl-2 protein family. As aresult, caspase-3 and its downstream effector proteinssuch as PARP were activated, and apoptosis was initiated[83, 179]. Detailed information regarding the MAPK-,
TGF-β- and TNF-α-related signalling pathways, which
induce inflammation, apoptosis and necrosis, are summarized in Fig. 4.
Mitochondrial damage

Mitochondria are energy production centres involved invarious signalling pathways in cells and are also a key
point of apoptotic regulation [83]. After exposure to GO
and carboxyl graphene (GXYG), the mitochondrial
membrane was depolarized, and the amount of mitochondria decreased in HepG2 cells [180]. Exposure to
GFNs resulted in significantly increased coupled anduncoupled mitochondrial oxygen consumption, dissipation of the mitochondrial membrane potential, andeventual triggering of apoptosis by activating the mitochondrial pathway [181]. For instance, GO increased theactivity of mitochondrial electron transport complexes I/
III and the supply of electrons to site I/II of the electrontransport chain, accelerating the generation of ROS during mitochondrial respiration in MHS cells [99]. The

Page 15 of 24

formation of •OH mediated by GO and the cytochromec/H2O2 electron-transfer system could enhance oxidative
and thermal stress to impair the mitochondrial respiration system and eventually result in dramatic toxicity[151]. Additionally, the oxygen moieties on GO might
accept electrons from cellular redox proteins, supporting
the redox cycling of cytochrome c and electron transport
proteins, and cytochromes MtrA, MtrB, and MtrC/
OmcA might be involved in transferring electrons toGO [182]. Therefore, except for the plasma membrane
damage and oxidative stress induction, GFNs can cause
apoptosis and/or cell necrosis by direct influencing cell
mitochondrial activity [183, 184].
DNA damage

Due to its small size, high surface area and surface
charge, GO may possess significant genotoxic properties
and cause severe DNA damage, for example, chromosomal fragmentation, DNA strand breakages, point
mutations, and oxidative DNA adducts and alterations[87, 122, 185, 186]. Mutagenesis was observed in mice
after intravenous injection of GO at a dose of 20 mg/kg
compared with cyclophosphamide (50 mg/kg), a classic
mutagen [112]. Even if GO cannot enter into the nucleus
of a cell, it may still interact with DNA during mitosis
when the nuclear membrane breaks down, which increases
the opportunity for DNA aberrations [87, 147, 187, 188].
The π stacking interaction between the graphene carbon
rings and the hydrophobic DNA base pairs can make a
DNA segment ‘stand up’ or ‘lay on’ the surface of graphenewith its helical axis perpendicular or parallel, respectively.
The intermolecular forces severely deform the end basepairs of DNA, which potentially increases the genotoxicity[189]. GO may also induce chromosomal fragmentation,
DNA adducts and point mutations by promoting oxidative
stress or triggering inflammation through the activation of
intracellular signalling pathways such as MAPK, TGF-βand NF-κB [110, 112, 146]. Graphene and rGO can also
elevate the expression of p53, Rad51, and MOGG1-1,
which reflect chromosomal damage, and decrease theexpression of CDK2 and CDK4 by arresting the cell cycletransition from the G1 to the S phase in various cell lines[112]. DNA damage can not only initiate cancer development but also possibly threaten the health of the next
generation if the mutagenic potential of GO arises in reproductive cells, which impacts fertility and the health ofoffspring [112, 190].
Inflammatory response

GFNs can cause a significant inflammatory response including inflammatory cell infiltration, pulmonary edema
and granuloma formation at high doses via intratracheallyinstillation or intravenous administration [30, 49]. Platelets are the important components in clot formation toOu et al. Particle and Fibre Toxicology (2016) 13:57

