Case Briefing Document and Lab Report Ref AUC 101 Report

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 of
eight 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 died
within two weeks of COVID19 experimental treatment, approximately one-third of those deaths were
caused by the experimental treatment itself.
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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 the
human 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 in
suspicious 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 the
presence 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 Police
and Lawyers around the UK.
It is incumbent upon medical staff in the UK to report any injury, adverse reaction or death following the
injection 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 been
reported.
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 as
a 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.
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The contents of the injection vials have been forensically examined and a laboratory report is attached to
this 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 and
mechanisms’
● Layman's summary of the article ‘Toxicity of graphene-family nanoparticles: a general review of the
origins 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
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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 system
c. destroys blood health
d. damages and destroys cells
e. can trigger cancer and accelerate ageing
f. damages mitochondria and DNA
g. triggers an inflammatory response and three different kinds of cell death
h. 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 that
they 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 by
a 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 to
mention 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 the
4
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 are
managed 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 a
breach of that duty falls far below what can reasonably be expected of the organisation in the
circumstances;
(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 or
organised, 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 involuntary
manslaughter. Since the decision in Andrews was a decision of your Lordships' house, it remains the most
authoritative 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 the
defendant has been in breach of a duty of care towards the victim who has died. If such breach of duty is
established 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 and
therefore 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; and
d) 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 taken
from 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 be
summarised as being the breach of an existing duty of care which it is reasonably foreseeable gives rise to
a 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 a
criminal 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
8
6
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 reasonable
grounds 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 procedure
material; 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 of
premises 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 “specific
premises warrant”); or
(b)
any premises occupied or controlled by a person specified in the application, including such sets of
premises 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 are
reasonable 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 in
paragraph (b) of that subsection; and
(b)
that it is not reasonably practicable to specify in the application all the premises which he occupies or
controls 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 the
application, the justice of the peace is satisfied that it is necessary to authorise multiple entries in order to
achieve 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 not
practicable 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 the
premises can secure immediate entry to them.
(4)
In this Act “relevant evidence”, in relation to an offence, means anything that would be admissible in
evidence 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 of
forces 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, in
most 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;
can telephone incidents to police control rooms;
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 an
incident 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 the
nature 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, be
that by phone, etc. or in person. If an alleged or possible victim cannot be contacted or later refuses to
provide 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
Page 1 of 48
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).
Page 2 of 48
TABLE OF CONTENTS
1. 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
2. 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
3.2.3. Counts .................................................................................................................... 26
3.4. AstraZeneca ............................................................................................................. 28
3.4.1. Microscopy ........................................................................................................ 28
3.4.3. Counts ................................................................................................................ 34
3.5. Pfizer ......................................................................................................................... 35
3.5.1. Microscopy ........................................................................................................ 35
3.5.3. Counts ................................................................................................................ 39
4. Interpretation, Discussions and Conclusion ................................................................ 41
5. Bibliography ................................................................................................................... 45
Page 3 of 48
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 STRICTLY
PROHIBITED.
Any reproduction and distribution queries should be directed

to
UNIT
At
[email protected]
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 and
quantitative report.
UNIT takes no responsibility for interpretations drawn by readers of the report.
Page 4 of 48
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 of
four 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 to
Moderna, 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

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
Page 5 of 48
Covid-19 Vaccine 4 ml, Astrazeneca Injection. Covid-19 Vaccine:

(ChAd0x1-S (recombinant)), Intramuscular use. Multidose vial (8x0.5ml doses). 108439/2 Lot-
PW40167 Expiry- 01-22. (Figure 2.1)
The bottle weight prior to the breaking of the seal was 12.184g and after the breaking of the
seal 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 be
interpreted 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.
Page 6 of 48
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 and
analysis of the methods used for the evaluation. Chapter three presents the results of the
analysis, 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.
Page 7 of 48
2. Methodology
2.1. Vial Descriptions
The secured vials were made of glass and the vaccines contained within the vials were in a
liquid form. The different parts of the vial are shown in figure 2.1. Each of these vials were
stored 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 for
posterity. 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 the
objective 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, that
during the penetration of the vial, the syringe would carry nanoscopic amounts of rubber seal
material, 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.
Page 8 of 48
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 prior
study 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 rich
medium. The optical and physical properties of the preserving medium posed a challenge to
the 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 rendered
an additional impediment to the task of their isolation.
During, wet microscopic evaluation at the dry laboratories, it was noted from the onset, that
the denser material settled at the bottom of the slide and got impregnated and encrusted with
the 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 the
study, 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 were
stored 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. It
should 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 then
represented 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 glass
chambers 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 source
of 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 of
nomenclature and related descriptions per vaccine, these inclusions are illustrated and defined
individually below-
The identified inclusions were-

1. 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 fluorescence
a. 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 and
embedded 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 the
Raman requires to be shot for a better grasp on the distribution of defects and other
characteristics 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 is
transparent 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 quite
distinct in their lamellar, structure.
The signature for carbon-carbon bond is quite distinct at 1600 cm-1 , 1100 cm-1 fpeak is
picked 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 (figure
3.24).
Nano forms of Graphene dominated the counts in all the samples. They were found to be
present 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 the
bottom of all the samples that were examined (figure 3.5).
These nano Graphene spicules were impossible to be evaluated by Raman, as their radius was
measurably 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 of
these 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 of
these 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 and
were 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 of
these inclusions took place at multiple levels within the solidified medium. Lighter material
came 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 the
edges 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 structure
of 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 a
quadrilateral 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 other
minor 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 this
form 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 establishment
of 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 abundance
is 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 was
necessary 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 particulate
material 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 material
sank below as traction bedload and salted across the slide while the medium evaporated under
the 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 self-
assemblage 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 competitive
environment 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), the
growth of the particle relies on the pH neutralisation and migration of neutral, unbound
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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 these
structures 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 of
moisture. The relatively denser material settled to the bottom, while, the lighter material was
on the top of the solidified solution with an additional detrital layer in the middle of the
medium.
General overview of the dried slide at low resolution exhibited a translucent medium with
forms 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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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 fluorescent
background, 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. These
chemistries 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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at lower resolution and at higher resolutions, Graphene Nano objects are present in great
abundance.
It is clear from the counts, that the nanoscopic structures far exceed the density counts than
microscopic structures.
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 wet microscopic 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 in
a 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, the
sedimentation 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. This
difference 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 clearly
visible 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 this
mechanism 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 typical
signatory movements attributed to the inter-particle forces. While the solution dried up, the
heavier carbon material that had sunk to the bottom of the slide was released from the nucleus
of the nanoparticles and these now began to exhibit traction movement. Figure 3.22, shows the
relative 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 (figure
3.24).
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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 self-
movement against the growing viscosity of the drying solution. When the solution dried, they were found
to 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 impregnated
with polyethylene glycol, Graphene Nano Objects including a vast number of nano scrolls and
a 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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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 other
forms 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 be
minimal in AstraZeneca’s selected inclusions.
Calcium composites with carbon also showed the same signature as the ones present in the two
Moderna 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 nano
objects 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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magnification. 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 however
these are present in significant numbers at higher magnifications. It is clear from the graph that
graphene nano objects dominate the count at higher resolutions.
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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 the
slide 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 found
when the slide dried. As the material was sucked into the pipette, distinct translucent to
transparent 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 self-
assembly nano particles that will encapsulate the mRNA.
The two objects of interest that were clearly noted to be floating about but could not be located
once the slide had dried, were: (1) an extremely pointy transparent spicule like object (figure
3.30) 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 floating
transparent 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 are
often 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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Figure 3.33. Raman spectra of various inclusions in AstraZeneca.
Raman spectroscopy was conducted on four selected representative samples for Pfizer (figure
3.32). Three of these samples showed carbon composite signatures with possible graphene in
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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 is
graphene with polyethylene glycol signal forming the bulk of the spectrum. Though, for initial
assessments, 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 this
sample at longer exposures is important to separate the signal from the article of interest with
the signal of the background.
In summary, Raman helped identify the inclusions within Pfizer and these identifications were
classed 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 High
magnifications (figure 3.34). The relative numbers of carbon composite 2 is quite high in one
of the tracks whereas it is completely absent in the first track. At higher magnifications the
counts are dominated by the sheer number of nano graphene objects.
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4. Interpretation, Discussions and Conclusion

Moderna
Active Ingredients Table 1
• mRNA List of declared ingredients for
Vehicles the vaccines.
• 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
• Magnesium Chloride Hexahydrate
• Polysorbate 80
• Ethanol
• Sucrose
• Sodium Chloride
• Disodium edetate dihydrate (EDTA)
• Water
Pfizer
Active 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
• 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 and
related products as well as any biological forms.
It should be noted, that all three vaccines have very different chemical ingredients as declared
by 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 in
form 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
categories-
1. 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, the
tubular forms along with nano dots and scrolls seem to be aimed at enhancing cell acceptance
for 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 presence
of 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 in
itself, 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 the
thermodynamic environment. However, based on the observations within this study, to
kickstart 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 high
concentration of graphene nano objects in all four vaccines, as the nanoparticles move through
the 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 in
determining 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 were
being 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 ingredients
of these vaccines. Given the context of the increasing use of graphene in drug delivery, the
findings of this project seem to fit in the reference frame of attempts towards enhanced and
tailored 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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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 have
been 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 will
continue on the Raman spectroscopic investigations to attain cleaner spectra and to isolate the
individual 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 are
absent from the list of ingredients for the vaccines.
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5. Bibliography
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Ou et al. Particle and Fibre Toxicology (2016) 13:57
DOI 10.1186/s12989-016-0168-y
REVIEW Open Access
Toxicity of graphene-family nanoparticles:

a general review of the origins and
mechanisms
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 and
toxicity 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 the
toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and
corona 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 role
in 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 of
completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and
facilitate their wide application.
Keywords: Graphene-family nanomaterials, Toxicity, Toxicokinetics, Mechanisms, Physicochemical properties, Future
prospects
Background attracts increasing attention for its potential biomedical

Graphene, which is isolated from crystalline graphite, applications. Graphene-based materials usually have sizes
is a flat monolayer composed of single-atom-thick, ranging from several to hundreds of nanometer and
two-dimensional sheets of a hexagonally arranged are 1-10 nm thick [8, 9], which is also the definition
honeycomb lattice [1]. Because of its unique structural, of ‘nanoparticles’ or ‘nanomaterials’. Due to their ex-
specific surface area and mechanical characteristics, the ceptional physical and chemical properties, graphene
functions and applications of graphene have gained materials have been widely used in various fields, inclu-
considerable attention since the discovery of the material ding energy storage; nanoelectronic devices; batteries
in 2004 [2, 3]. Graphene and its derivatives include mono- [10–12]; and biomedical applications, such as antibac-
layer graphene, few-layer graphene (FLG), graphene oxide terials [13, 14], biosensors [15–18], cell imaging [19, 20],
(GO), reduced graphene oxide (rGO), graphene nano- drug delivery [8, 21, 22], and tissue engineering [23–25].
sheets (GNS), and graphene nanoribbons, etc. [4–7]. Along with the application and production of GFNs
GO is one of the most vital chemical graphene derivatives increasing, the risk of unintentional occupational or
of the graphene-family nanomaterials (GFNs), which environmental exposure to GFNs is increasing [26]. And
recently, there are some investigation on GFNs exposure
* Correspondence: [email protected] in occupational settings and published data showed that
1
Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
Full list of author information is available at the end of the article
© 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, and
reproduction 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 Page 2 of 24
the occupational exposure of GFNs had potential tox- toxicity of GFNs [44–46]. So to make them clear may be
icity to the workers and researchers [27–29]. GFNs can helpful to better understand the laws of the occurrence
be delivered into bodies by intratracheal instillation [30], and development of GFNs toxicity.
oral administration [31], intravenous injection [32], in-
traperitoneal injection [33] and subcutaneous injection
[34]. GFNs can induce acute and chronic injuries in Administration route
tissues by penetrating through the blood-air barrier, The common administration routes in animal models
blood-testis barrier, blood-brain barrier, and blood- include airway exposure (intranasal insufflation, intratra-
placenta barrier etc. and accumulating in the lung, liver, cheal instillation, and inhalation), oral administration,
and spleen etc. For example, some graphene nanomate- intravenous injection, intraperitoneal injection and sub-
rials aerosols can be inhaled and substantial deposition cutaneous injection. The major exposure route for GFNs
in the respiratory tract, and they can easily penetrate in the working environment is airway exposure, thus
through the tracheobronchial airways and then transit inhalation and intratracheal instillation are used mostly
down to the lower lung airways, resulting in the subse- in mice to simulate human exposure to GFNs. Though
quent formation of granulomas, lung fibrosis and adverse the inhalation method provides the most realistic simu-
health effects to exposed persons [2, 29]. Several reviews lation to real life exposure, instillation is more effective
have outlined the unique properties [35, 36] and summa- and time-saving method, and GFNs was found that caus-
rized the latest potential biological applications of GFNs ing longer inflammation period using instillation (intratra-
for drug delivery, gene delivery, biosensors, tissue engin- cheal instillation, intrapleural installation and pharyngeal
eering, and neurosurgery [37–39]; assessed the biocom- aspiration) than inhalation [24, 30, 47, 48]. GFNs were in-
patibility of GFNs in cells (bacterial, mammalian and vestigated to deposit in the lungs and accumulate to a
plant) [7, 40, 41] and animals (mice and zebrafish) [42]; high level, which retained for more than 3 months in the
collected information on the influence of GFNs in the soil lungs with slow clearing after intratracheal instillation
and water environments [43]. Although these reviews [49]. Intravenous injection is also widely used to assess the
discussed the related safety profiles and nanotoxicology of toxicity of graphene nanomaterials, and graphene
GFNs, the specific conclusions and detailed mechanisms circulates through the body of mice in 30 min, accumula-
of toxicity were insufficient, and the mechanisms of tox- ting at a working concentration in the liver and bladder
icity were not summarized completely. The toxicological [32, 50–52]. However, GO derivatives had rather finite
mechanisms of GFNs demonstrated in recent studies intestinal adsorption and were rapidly excreted in adult
mainly contain inflammatory response, DNA damage, mice via oral administration [31, 53]. Nano-sized GO
apoptosis, autophagy and necrosis etc., and those mecha- (350 nm) caused less mononuclear cells to infiltrate sub-
nisms can be collected to further explore the complex cutaneous adipose tissue after subcutaneous injection in
signalling pathways network regulating the toxicity of the neck region compared to micron-sized GO (2 μm)
GFNs. It needs to point out that there are several fac- [34]. GO agglomerated near the injection site after intra-
tors which largely influence the toxicity of GFNs, such peritoneal injection, and numerous smaller aggregates
as the concentration, lateral dimension, surface struc- settled in the proximity of the liver and spleen serosa
ture and functionalization etc. Herein, this review presents [31, 33]. Experiments on skin contact with or skin perme-
a comprehensive summary of the available information on ation of GFNs were not found in the papers reviewed
the mechanisms and regulating factors of GFNs toxicity in here, and there is insufficient evidence available to con-
vitro and in vivo via different experimental methods, with clude that graphene can penetrate intact skin or skin
the goals of providing suggestions for further studies of lesions. The route of nasal drops, which has been widely
GFNs and completing the toxicology mechanisms to used to test the neurotoxicity or brain injury potential of
improve the biological safety of GFNs and facilitate other nanomaterials, was not mentioned in the papers
their wide application. reviewed here.
Toxicity of GFNs (in vivo and in vitro) GFNs entry paths

GFNs penetrate through the physiological barriers or GFNs reach various locations through blood circulation
cellular structures by different exposure ways or admi- or biological barriers after entering the body, which
nistration routes and entry the body or cells, eventually results in varying degrees of retention in different
resulting in toxicity in vivo and in vitro. The varying organs. Due to their nanosize, GFNs can reach deeper
administration routes and entry paths, different tissue organs by passing through the normal physiological
distribution and excretion, even the various cell uptake barriers, such as the blood-air barrier, blood-testis
patterns and locations, may determine the degree of the barrier, blood-brain barrier and blood-placental barrier.
Blood-air barrier Blood-placenta barrier