Page 16 of 24

Fig. 4 Schematic diagram of MAPKs, TGF-beta and TNF-α dependent pathways involved in GFNs toxicity. ROS was the main factors activating the
MAPKs and TGF-beta signaling pathways to lead to the activation of Bim and Bax, triggering the cascade of caspases and JNK pathway. The
activation of caspase 3 and RIP1 resulted in apoptosis and necrosis finallyattack pathogens and particulate matter during the inflammatory response, and GO could directly activate
platelet-rich thrombi formation to occlude lung vessels
after intravenous injection [98, 191]. A strong inflammatory response was induced by subcutaneously injection with GO for 21 days, along with the secretion of
key cytokines, including IL-6, IL-12, TNF-α, MCP-1,
and IFN-g [34, 192]. GFNs can trigger an inflammatory
response and tissue injury by releasing cytokines andchemokines that lead to the recruitment of circulating
monocytes and stimulating the secretion of Th1/Th2
cytokines and chemokines [124, 193]. Additionally,
pristine graphene [193] and rGO [110] evoke an inflammatory response by binding to toll-like receptors(TLRs) and activating the NF-κB signalling pathway incells. The NF-κB signalling cascade is triggered by
TLRs and pro-inflammatory cytokines such as IL-1 andTNF-α. Upon activation, NF-κB shifts from the cytoplasm
to the nucleus, facilitating the binding of degrading IκB
and acting as a transcription factor to synthesize numerous pro-inflammatory cytokines [194]. A schematic of thesignalling pathway of TLR4 and TLR9 activated by GFNs
is shown in Fig. 5.

Apoptosis

Apoptosis is defined as the self-destruction of a cell
regulated by genes through complicated programmes[83, 195]. GO and rGO caused apoptosis and inflammation in mice lungs after inhalation [99], and GFNs also
had pro-apoptotic effects in cells [111, 113, 124, 196].
Additionally, graphene and GO physically damaged cell
membranes [166], increased the permeabilization of
the outer mitochondrial membrane and changed themitochondrial membrane potential; the increased ROS
triggered the MAPK and TGF-β signalling pathways
and activated caspase-3 via mitochondrial-dependent
apoptotic cascades, prompting the execution of apoptosis [83, 99]. Similarly, rGO caused apoptosis at alow dose and an early time point, triggered by thedeath-receptor and canonical mitochondrial pathway[110]. Another study showed three different apoptosis
pathways by GFNs: GO led to ROS-dependent apoptosis through direct interaction with protein receptors
and subsequent activation of the B-cell lymphoma-2(Bcl-2) pathway; GO-COOH transmitted a passive apoptosis signal to nuclear DNA by binding to protein receptors and activating a ROS-independent pathway; However,Ou et al. Particle and Fibre Toxicology (2016) 13:57

Page 17 of 24

Fig. 5 A schematic diagram elucidating signalling pathway of TLR4 and TLR9 responsible for GFNs-induced cytotoxicity. GFNs can be recognized
by TLRs, thus activate IKK and IκB by a MyD88-dependent mechanism, resulting in the release of NF-κB subunits and initiating the translocation
into the nucleus. Thus, pro-inflammatory factors were transcribed and secreted out of nucleus, modulating the immune responses initiating
programmed autophagy, apoptosis and necrosis

GO-PEI severely damaged the membranes of T lymphocytes to trigger apoptosis [105, 197].

Autophagy

Autophagy is the process of self-degradation of cellularcomponents and recently recognized as non-apoptotic
cell death [198–200]. Autophagy activation requires
autophagosome formation containing Beclin 1, multiple
autophagy-related proteins (ATG), microtubule-associatedprotein light chain 3 (LC3) and p62 [201]. Autophagosome accumulation is associated with exposure to various
nanoparticles [202–205], and autophagy can remove
extracellular organisms and destruct the organisms in thecytosol [206]. GO and GQDs was shown to induce autophagosome accumulation and the conversion of LC3-I to
LC3-II; inhibit the degradation of the autophagic substrate
p62 protein [207, 208]. Furthermore, GO can simultaneously trigger TLR4 and TLR9 responses in macrophages[34, 192] and colon cancer cells CT26 [206]. The autophagy pathway is linked to phagocytosis by TLR signalling inmacrophages [206, 209].