The lungs are a potential entrance for graphene nano- The placental barrier is indispensable in maintaining
particles into the human body through airway. The pregnancy, as it mediates the exchange of nutrients and
inhaled GO nanosheets can destroy the ultrastructure metabolic waste products, exerts vital metabolic func-
and biophysical properties of pulmonary surfactant (PS) tions and secretes hormones [67]. A recent review
film, which is the first line of host defense, and emerge suggested that the placenta does not provide a tight
their potential toxicity [54]. The agglomerated or dis- barrier against the transfer of nanoparticles to foetuses,
persed particles deposit on the inner alveolar surface specifically against the distribution of carbonaceous
within the alveoli and then be engulfed by alveolar nanoparticles to and in the foetus [42]. It was suggested
macrophages (AMs) [55]. Clearance in the lungs is facili- that rGO and gold particles (diameter of 13 nm) are
tated by the mucociliary escalator, AMs, or epithelial barely present or are absent in the placenta and foetus
layer [56–58]. However, some small, inhaled nanoparti- in late gestation after intravenous injection [44, 68].
cles infiltrate the intact lung epithelial barrier and can However, other reports showed that transplacental trans-
then transiently enter the alveolar epithelium or the fer does occur in late gestational stages [69, 70]. Much
interstitium [59, 60]. Intratracheally instilled graphene attention had been paid to the developmental toxicity of
can redistribute to the liver and spleen by passing nanomaterials, and reports showed that many nanopar-
through the air-blood barrier [61]. The study of blood- ticles did cross the placental barrier and strongly influ-
air barrier may draw an intensive attention, since the enced the development of embryos [71–75]. But studies
researchers and workers occupational exposure of GFNs of the exposure to graphene materials through the
usually through inhalation. To make clear how the placenta barrier are deficient, and how these particles
blood-air barrier plays a role in the toxicity of GFNs transfer to embryos should be evaluated in detail in the
may become a research hot topic. future.
These four barriers were the most frequently men-
tioned barriers in the literature, and other barriers have
Blood-brain barrier
not been evaluated in recent studies, such as skin
The intricate arrangement of the blood-brain barrier,
barriers, which have not been mentioned in any of the
consisting of numbers of membrane receptors and
hundreds of GFNs toxicity studies searched. Moreover,
highly selective carriers, only exerts subtle influence on
the mechanism by which GFNs pass through these
blood circulation and the brain microenvironment com-
barriers is not well understood, and more systematic
pared to the peripheral vascular endothelium [62]. The
investigations are urgently needed.
research on the mechanism of blood-brain barrier had
made some progress involved in diseases and nano-
Distribution and excretion of GFNs in tissue
toxicity. Matrix-assisted laser desorption/ionization
The absorption, distribution, and excretion of graphene
(MALDI) mass spectrometry imaging (MSI) revealed
nanoparticles may be affected by various factors inclu-
that rGO, with an average diameter of 342 ± 23.5 nm,
ding the administration routes, physicochemical proper-
permeated through the paracellular pathway into the
ties, particle agglomeration and surface coating of GFNs.
inter-endothelial cleft in a time-dependent manner by
The different administration routes influence the dis-
decreasing the blood-brain barrier paracellular tight-
tribution of GFNs, for example, intratracheally instilled
ness [63]. In addition, graphene quantum dots (GQDs),
FLG passing through the air-blood barrier mainly accu-
with a small size of less than 100 nm, can cross through
mulated and was retained in the lungs, with 47 %
the blood-brain barrier [64]. Studies on how graphene
remaining after 4 weeks [61]. Intravenously administered
materials pass through the blood-brain barrier and cause
GO entered the body through blood circulation and was
neurotoxicity are very rare, and more data are needed
highly retained in the lung, liver, spleen and bone
to draw a conclusion.
marrow, and inflammatory cell infiltration, granuloma
formation and pulmonary edema were observed in the
Blood-testis barrier lungs of mice after intravenous injection of 10 mg kg/
The blood-testis and blood-epididymis barriers are body weight GO [49]. Similarly, high accumulation of
well known for being some of the tightest blood- PEGylated GO derivatives was observed in the reticulo-
tissue barriers in the mammalian body [65]. GO parti- endothelial (RES) system including liver and spleen after
cles with diameters of 54.9 ± 23.1 nm had difficulty intraperitoneal injection. In contrast, GO-PEG and FLG
penetrating the blood-testis and blood-epididymis did not show detectable gastrointestinal tract absorption
barriers after intra-abdominal injection, and the sperm or tissue uptake via oral administration [31].
quality of the mice was not obviously affected even at The different properties of GFNs, such as their size,
300 mg/kg dosage [66]. dose and functional groups, always lead to inconsistent
results in the distribution profiles of graphene. For in- Uptake and location of GFNs in cells
stance, Zhang et al. found that GO was mainly entrapped The uptake and location of GFNs have also been
in mouse lungs [49]; however, Li et al. observed that GO observed to exert different effects in different cell lines.
accumulated in mouse liver [76]. Notably, small GO Graphene is taken up into cells via various routes [82, 83].
sheets, with diameters of 10–30 nm, were mainly distri- Basically, the physicochemical parameters such as the
buted in the liver and spleen, whereas larger GO sheets size, shape, coating, charge, hydrodynamic diameter,
(10–800 nm) mainly accumulated in the lungs [49, 52, isoelectric point, and pH gradient are important to allow
77]. If the size of GO is larger than the size of the vessels, GO to pass through the cell membrane [84]. As stated
GO usually becomes stuck in the arteries and capillaries previously, nanoparticles with diameters <100 nm can
in the proximity of the injection site. The accumulation of enter cells, and those with diameters <40 nm can enter
GO in the lungs was shown to increase with an increase the nucleus [85]. For example, GQDs possibly penetrate
in the injected dose and size, but that in the liver sig- cell membranes directly, rather than through energy-
nificantly decreased [78]. Coating biocompatible polymers dependent pathways [86, 87]. Larger protein-coated
onto GO also affects the biodistribution, for instance, the graphene oxide nanoparticles (PCGO) (~1 μm) enter cells
intravenous injection of GO-PEG and GO-dextran (GO- mainly through phagocytosis, and smaller PCGO nano-
DEX) accumulate in the reticuloendothelial system (RES), particles (~500 nm) enter cells primarily through clathrin-
including the liver and spleen, without short-term toxicity mediated endocytosis [88]. GO sheets could adhere and
[31, 79]. Moreover, the charge of plasma proteins and ad- wrap around the cell membrane, insert in the lipid bilayer
sorption of GO by plasma proteins also affects the biodis- or be internalized into the cell as a consequence of inter-
tribution [34]. actions with cells [89]. Similarly, PEGylated reduced
The excretion and clearance of GFNs vary in different graphene oxide (PrGO) and rGO were shown to adhere
organs. In the lungs, observations indicated that NGO is onto the lipid bilayer cell membrane prominently due to
drawn into and cleared by AMs, which might be elimi- the interaction of hydrophobic, unmodified graphitic
nated from the sputum through mucociliary clearance domains with the cell membrane [90, 91]. Consequently,
or other ways [57], and 46.2 % of the intratracheally it was suggested that prolonged exposure to or a high
instilled FLG was excreted through the faeces 28 d after concentration of graphene induces physical or biological
exposure [61]. In the liver, nanoparticles can be elimi- damage to the cell membrane, along with destabilization
nated thorough the hepato-biliary pathway following the of actin filaments and the cytoskeleton [92].
biliary duct into the duodenum [80]. In addition, PEGy- Current data demonstrates that GO sheets interact
lated GNS that mainly accumulates in the liver and with the plasma membrane and are phagocytosed by
spleen can be gradually cleared, likely by both renal and macrophages. Three major receptors on macrophages
faecal excretion. As recently reviewed, GO sheets larger take part in the phagocytosis of GNS: the Fcg receptor
than 200 nm are trapped by splenic physical filtration, (FcgR), mannose receptor (MR), and complement recep-
but small sizes (approximately 8 nm) can penetrate the tor (CR). Furthermore, FcgR is a key receptor in the
renal tubules into the urine and be rapidly removed mediated phagocytic pathway [90, 93, 94]. The protein
without obvious toxicity [81]. The excretion paths of corona of GO promotes the recognition by macrophage
GFNs have not yet been clearly explained, but renal and receptors, especially the IgG contained within the pro-
faecal routes appear to be the main elimination routes tein corona. Macrophages were observed to undergo
for graphene. prodigious morphological changes upon contact with
Recently, the distribution and excretion/toxicity strat- GO [34]. After internalization, graphene accumulated in
egy has become an important part of nano-toxicological the cell cytoplasm, perinuclear space, and nucleus,
studies. To date, several controversial results regarding which induced cytotoxicity in murine macrophages by
the distribution and excretion of graphene in vivo have increasing intracellular ROS through depletion of the
been reported in several papers, and a systematic evalu- mitochondrial membrane potential and by triggering
ation of the toxicokinetics of GFNs is still needed. The apoptosis through activation of the mitochondrial
metabolism and excretion of nanomaterials are long- pathway [83]. The possible interactions and accumula-
period processes, however, the recent studies of GFNs tion sites of GFNs are summarized in Fig. 1.
had been limited to short-term toxicological assess-
ments, and the long-term accumulation and toxicity of Toxicity of GFNs in organs
GFNs on different tissues remain unknown. Therefore, The toxicity and biocompatibility of GFNs has been
long-term studies on the deposition and excretion of observed and assessed through theoretical and animal
GFNs need to be performed using different cells and model studies. At present, there are a mass of data dem-
animals to ensure the materials’ biosafety before utilization onstrating the toxicity of GFNs in different organs or
in human biomedical applications. systems in animals, so that it is hard to list all the data
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 exfoliated
graphene; 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 the
plasma 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 from
such 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 Science
in this review. Thus we summarized a certain number suggested to reduce the likelihood of lung fibrosis for-
literature and chose some in vivo toxicological studies of mation in cells or mice, whereas lung fibrosis was ob-
GFNs listed in Table 1. served when graphene was suspended with bovine
serum albumin (BSA) [100]. In addition, radioactive
Toxicity in internal organs isotopes can be delivered into the lungs, accompanied
GO can result in acute inflammation response and by a depth distribution of 125I-NGO in the lungs, and
chronic injury by interfering with the normal physio- the isotopes might deposit there and result in muta-
logical functions of important organs [32, 81]. Oral tions and cancers [30]. However, recent publications
gavage experiments did not show detectable absorption claimed no obvious pathological changes in mice ex-
of GO through the gastrointestinal tract [95]. Interes- posed to low dosages of GO and functionalized gra-
ting, a low dose of GO caused serious damage to the phene by intravenous injection, including aminated
gastrointestinal tract after maternal mice drank a GO GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-
suspension rather than a high-dose of GO because a low PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and
dose of GO without agglomeration can easily attach to GO-PEG; only GO-PEG and GO-PAA induced less toxicity
the gastrointestinal surface and cause destruction than pristine GO in vivo [31, 79, 89]. So the functional
through its abundant sharp edges [53]. GFNs caused groups of GFNs and the working concentration or ag-
inflammation and remained in the lung on day 90 after a gregate state largely influence the toxicity of GFNs.
single intratracheal instillation, and even translocated to Recently, the ways to modify the functional group of
lung lymph nodes by a nose-only inhalation [96, 97]. A GFNs, decrease the working concentration or change
high dose of GO that forms aggregations can block pul- the aggregate condition are usually used to decrease
monary blood vessels and result in dyspnea [50, 98], and the toxicity of GFNs.
platelet thrombi were observed at high concentrations
of 1 and 2 mg/kg body weight via intravenous injec- Toxicity in the central nervous system
tion [89]. GO reportedly disrupted the alveolar- Graphene has largely benefited neurosurgery with the
capillary barrier, allowing inflammatory cells to infil- application of drug/gene delivery for brain tumour treat-
trate into the lungs and stimulate the release of pro- ment, intracranial and spinal biocompatible devices,
inflammatory cytokines [99]. Fibrosis and inflamma- biosensing and bioimaging techniques. Studies regarding
tion could be verified by the increased levels of the the potentialities or risks of graphene in the brain have
protein markers collagen1, Gr1, CD68 and CD11b in emerged. In the chicken embryo model, pristine gra-
the lungs. The use of Tween 80 to disperse FLG or a phene flakes decreased the ribonucleic acid level and the
pluronic surfactant to disperse graphene was rate of deoxyribonucleic acid synthesis, leading to
Table 1 Toxicity of GFNs in organs
Graphene family Physiochemial properties Animals Dose and time incubation Effects Reference
nanomaterials and functionalization
Nanoscale graphene oxide No information C57BL/6 mice 0, 1, 5, 10 mg/kg, intratracheal instillation Result in acute lung injury (ALI) and chronic [30]
(NGO) 0 h, 24 h, 48 h, 72 h and 1 week pulmonary fibrosis
Few layer graphene (FLG) No information ICR mice 0.1, or 1 mg/mL, oral gavage or intratracheal Intratracheally instilled FLG resulted in acute [61]
instillation 3 or 28 days lung injury and pulmonary edema, FLG didn’t
show detectable absorption through the
gastrointestinal tract by oral gavage.
Graphene platelets (GPs) No information Mice inhalation exposure, 1 day-6 weeks GP caused acute inflammation in lung at [48]
1 day, and alleviated inflammation in lung
after 6 weeks
Graphene nanoplatelets Thickness of 10 nm Female C57BL/6 50 μg per mouse, pharyngeal aspiration or Large GP were inflammogenic in both the [24]
(GPs) Size of 5–30 μm strain mice intrapleural installation, 24 h- 7 days lung and the pleural space
GO Thickness of 0.93 nm Sprague-Dawley rats 0.5 or 4 mg/m3, inhalation exposure, The single inhalation exposure to GO induce [235]
Size of 150–250 nm single 6 h minimal toxic responses in rat lungs
GO Thickness of 0.9 nm Male ICR mice 1.0 mg/kg, intravenous injected, 24 h Accumulated mainly in the liver and lungs [78]
size of l-GO: 1–5 μm
size of s-GO:100–500 nm
GO Thickness of < 4 nm Male and female 24 mg/kg, tail vein injected, 5 days Didn’t effect pup numbers, sex ratio, weights, [66]
size of l-GO:237.9 ± 79.3 nm; size of ICR-strain mice pup survival rates or pup growth, low toxicity
s-GO: 54.9 ± 23.1 nm for male reproduction
GO Thickness of ~1.0 nm Kun Ming mice 1,10 mg/ kg, intravenous injection 14 days Led to high accumulation, long-time retention, [49]
sizes of 10–800 nm pulmonary edema and granuloma formation
NGO-PEG Thickness of 1 nm Male Kunming mice 5 mg/kg, tail intravenous injection NGO-PEG alleviated acute tissue injuries, [81]
size of 10–800 nm 10 min-24 h decreased the early weight loss
GO Thickness of 0.94,1.22, 4.43 and Balb/c mice 4 mg/kg, intraperitoneal injection Accumulated in the reticuloendothelial (RES) [31]
GO-PEG 5.66 nm, 1, 7 and 30 days system including liver and spleen over a
RGO-PEG size of 450, 25, 50 and 27 nm long time
nRGO-PEG
GO Thickness of GO, GQD: 0.5–1 nm Balb/c mice 20 mg/kg intravenous injection or GO appeared toxic and caused death [176]
Graphene quantum dots sizes of GO, GQD: 3–5 nm intraperitoneal injection 14 days GQD revealed no accumulation in organs and
(GQD) caused low cytotoxicity
Purified graphene oxide Thickness of 1–2 nm, Female C57Bl/6 mice 50 μg/animal, intraperitoneal injection Induced moderate inflammation and granuloma [99]
(pGO) lateral dimension of 24 h, 7 days, formation following
100–500 nm
GO Thickness of 3.9 and 4.05 nm, C57BL/6 male mice Series concentrations, subcutaneous The micro-size of GO induced much stronger [34]
size of 350 nm and 2 μm injection21 days inflammation responses than the nanosized GO
GO Size of 1110 to 16 200 nm C57BL/6 J mice 2 or 20 mg/kg, subcutaneous and Both GO and a reduction of GO result in immune [84]
intraperitoneal injection cell infiltration, uptake, and clearance.
Page 6 of 24
RGO-iron oxide Thickness of ˂10 nm Female Balb/c mice 400 μg, subcutaneous injection, RGO–IONP can effectively inactivate multiple-drug- [236]
nanoparticles (rGO-IONP) Size of 15.0 ± 2.0 nm resistant bacteria in subcutaneous abscesses
Table 1 Toxicity of GFNs in organs (Continued)