Necrosis

Necrosis is an alternate form of cell death induced byinflammatory responses or cellular injury. The exposure of cells to pristine graphene causes apoptosis andnecrosis at high doses (50 mg/mL) [83]. Reportedly,
LDH leakage and the opening of the mitochondrial
permeability transition pore, induced by elevated level ofcytoplasmic Ca2+, lead to apoptosis/necrosis [210]. GO
treatment was revealed to induce macrophagic necrosis by activating TLR4 signalling and subsequently
partly triggering autocrine TNF-α production [93].
GO combined with CDDP (GO/CDDP) triggered necrosis
by decreasing RIP1 and increasing RIP3 proteins, accompanied with the release of high mobility group B1(HMGB1) into the cytosol from the nucleus and out of
CT26 cells [205, 211, 212].
Epigenetic changes

Epigenetics involve DNA methylation, genomic imprinting, maternal effects, gene silencing, and RNA editing[213–215]. DNA methylation, which is one of the beststudied epigenetic modifications, includes phosphorylation,

Ou et al. Particle and Fibre Toxicology (2016) 13:57

ubiquitination, and ATP-ribosylation and can lead tochromatin remodelling [197, 216, 217]. A recently paper
reported that SL-GO/FL-GO exposure resulted in global
DNA hypermethylation through upregulating DNMT3B
and MBD1 genes; GNP treatment caused hypomethylation
by decreasing the expression of DNMT3B and MBD1
genes [216]. GO could activate the miRNA-360 regulation
pathway to suppress the DNA damage-apoptosis signallingcascade by affecting the component of CEP-1 [218]. Taken
together, these data suggest that GFNs could cause subtle
changes in gene expression programming by modulating
epigenetic changes. However, studies of GFNs-induced
epigenetic changes are few, and the epigenetic mechanism
caused by GFNs exposure is not fully understood.
To conclude, many studies have discussed representative mechanisms of GFNs toxicity involving four signalling pathways: TLRs, TGF-β, TNF-α and MAPKs.
These four signalling pathways are correlative andcross-modulatory, making the inflammatory response,
autophagy, apoptosis and other mechanisms independentand yet connected to each other. Additionally, oxidative
stress appears to play the most important role in activating these signalling pathways. It has been reported that
there are intersections of apoptosis, autophagy and necrosis in the studies of other nanomaterials toxicity, they
inhibit or promote mutually in some conditions. However,the signalling pathways of GFNs toxicity investigated in
papers to date are only a small part of an intricate web,and the network of signalling pathways needs to be
explored in detail in the future.

Data gaps and future studies
Currently, the literature is insufficient to draw conclusions about the potential hazards of GFNs. Two opposite
opinions have begun to emerge: some researchers
suggested that graphene materials are biocompatible in a
number of studies focused on biomedical applications[119, 154, 162, 219], and other studies reported adverse
biological responses and cytotoxicity [32, 118, 135, 138,
192]. These inconsistent results might have been causedby several factors, including the different researchgroups, various cellular or animal models, and varying
physicochemical characterizations of GFNs. When GFNs
are explored for in vivo applications in the human bodyor some other biomedical applications, biocompatibility
must be considered, and more detailed and accurate
studies of GFNs toxicity are needed.
First, detailed physicochemical characterization is imperative in all future studies of GFNs toxicity. In theexperiments, feature descriptions of GFNs should include their size, morphology, surface area, charge, surface modifications, purity, and agglomeration [88, 141,
148, 162]. Because these physicochemical factors largely

Page 18 of 24

influence the toxicity and biocompatibility of GFNs,
single-factor experimental designs and the exclusion ofother interfering factors should be considered. Detailsof the fabrication process should also be provided because
the formed oxidative debris could largely alter the surface
structure of graphene and GO during functionalization[151]. Importantly, a single, universal method needs to beestablished in graphene technology, which will allow for
better comparison of data from different studies or different laboratories.
Second, different observational criteria, parametersand selection of experimental methods might induce
large inter-laboratory variations [220, 221]. For example,
the MTT assay always fails to accurately predict graphene
toxicity because the spontaneous reduction results in a
false positive signal. Therefore, appropriate alternative
assessments should be utilized, such as the water-soluble
tetrazolium salt reagent (WST-8), ROS assay, and trypan
blue exclusion test [106, 222]. Additionally, the cometassay often shows higher levels of DNA damage than the
micronucleus assay because the former measures therepairable injury and the latter measures the gene damage
that remains after cell division [159, 223]. Therefore,
caution is required in choosing the most appropriate assay
to evaluate the toxicity of graphene materials to avoid
false-positive results.
Third, the selection of cell lines is of vital importancebecause cancer cell lines tend to be sensitive or resistantdepending upon their genetic background. The samegraphene nanoparticles can cause different reactions
depending on their various cells origins. Suitable celllines with good stability must be used to avoid false positive or negative results. Primary cells derived fromhumans or animals can better simulate the health conditions of humans. A large amount of primary cells havebeen utilized to test the toxicity of other nanomaterials[224–228], but the culturing of primary cells is extremelyrare in the experiments with GFNs to date [210, 229].
Various cell experiments combined with primary cells
should be performed to comprehensively evaluate thephysicochemical properties and toxicity of GFNs.
Fourth, the administration route of GFNs plays a very
important role in toxicity studies, and different delivery
methods will result in different toxicological reactions[32, 53]. Thus, the route and period of exposure should
be carefully chosen according to the aim of the study.
Nasal drug delivery is often used to study the neurotoxicity of nanomaterials [230, 231], but this administration
method has rarely been applied in the testing of GFNs
toxicity. Toxicological studies of GFNs in the nervous
system are rare, and the mechanism is unclear and needs
to be studied further in the future. Recent toxicokinetic
studies involving the absorption, distribution, metabolism,
accumulation, and excretion of GFNs through different

Ou et al. Particle and Fibre Toxicology (2016) 13:57

exposure routes have yielded some results but are far from
sufficient to clarify the internal complex mechanisms. For
instance, further studies are needed to understand the specific molecular mechanisms of GFNs passing through thephysiological barriers and the amount of accumulation or
the excretion period of GFNs in tissues. In addition, given
the increased exposure of humans to GFNs, the assessment of systemic toxicity in the human body is indispensable in future studies.
Fifth, another important issue requiring attention is
the long-term fate of GFNs after entering the body or
being taken up by cells. Most recent studies have consisted of short-term toxicity assessments [89, 232], andlong-term toxic injury has not received much attention
since the widespread application of GFNs in 2008. Moreover, a functionalized graphene surface can improve its
biocompatibility, but the long-term stability of the surface
coatings should be considered [233]. If the surface coatings eventually break down, their toxicity may be significantly different from the short-term exposure results.
Extended studies are needed to determine if longer
treatment times influence the nanotoxic potential of
GFNs.
Sixth, more specific signalling pathways in the mechanism of GFNs toxicity need to be discovered and elucidated. Currently, several typical toxicity mechanisms of
GFNs have been illustrated and widely accepted, such asoxidative stress, apoptosis, and autophagy. However,these mechanisms have only been described in generalterms, and the specific signalling pathways within thesemechanisms need to be investigated in detail. The signalling pathways involved in the toxicity of other nanomaterials may also be relevant to the study of GFNs.
Therefore, more signalling pathways should be detectedin future research. For instance, nano-epigenetics has
been considered in numerous studies of nanomaterials,
which is also helpful in assessing the limited toxicity andside effects of GFNs. Recent studies have shown that
GFNs could cause epigenetic and genomic changes thatmight stimulate physical toxicity and carcinogenicity[234]. GFNs have high surface areas, smooth continuous
surfaces and bio-persistence, similar to the properties oftumorigenic solid-state implants. It is unknown whether
GFNs have the potential to induce foreign body sarcomas, and definitive studies of tumour potentialities orrisks of graphene should therefore be conducted assoon as possible.

Conclusions
In the past few years, GFNs have been widely utilized ina wide range of technological and biomedical fields.
Currently, most experiments have focused on the toxicity of GFNs in the lungs and livers. Therefore, studiesof brain injury or neurotoxicity deserve more attention