GO Thickness of 0.94, 1.22, 4.43 Female balb/c mice 100 mg/kg, Oral administration; 50 mg/kg, No obvious tissue uptake via oral administration, [237]
GO-PEG and 5.66 nm, intraperitoneal injection, 1, 7 and 30 days indicating the rather limited intestinal
size of 450, 25, 50 and 27 nm adsorption of those nanomaterials
RGO sizes of small rGO: 87.97 ± 30.83, Male C57black/6 60 mg/kg, oral gavage, 5 days RGO affected general locomotor activity, balance, [31]
sizes of large rGO:472.08 ± 249.17 nm mice and neuromuscular coordination, but showed
little change in exploratory, anxiety-like, or
learning and memory behaviors.
Page 7 of 24
harmful effects on brain tissue development and the demonstrated haemolytic effect up to 75 μg/mL [104].
atypical ultrastructure was observed in the brain [101]. GO-polyethylenimine (GO-PEI) exhibited notable toxicity
The recent researches of GFNs in the central nervous by binding to HSA, even at 1.6 μg/mL [105]. Carboxylated
system are mostly involved in the application rather than graphene oxide (GO-COOH) showed significant cytoto-
the toxicity. The data of the toxic study on GFNs is xicity toward T lymphocytes at concentrations above
underway. 50 μg/mL and had good biocompatibility below 25 μg/
mL, whereas GO-chitosan nearly inhibited haemolytic
Toxicity in reproduction and development system activity [106]. Until now, the corresponding risk of hae-
Pristine graphene reduced the vascularization of the mocompatibility has remained largely unknown.
heart and the density of branched vessels after injection In conclusion, the lung injury induced by GFNs has
into fertilized chicken eggs followed by incubation for 19 been studied in several studies, the results of which have
d [101]. GO and rGO damage zebrafish embryos by demonstrated inflammatory cell infiltration, pulmonary
influencing the embryo hatching rate and body length in edema and granuloma formation in the lungs. However,
a concentration-dependent manner. Although no obvi- only a few specific studies have evaluated in other or-
ous malformation or mortality was observed in exposed gans, such as the liver, spleen, and kidney, and the injury
zebrafish embryos [102], GO adhered to and was wrapped symptoms, damage index and level of damage to these
in the chorion of the zebrafish embryos, causing remar- internal organs were not fully investigated. Moreover,
kable hypoxia and hatching delay. GO aggregates were studies on the neurotoxicity of GFNs are quite rare; no
retained in many organelles, such as the eyes, heart, yolk data has revealed which nerves or brain areas experience
sac, and tail of the embryos, and apoptosis and reactive damage, nor have the related behavioural manifestations
oxygen species (ROS) generation were observed in these been studied. The developmental toxicity of GFNs may
regions [103]. induce structural abnormalities, growth retardation, be-
The GFNs exert different toxicological effects on male havioural and functional abnormalities, and even death.
or female reproductive system. Data showed that GO A study on the reproductive and developmental toxicity
exerted very low or nearly no toxic effects on male of GFNs will be extremely significant and gain extensive
reproduction even at a high dose via intra-abdominal attention in the future. Almost all the GFNs toxicity
injection [66]. Additionally, rGO did not change the studies were short-period experiments, and no studies
serum estrogen levels of non-pregnant female mice. The have investigated long-term chronic toxic injury. How-
condition is different in the female mouse: mouse dams ever, based on studies of other nanomaterials toxicity,
could give birth to healthy offspring after rGO injection long-term GFNs exposure may be an important factor
before mating or during early gestation, and only a few harming health [107–109]. Therefore, the long-term
abnormal foetuses were present among the rGO-injected study of GFNs is necessary.
dam litters. However, the pregnant mice had abortions
at all dose, and most pregnant mice died when the high
dose of rGO was injected during late gestation [44]. Not- Toxicity of GFNs in cell models
ably, the development of offspring in the high dosage The cytotoxicity of GFNs in vitro has been verified in
group was delayed during the lactation period. The high various cells to change the cell viability and morphology,
dose of GO decreased the maternal mice’s water con- destroy the membrane integrity, and induce DNA
sumption by oral exposure, which reduced milk produc- damage [110–112]. GO or rGO decrease cell adhesion;
tion and thus postponed the growth of offspring [53]. induce cell apoptosis; and enter lysosomes, mitochon-
Though the findings indicate that GFNs are potentially dria, cell nuclei, and endoplasm [113]. GQDs entered
harmful to development, but data on reproductive and cells and induced DNA damage by the increased expres-
developmental toxicity are still deficient. Studies of the sion of p53, Rad 51, and OGG1 proteins in NIH-3 T3
influence of GFNs on male and female reproduction cells [87]. However, GQDs did not pose significant tox-
and development are still required to elucidate the icity to human breast cancer cell lines (at a dose of
underlying toxicity mechanism. 50 μg/mL) or human neural stem cells (at a dose of
250 μg/mL) [114, 115]. GO derivatives dramatically
Influence of haemocompatibility decreased the expression of differential genes that are
GO release into the blood is ineluctable. The haemo- responsible for the structure and function of the cell
compatibility of GO was found to be dependent on the membrane, such as regulation of the actin cytoskeleton,
functional coating and the exposure conditions. GO focal adhesion and endocytosis [89]. In rat pheochromo-
with submicron size resulted in the greatest haemo- cytoma cells (PC12 cells), graphene and rGO caused
lytic activity, while aggregated graphene induced the cytotoxic effects and mitochondrial injury, such as the
lowest haemolytic reaction. Pristine graphene and GO release of lactate dehydrogenase (LDH), an increase in
the activation of caspase-3, and the generation of ROS uptake. Moreover, an in vitro study reported that 20 μg/mL
[82, 116]. GO nanosheets exhibited no cytotoxicity in A549 within
Graphene can increase cell viability [117] or cause cell 2 h of incubation, but a higher concentration (85 μg/mL)
death [118] depending on the cell line, type of graphene decreased the cell viability to 50 % within 24 h [136, 137].
material and the doseage. GO cytotoxicity was observed Lü et al. also demonstrated that GO had no obvious
in human fibroblasts and lung epithelial cells at concen- cytotoxicity at low concentrations for 96 h in a human
trations above 20 μg/mL after 24 h, but minimal toxicity neuroblastoma SH-SY5Y cell line, but the viability of cells
was found in A549 cells at concentrations higher than sharply decreased to 20 % after treatment with 100 mg/mL
50 μg/mL [119]. The biological responses induced by GO for 96 h of incubation [123]. The results in HeLa cells,
GO such as ROS, malondialdehyde (MDA), and LDH in- NIH-3 T3 cells, and breast cancer cells (SKBR3, MCF7)
creased, whereas superoxide dismutase (SOD) decreased treated with graphene nanoribbons also showed a dose-
dose-dependently in HeLa cells [120]. However, GO- (10–400 mg/ml) and time-dependent (12–48 h) decrease in
molecular beacon (GO-MB) showed low cytotoxicity cell viability [138]. Increasing concentrations of GO entered
even at 20 μg/mL in HeLa cells [121]. GO decreased the the lysosomes, mitochondria, endoplasm, and cell nucleus
viability of A549 cells, while the same concentration and [119]. Several data indicated that rGO caused apoptosis-
time of exposure increased the cell viability of CaCo2 mediated cell death at a lower dose and early time point
colorectal carcinoma cells [122]. Another study reported but that necrosis was prevalent with the increase in time/
that GO dramatically enhanced the differentiation of dose [110, 135].
SH-SY5Y, accompanied by increasing neurite length and
the expression of neuronal marker MAP2 at low concen- Lateral dimension
trations but that GO suppressed the viability of SH- Nanoparticles with sizes <100 nm can enter the cell,
SY5Y cells at high doses (≥80 mg/mL) [123]. Functiona- <40 nm can enter nucleus, and smaller than <35 nm can
lized coatings on GO, such as GO-PEG [124] and GO- cross the blood brain barrier [85]. One study showed
chitosan [125], can profoundly attenuate the particles’ that GO (588, 556, 148 nm) did not enter A549 cells
cytotoxicity by inhibiting the interactions between cells. and had no obvious cytotoxicity [112]. When the diam-
The toxicity of GFNs in vitro is summarized in Table 2. eter of graphene is between 100 ~ 500 nm, the smallest
Data on the cytotoxicity of graphene nanomaterials are size may cause the most severe toxicity, and when the
contrasting, and varying characteristics influence the re- diameter is below 40 nm, the smallest sizes may be the
sults. The mechanisms and influencing factors of safest. For instance, rGO with a diameter of 11 ± 4 nm
toxicity need to be elucidated in detail. could enter into the nucleus of the hMSCs and cause
chromosomal aberrations and DNA fragmentation at
Origins of GFNs toxicity very low concentrations of 0.1 and 1.0 mg/mL in 1 h.
Reportedly, the characteristics of graphene, including its However, rGO sheets with diameters of 3.8 ± 0.4 nm
concentration, lateral dimension, surface structure, func- exhibited no notable genotoxicity in hMSCs even at a
tional groups, purity and protein corona, strongly influ- high dose of 100 mg/mL after 24 h [118].
ence its toxicity in biological systems [2, 7, 104, 126–129]. In an in vivo study, s-GO (100–500 nm) preferentially
accumulated in the liver, whereas l-GO (1–5 μm) was
Concentration mainly located in the lungs because l-GO formed larger
Numerous results have shown that graphene materials GO-protein complexes that were filtered out by the
cause dose-dependent toxicity in animals and cells, such pulmonary capillary vessels after intravenously injection
as liver and kidney injury, lung granuloma formation, [31]. Given the relative lateral sizes (205.8 nm, 146.8 nm
decreased cell viability and cell apoptosis [130–134]. In and 33.78 nm) of the three GO nanosheets at the same
vivo studies, GO did not exhibit obvious toxicity in mice concentration, smaller GO experiences much greater up-
exposed to a low dose (0.1 mg) and middle dose take than larger GO in Hela cells [139]. The high uptake
(0.25 mg) but induced chronic toxicity at a high dose of s-GO changed in the microenvironment of cells and
(0.4 mg). The high content of GO mainly deposited in consequently induced the greatest viability loss and most
the lungs, liver, spleen, and kidneys and was difficult to serious oxidative stress among three sizes of GO samples
be cleaned by the kidneys via a single tail vein injection [119]. As a result, one study delineated that GO size-
[135]. Intriguingly, increasing the dose resulted in a dra- dependently induced the M1 polarization of macro-
matic decrease in the hepatic uptake but an increase in phages and pro-inflammatory responses in vitro and in
the pulmonary uptake of s-GO by intravenous injection vivo. Larger GO showed stronger adsorption onto the
[31], because the high dose of GO potentially surpassed plasma membrane with less phagocytosis, eliciting
the uptake saturation or depleted the mass of plasma robust interactions with TLRs and activating NF-κB
opsonins, which consequently suppressed the hepatic pathways, compared to smaller GO sheets, which were
Table 2 Toxicity of GFNs in cell models
Graphene family Physiochemial properties and Cells Dose and time incubation Effects Reference
nanomaterias Functionalization
Pristine graphene Thickness of 2–3 nm, size of Murine RAW 264.7 macrophages 5, 10, 20, 40, 80 and 100 mg/ Depleted of the mitochondrial membrane [83]
500–1000 nm mL, 48 h potential, increased ROS, triggered apoptosis
Pristine graphene Thickness of 3–5 nm, size of Rat pheochromocytoma cells 10–100 μg/mL 1–48 h Increased LDH release, ROS levels and [82]
100–110 nm PC12 cells caspase3 activation, induced apoptosis
Graphene oxide(GO) Four different diameters Human Erythrocytes 3.125-200 μg/mL 24 h Hemolytic activity, ROS generation, LDH [106]
(342–765 nm) Human skin release, decreased cell viability
fibroblasts CRL-2522
GO Thickness of 0.9 nm Human lung epithelial A549 10, 25, 50, 100 and 200 μg/mL Dose-dependent oxidative stress, cell viability [119]
lateral size: s-GO, 160 ± 90 nm; cells 24 h decreased at high concentration
m-GO, 430 ± 300 nm;
l-GO, 780 ± 410 nm
GO Thickness of 1 nm, lateral Human lung fibroblast cells HLF 10–500 μg/mL 2–24 h Oxidative stress induced, concentration- [148]
dimension of 200–500 nm cells dependent cytotoxicity and genotoxicity
GO Size distribution: 592 ± 10.9 nm HeLa cells 0–80 μg/mL 24 h Released LDH, increased MDA and ROS [120]
in PBS, 1272 ± 56.2 nm in FBS generation, decreased SOD, reduction of
cell viability,
GO smaller-sized GO: 50–350 nm Macrophage cell J774A.1 20 μg/mL 1-24 h Size-dependent M1 induction of [94]
intermediate-sized GO: 350–750 nm THP-1 cells macrophages,
larger-sized GO: 750–1,300 nm HEK293 cells pro-inflammatory responses
MEL cells
HUT102 cells
GO thickness: < 2 nm, Mouse CT26 colon carcinoma 50–100 μg/mL 18 h Triggered autophagy, enhances cell death [206]
lateral size: 450 nm cell
Reduced graphene oxide (rGO) Thickness of 11 ± 4 nm Human mesenchymal stem 0.01–100 μg/mL 1–24 h Induced DNA fragmentations and [118]
lateral size of 3.8 ± 0.4 μm cells (hMSCs) chromosomal aberrations
RGO Thickness of 7 nm human liver carcinoma 1–200 mg/L 4–72 h Dose-dependent DNA damage, oxidative [31]
lateral size of 40 nm cells (HepG2 cells) stress, cytotoxicity
RGO Lateral size of 100–1500 nm U87 and U118 glioma 0–100 μg/mL 24 h Reduction of cell proliferation and cell [238]
cell lines viability, induced apoptosis
Bacterially reduced graphene oxide Thickness of 4.23 nm MCF-7 cells 20–100 μg/mL 24–72 h Increased ROS generation, released LDH, [181]
(B-rGO) average size of 3833 nm dose-dependent toxicity
Reduced graphene oxide Thickness of 1 nm, hMSCs 0.01, 0.1, 1.0, 10, 100 μg/mL Caused DNA fragmentations and [239]
Nanoribbons(rGONR) length of 10 μm, 96 h chromosomal aberrations
width of 50–200 nm,
Reduced graphene oxide sheets Thicknesses of ~1.2 nm, hMSCs 0.01, 0.1, 1.0, 10, 100 μg/mL Caused slight cell membrane damage and [239]
(rGOSs) lateral sizes of ~2 μm 96 h cytotoxicity
Graphene-dextran Thickness of 2.8 nm HeLa cells 10, 50,200 mg/L 24, 48, 72 h GO-DEX remarkably reduced cell toxicity [91]
Page 10 of 24
(GO-DEX) size of 50–100 nm
GNP-COOH Thickness of GNP-COOH: 735.9 nm Human bronchial epithelial 10, 50 mg/L 24 h Caused single stranded DNA damage, [240]
GNP-NH2 thickness of GNP-NH2: 945.5 nm cells (BEAS-2B cells) genotoxicity and hypomethylation
Table 2 Toxicity of GFNs in cell models (Continued)

PEG-DSPE (O-GNR-PEG-DSPE) Width of 125–220 nm, lengths HeLa cells 10–400 μg/mL 24–48 h Dose-dependent and time-dependent [138]
between of 500–2500 nm NIH-3 T3 cells decrease in cell viability
SKBR3 cells
MCF7 cells
PEI-GO, Thickness of 1–2 nm lateral width of Human lung fibroblast cells 1, 10, 50, 100 μg/ml 24 h Caused concentration-dependent [15]
PEG-GO, 100–500 nm cytotoxicity and genotoxicity
LA-PEG-GO
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 mitigated [166]
at 10 % FBS
Page 11 of 24
more likely taken up by cells [94]. To further uncover (HP), F10, W23, and F35. The protein’s secondary and ter-
the detailed mechanism underlying these effects, more tiary structures are largely lying on the graphene surface,
studies are needed to illustrate the vital mechanism of disrupting the structure and function of the protein [41]
the lateral size of graphene materials. (Fig. 2). In addition, GO can insert between the base
pairs of double-stranded DNA and disturb the flow of
Surface structure genetic information at the molecular level, which
GFNs possess widely varying surface chemistries. For might be one of the main causes of the mutagenic
example, the pristine graphene surface is hydrophobic, effect of GO [7, 112, 146, 147].
GO surface is partially hydrophobic with carboxylate
groups [140–142], and rGO has intermediate hydrophi- Charge
licity [143]. GFNs were observed to disrupt the function A number of studies have highlighted the importance of
and structure of cell membranes and proteins probably the GO surface charge because of its ability to affect the
by exceptionally strong molecular interactions with cells internalization and uptake mechanism of cells [148–150].
[2, 91]. For instance, rGO bonded to cell membranes, GO internalization was negligible in non-phagocytes,
stimulated receptors and activated mitochondrial path- which was likely due to the strong electrostatic repulsion
ways, inducing apoptosis [110, 111, 144]. Limited evi- between the negatively charged GO and the cell surface
dence showed that GO is smaller and less toxic than [34]. However, others have suggested that negatively
rGO because of the high oxygen content, smoother charged nanoparticles can be internalized into non-
edges, and hydrophilic properties of the former species phagocytic cells by binding to available cationic sites
[104, 145, 146]. Because of the different surface oxida- on the cell surface and be taken up by scavenger re-
tion states of GO and rGO, GO possessing distinct ceptors [110, 146, 150]. GO/GS particles reportedly
hydrophilicity might be internalized and taken up by cause morphological changes and significant lysis,
HepG2 cells easily. On the contrary, rGO with evident leading to high haemolysis in red blood cells (RBCs).
hydrophobicity, could be adsorbed and aggregated at cell RBC membrane disruption is probably attributed to
surfaces without (or with lower) uptake [110]. Due to the strong electrostatic interactions between the nega-
strong π-π stacking interactions, graphene is highly tively charged oxygen groups on the GO/GS surface
capability of breaking many residues of the protein, and positively charged phosphatidylcholine lipids on
particularly the aromatic ones, such as the villin headpiece 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-π
stacking 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
Functionalization for the observed toxicity, rather than the nanomaterial