Page 19 of 24

in the future. Many experiments have shown that GFNs
have toxic side effects in many biological applications,but the in-depth study of toxicity mechanisms is urgently
needed. In addition, contrasting results regarding thetoxicity of GFNs need to be addressed by effective experimental methods and systematic studies. This review
provides an overview of the toxicity of GFNs by summarizing the toxicokinetics, toxicity mechanisms and influencing factors and aimed to provide information to facilitate
thorough research on the in vitro and in vivo haemo- andbiocompatibility of GFNs in the future. This review will
help address safety concerns before the clinical andtherapeutic applications of GFNs, which will be important for further development of GFNs in biologicalapplications.
Abbreviations
AMs: Alveolar macrophages; BBB: Blood-brain barrier; BEB: Blood-epididymis
barriers; BTB: Blood-testis barrier; CR: Complement receptor; FcgR: Fcg
receptor; FLG: Few-layer graphene; GFNs: Graphene family nanomaterials;
GNS: Graphene nanosheets; GO: Graphene oxide; GO-COOH: Carboxylated
graphene oxide; GO-DEX: GO-dextran; GO-MB: GO-molecular beacon; GONH2: Aminated GO; GO-PAA: Poly(acrylic acid)-functionalized GO; GOPAM: Poly(acrylamide)-functionalized GO; GO-PEG: PEGylated GO derivatives;
GO-PEI: GO-polyethylenimine; GQDs: Graphene quantum dots; GSHPX: Glutathione peroxidase; GXVG: Carboxyl graphene; LDH: Lactate anddehydrogenase; MALDI: Matrix-assisted laser desorption/ionization;
MAPKs: Mitogen-activated protein kinase; MDA: Malondialdehyde;
MØ: Macrophage; MR: Mannose receptor; MSI: Mass spectrometry imaging;
PC12 cells: Rat pheochromocytoma cells; PCGO: Protein-coated graphene
oxide nanoparticles; PrGO: PEGylated reduced graphene oxide;
RES: Reticuloendothelial system; rGO: Reduced graphene oxide; ROS: Reactive
oxygen species; SOD: Superoxide dismutase; TLRs: Toll-like receptor
Acknowledgements
Not applicable.
Funding
This review was supported by the National Natural Science Foundation ofChina (81550011, 51172283, 81400557), Natural Science Foundation ofGuangdong Province (2015A030313299) and Guangdong Provincial Medical
Research Foundation (A2016360).
Availability of data and materials
Databases/repositories and materials is not applicable in this review.
Authors’ contributions
All authors contributed to the design and concept of this article. LO drafted
the manuscript. BS and JL critically revised the manuscript. All authors readand approved the final manuscript.
Competing interest
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Author details
1
Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
2
The First Affiliated Hospital of Jinan University, Guangzhou, China. 3The
General Hospital of People’s Liberation Army, Beijing, China.
Received: 13 June 2016 Accepted: 13 October 2016

Ou et al. Particle and Fibre Toxicology (2016) 13:57

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A summary of the article ‘Toxicity
of graphene-family nanoparticles:
a general review of the origins
and mechanisms’.
Graphene was discovered in 2004. Isolated from graphite crystals, it is made up of atsheets, each an atom thick. Graphene oxide is one of several deriva ves of graphene and its
unique proper es have so far seen it used in a variety of "elds, including biomedical
applica ons such as drug delivery.
Data on its toxicity are based on a limited mix of animal and in vitro studies, where various
deriva ves of graphene have been administered in di%erent ways. This is important to noteas the type of graphene and the way it enters the body – e.g. through inhala on or via
subcutaneous injec on – a%ects where it ends up in the body and its degree of toxicity.
More research is needed to understand the impact of graphene oxide administeredsubcutaneously, as with the Covid-19 mRNA vaccines.
What makes graphene toxic?
The extent of its toxicity depends on its characteris cs, from the size of the material, to its
concentra on, surface structure and charge.


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Dosage: higher concentra ons of GO tend to cause more damage: in one study, ahigh dose caused chronic toxicity, with GO deposits found in the lungs, liver, spleen
and kidneys.
Size: where graphene nanopar cles end up in the body and the impact they havedepends on their size. More research on this is needed.
Surface structure: GFNs in general can disrupt the func on and structure of cell
membranes and proteins. GO can insert between the base pairs of double-stranded
DNA and disturb the ow of gene c informa on.
Charge: the surface charge of GO can cause it to be taken in by cells. It canreportedly alter cell structure and cause extensive destruc on of red blood cells.
Surface modi"ca on: studies con"rm that altering the surface of GFNs using
substances such as PEG (a process called func onalisa on) largely decreases toxicity.
However, given its high poten al to be taken in by cells, further studies are needed
to assess the possible long-term e%ects.
Impuri es: tradi onally prepared GO o;en contains high levels of Mn2+ and Fe2+,
which could lead to unusually high levels of cell toxicity and DNA fracturing.
Protein corona e%ect: graphene’s high free surface charge can cause it to form
‘coronas’ with proteins in the body. Several papers report that GO forms suchcoronas and these may regulate the adhesion of GO to certain cells and evenmi gate its toxicity to cells.