Studies confirmed that functionalization with PEG [52], itself, which has resulted in conflicting data on GFNs
PEGylated poly-L-lysine (PLL) [151], poly(ε-caprolactone) cytotoxicity [35, 160]. Traditionally prepared GO often
[152], polyvinyl alcohol [3], Pluronic [153], amine [98], contains high levels of Mn2+ and Fe2+, which are highly
carboxyl, and dextran [79] groups largely decreases the mutagenic to cells. The nonspecific release of these ions
toxicity and improves the biocompatibility of graphene. In from traditionally prepared GO might lead to unusually
vivo results revealed that only mild chronic inflammation high levels of cytotoxicity and DNA fracturing [39]. In
emerged after the subcutaneous injection of GO-Pluronic particular, Peng et al. [161] produced high-purity GO
hydrogel and no noticeable short-term toxicity was tested containing only 0.025 ppm Mn2+ and 0.13 ppm Fe2+,
after the intravenous injection of GO-DEX [79, 154]. and Hanene et al. [162] invented a new method to
PEGylated GS did not induce appreciable toxicity in mice prepare high-purity, single-layer GO sheets with good
exposed to 20 mg/kg for 3 months, as evaluated by blood aqueous dispersibility and colloidal stability. GO pro-
biochemistry and histological examinations, and showed duced by these new methods did not induce significant
relatively low retention in the RES [52, 155]. Coating GO cytotoxic responses (at exposure doses up to 100 μg/mL)
with chitosan almost eliminated the haemolytic activity in in vitro, and no obvious inflammatory response or
blood [39]. Moreover, the PEG coating effectively allevi- granuloma formation (exposure doses up to 50 μg/animal)
ated GO-induced acute tissue injuries; decreased GO were observed in vivo. Therefore, the purity of GFNs
aggregation and retention in the liver, lungs, and spleen; deserves attention and is a vital step towards the deter-
and promoted the clearance of GO [81], GO-DEX [79], mination of GFNs involved in bioapplications.
and fluorinated graphene oxide (FGO) [156].
In vitro, several cell function assays showed clear Protein corona effect
evidence that the surface functionalization of pristine Because of the high free surface charge, nanomaterials
graphene or GO was critical for reducing the strong can easily form “coronas” with proteins in biological
toxicity effects [91]. PEG-GO, PEI-GO and LA-PEG-GO systems [163, 164]. The protein corona is suggested to
damaged human lung fibroblast cells less than GO affect the circulation, distribution, clearance and toxicity
[148]. PEG-GO exhibited no cytotoxicity toward several of nanoparticles. Several papers reported that GO forms
cell cultures, such as glioblastoma cells (U87MG), breast GO-protein coronas with adsorbed plasma proteins in
cancer cells (MCF-7), human ovarian carcinoma cells serum and these GO-protein coronas play an important
(OVCAR-3), colon cancer cells (HCT-116), and lympho- role in deciding the fate of the GO biokinetic behaviour
blastoid cells (RAJI), at concentrations up to 100 μg/mL in vivo. Such GO-protein coronas can regulate the adhe-
[119, 157, 158]. GQDs-PEG exhibited very low or no sion of GO to endothelial and immune cells through
toxicity against lung and cervical cancer cells even at both specific and nonspecific interactions [165]. Basic-
very high concentrations (200 μg/mL) [159]. However, as ally, immunoglobulin G and complement proteins in
a non-biodegradable material with great potential for the protein corona help to reorganize nanoparticles in
cellular internalization, further investigation is needed to immune cells, causing the particles to be engulfed by
assess the possible long-term adverse effects of function- the RES, and IgG-coated GO was taken up by either
alized graphene. specific or nonspecific interactions with cell membrane
receptors [31, 165]. However, another study found that
Aggregations and sedimentation GO could not adhere to mucosal epithelial cells directly in
Reportedly, nanomaterials have a propensity to form the intestinal tract after the filial mice drank an aqueous
aggregates rather than individual units, particularly under GO solution because abundant proteins in the milk had
physiological conditions. GS surfaces allowed fewer RBCs adsorbed on the surface of the GO and thus inhibited
attach comparing to GO, and GS had the lower haemo- their direct interaction with the mucosal epithelial cells
lytic activity for more aqueous aggregations formation. In [53]. Protein corona mitigated the cytotoxicity of GO by
contrast, the fast sedimentation and aggregate formation limiting its physical interaction with the cell membrane
of GS greatly inhibited the nutrient availability of human and reducing the cellular morphological damage in HeLa,
skin fibroblast cells that were grown on the bottom of THP-1 and A549 cells [166–168]. The cytotoxic effect
wells [106]. Therefore, the aggregations and sedimentation was largely reduced when GO was pre-coated with FBS
of graphene particles exert varying effects on different and incubated with cells; nearly ∼ 90 % survival was
cells. observed with 100 μg/mL FBS-coated GO and 100 %
survival with 20 μg/mL FBS-coated GO. Similar trends
Impurities were observed for GO covered by BSA [166, 169]. Con-
Nanomaterial purity is an important consideration be- sistently, additional serum could neutralize the toxicity of
cause residual, contaminating metals may be responsible pristine GO in J774.A1 cells at a dose of 4 μg/mL, which
lead to a decrease in cell number of 52.5 % compared to Physical destruction

untreated cells [89]. Graphene is a unique nanomaterial compared with other
After reviewing many studies, it can be concluded that spherical or one-dimensional nanoparticles due to its
the toxicity of graphene is influenced by multiple factors. two-dimensional structure with sp2-carbons. The phy-
Those factors combined to largely change the toxicity of sical interaction of graphene nanoparticles with cell
GFNs in many cases. Scientific studies often need the membranes is one of the major causes of graphene cyto-
clear identification of cause and effect, which should toxicity [7, 170, 171]. Graphene has high capability to
keep only one factor different at a time, so that the effect bind with the α-helical structures of peptides because of
of that single factor can be determined. But in some its favourable surface curvature [172]. At concentration
papers, several factors influencing GFNs toxicity were above 75 μg/mL, pristine graphene largely adhered to
studied at the same time, which led to confused results. the surfaces of RAW 264.7 cells and resulted in abnor-
mal stretching of the cell membrane [104]. The strong
Possible toxicity mechanisms of GFNs hydrophobic interactions of GFNs with the cell mem-
Although some physicochemical properties and the brane lead to the morphological extension of F-actin
toxicity of GFNs have been well studied by many filopodial and cytoskeletal dysfunction. Furthermore, the
scholars, the exact mechanisms underlying the toxicity sharpened edges of GNS may act as ‘blades’, inserting
of GFNs remain obscure. A schematic of the main mecha- and cutting through bacterial cell membranes [173].
nisms of GFNs cytotoxicity is illustrated in Fig. 3. Moreover, GO also damaged the outer membrane of
Fig. 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
E. coli bacteria directly, resulting in the release of intracel- formation of •OH mediated by GO and the cytochrome-
lular components [173]. However, TEM imaging revealed c/H2O2 electron-transfer system could enhance oxidative
that pre-coating GO with FBS eliminated the destruction and thermal stress to impair the mitochondrial respi-
of cell membranes [166]. ration system and eventually result in dramatic toxicity
[151]. Additionally, the oxygen moieties on GO might
ROS production leading to oxidative stress accept electrons from cellular redox proteins, supporting
Oxidative stress arises when increasing levels of ROS the redox cycling of cytochrome c and electron transport
overwhelm the activity of antioxidant enzymes, including proteins, and cytochromes MtrA, MtrB, and MtrC/
catalase, SOD, or glutathione peroxidase (GSH-PX) OmcA might be involved in transferring electrons to
[174]. ROS act as second messengers in many intracellu- GO [182]. Therefore, except for the plasma membrane
lar signalling cascades and lead to cellular macromolecu- damage and oxidative stress induction, GFNs can cause
lar damage, such as membrane lipid breakdown, DNA apoptosis and/or cell necrosis by direct influencing cell
fragmentation, protein denaturation and mitochondrial mitochondrial activity [183, 184].
dysfunction, which greatly influence cell metabolism and
signalling [175–177]. The interactions of GO with cells DNA damage
can lead to excessive ROS generation, which is the first Due to its small size, high surface area and surface
step in the mechanisms of carcinogenesis, ageing, and charge, GO may possess significant genotoxic properties
mutagenesis [83, 122]. Oxidative stress had a significant and cause severe DNA damage, for example, chromo-
role in GO-induced acute lung injury [30], and the somal fragmentation, DNA strand breakages, point
inflammatory responses caused by oxidative stress often mutations, and oxidative DNA adducts and alterations
emerged upon exposure to GFNs [133, 177, 178]. The [87, 122, 185, 186]. Mutagenesis was observed in mice
activity of SOD and GSH-PX decreased after exposed to after intravenous injection of GO at a dose of 20 mg/kg
GO in a time- and dosage-dependent manner [82, 106, compared with cyclophosphamide (50 mg/kg), a classic
119]. Similarly, oxidative stress was the key cause of mutagen [112]. Even if GO cannot enter into the nucleus
apoptosis and DNA damage after HLF cells were ex- of a cell, it may still interact with DNA during mitosis
posed to GO [148]. Both the mitogen-activated protein when the nuclear membrane breaks down, which increases
kinase (MAPK) (JNK, ERK and p38) and TGF-beta- the opportunity for DNA aberrations [87, 147, 187, 188].
related signalling pathways were triggered by ROS gen- The π stacking interaction between the graphene carbon
eration in pristine graphene-treated cells, accompanied rings and the hydrophobic DNA base pairs can make a
by the activation of Bim and Bax, which are two pro- DNA segment ‘stand up’ or ‘lay on’ the surface of graphene
apoptotic members of the Bcl-2 protein family. As a with its helical axis perpendicular or parallel, respectively.
result, caspase-3 and its downstream effector proteins The intermolecular forces severely deform the end base
such as PARP were activated, and apoptosis was initiated pairs of DNA, which potentially increases the genotoxicity
[83, 179]. Detailed information regarding the MAPK-, [189]. GO may also induce chromosomal fragmentation,
TGF-β- and TNF-α-related signalling pathways, which DNA adducts and point mutations by promoting oxidative
induce inflammation, apoptosis and necrosis, are sum- stress or triggering inflammation through the activation of
marized in Fig. 4. intracellular signalling pathways such as MAPK, TGF-β
and NF-κB [110, 112, 146]. Graphene and rGO can also
Mitochondrial damage elevate the expression of p53, Rad51, and MOGG1-1,
Mitochondria are energy production centres involved in which reflect chromosomal damage, and decrease the
various signalling pathways in cells and are also a key expression of CDK2 and CDK4 by arresting the cell cycle
point of apoptotic regulation [83]. After exposure to GO transition from the G1 to the S phase in various cell lines
and carboxyl graphene (GXYG), the mitochondrial [112]. DNA damage can not only initiate cancer develop-
membrane was depolarized, and the amount of mito- ment but also possibly threaten the health of the next
chondria decreased in HepG2 cells [180]. Exposure to generation if the mutagenic potential of GO arises in repro-
GFNs resulted in significantly increased coupled and ductive cells, which impacts fertility and the health of
uncoupled mitochondrial oxygen consumption, dissipa- offspring [112, 190].
tion of the mitochondrial membrane potential, and
eventual triggering of apoptosis by activating the mito- Inflammatory response
chondrial pathway [181]. For instance, GO increased the GFNs can cause a significant inflammatory response in-
activity of mitochondrial electron transport complexes I/ cluding inflammatory cell infiltration, pulmonary edema
III and the supply of electrons to site I/II of the electron and granuloma formation at high doses via intratracheally
transport chain, accelerating the generation of ROS dur- instillation or intravenous administration [30, 49]. Plate-
ing mitochondrial respiration in MHS cells [99]. The lets are the important components in clot formation to
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 finally
attack pathogens and particulate matter during the in- Apoptosis