What does it do to the body?
Again, studies here are lacking but here is what data have shown so far:Graphene nanomaterials (GFNs) can penetrate the body’s natural barriers
Their small size means graphene nanomaterials – including graphene oxide (GO) – canpenetrate the body’s exis ng protec ve barriers and in"ltrate the brain, placenta andpoten ally the testes. In terms of the placenta, some studies have found that the placenta
does not prevent nanopar cles from transferring to foetuses; this has a signi"cant impact
on foetal development.
Graphene oxide can damage internal organs
GO can cause acute inamma on and injury to vital organs. In the lungs, it was found tocause inamma on, pulmonary oedema and the forma on of granulomas. There has beenvery li@le research into the toxicity of GFNs to other organs such as the liver, spleen andkidneys.
GFNs can damage the central nervous system
Graphene nanomaterials are increasingly used in neurosurgery, and studies have mainly
focused on its applica ons rather than its toxicity. However, studies do show that thesematerials can decrease the level of ribonucleic acid and its rate of synthesis, which harms
brain ssue development. More studies on its toxicity are underway.
GO damages the reproduc on and development system
Animal studies show that GO and reduced GO (rGO) damage embryos. One study on micefound that all pregnant females injected with rGO had abor ons, regardless of dose. Mostof the mice given a high dose died, and their o%spring su%ered delayed development.
GO destroys blood health
It is impossible to prevent GO from entering the blood. Its impact on the blood depends on
its coa ng, size and dosage but it has been found to damage red blood cells and T
lymphocytes.
GO damages and destroys cells
Graphene nanomaterials in general alter the viability, shape, size and structure of cells. GO
speci"cally decreases cell adhesion, which can disrupt important processes and lead todisease. It also causes apoptosis – a form of programmed cell death – and enters into thelysosomes, mitochondria, nucleus and endoplasm of cells. GO deriva ves drama callydecrease the expression of di%eren al genes that are responsible for the structure andfunc on of the cell membrane.
GO can trigger cancer and accelerate ageing
GO’s interac on with cells causes extensive oxida ve stress which can trigger thegenera on of cancer cells, cause cells to mutate, and accelerates ageing. GO-induced
oxida ve stress has been implicated in acute lung damage, cell death and DNA damage.

GO damages mitochondria
Mitochondria are the vital energy centres within cells. Studies have found that exposure to
GO can both reduce the number of mitochondria in par cular cells, and impair
mitochondrial ac vity in such a way that it generates oxida ve stress and causes cell death.
GO damages DNA
GO’s small size, high surface area and surface charge could poten ally cause severe DNA
damage. Even if GO is unable to enter a cell’s nucleus, it can s ll interact with DNA at the
point in the cell’s cycle when the nuclear membrane breaks down (mitosis). This increases
the poten al for DNA aberra ons. Interac on between graphene and the DNA base pairs
can severely deform the end base pairs of DNA. GO may also cause muta ons that could
lead to the development of cancer – and if those muta ons happen in reproduc ve cells,
they could impact fer lity and threaten the health of the next genera on.
GO triggers an in#ammatory response and three di$erent kinds of cell death
GFNs (including GO) can cause signi"cant inammatory responses and ssue injury. GO has
been found to trigger apoptosis and autophagy – both forms of programmed cell death – aswell as necrosis, a form of cell death that occurs as a result of infec on or injury.
GO causes epigene c changes
Data suggests that GFNs including GO could modulate epigene c changes that cause subtle
changes in gene expression programming. However, this is not fully understood.
A lack of data
There simply isn’t suAcient data with which to fully understand the toxicity of GFNs to thebody. Recent studies have focused on short-term impact but li@le is known about long-term
e%ects. For example, while func onalised graphene does seem to have lower toxicity, what
happens if the surface coa ngs that lower the toxicity break down over me? Also, it isunknown as to whether GFNs could induce foreign body sarcomas, so more studies on thisare urgently needed.