flammatory response, and GO could directly activate Apoptosis is defined as the self-destruction of a cell
platelet-rich thrombi formation to occlude lung vessels regulated by genes through complicated programmes
after intravenous injection [98, 191]. A strong inflam- [83, 195]. GO and rGO caused apoptosis and inflamma-
matory response was induced by subcutaneously injection in mice lungs after inhalation [99], and GFNs also
tion with GO for 21 days, along with the secretion of had pro-apoptotic effects in cells [111, 113, 124, 196].
key cytokines, including IL-6, IL-12, TNF-α, MCP-1, Additionally, graphene and GO physically damaged cell
and IFN-g [34, 192]. GFNs can trigger an inflammatory membranes [166], increased the permeabilization of
response and tissue injury by releasing cytokines and the outer mitochondrial membrane and changed the
chemokines that lead to the recruitment of circulating mitochondrial membrane potential; the increased ROS
monocytes and stimulating the secretion of Th1/Th2 triggered the MAPK and TGF-β signalling pathways
cytokines and chemokines [124, 193]. Additionally, and activated caspase-3 via mitochondrial-dependent
pristine graphene [193] and rGO [110] evoke an inflam- apoptotic cascades, prompting the execution of apop-
matory response by binding to toll-like receptors tosis [83, 99]. Similarly, rGO caused apoptosis at a
(TLRs) and activating the NF-κB signalling pathway in low dose and an early time point, triggered by the
cells. The NF-κB signalling cascade is triggered by death-receptor and canonical mitochondrial pathway
TLRs and pro-inflammatory cytokines such as IL-1 and [110]. Another study showed three different apoptosis
TNF-α. Upon activation, NF-κB shifts from the cytoplasm pathways by GFNs: GO led to ROS-dependent apop-
to the nucleus, facilitating the binding of degrading IκB tosis through direct interaction with protein receptors
and acting as a transcription factor to synthesize numer- and subsequent activation of the B-cell lymphoma-2
ous pro-inflammatory cytokines [194]. A schematic of the (Bcl-2) pathway; GO-COOH transmitted a passive apop-
signalling pathway of TLR4 and TLR9 activated by GFNs tosis signal to nuclear DNA by binding to protein recep-
is shown in Fig. 5. tors and activating a ROS-independent pathway; However,
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 lympho- Necrosis

cytes to trigger apoptosis [105, 197]. Necrosis is an alternate form of cell death induced by
inflammatory responses or cellular injury. The expos-
ure of cells to pristine graphene causes apoptosis and
Autophagy necrosis at high doses (50 mg/mL) [83]. Reportedly,
Autophagy is the process of self-degradation of cellular LDH leakage and the opening of the mitochondrial
components and recently recognized as non-apoptotic permeability transition pore, induced by elevated level of
cell death [198–200]. Autophagy activation requires cytoplasmic Ca2+, lead to apoptosis/necrosis [210]. GO
autophagosome formation containing Beclin 1, multiple treatment was revealed to induce macrophagic necro-
autophagy-related proteins (ATG), microtubule-associated sis by activating TLR4 signalling and subsequently
protein light chain 3 (LC3) and p62 [201]. Autophago- partly triggering autocrine TNF-α production [93].
some accumulation is associated with exposure to various GO combined with CDDP (GO/CDDP) triggered necrosis
nanoparticles [202–205], and autophagy can remove by decreasing RIP1 and increasing RIP3 proteins, accom-
extracellular organisms and destruct the organisms in the panied with the release of high mobility group B1
cytosol [206]. GO and GQDs was shown to induce autop- (HMGB1) into the cytosol from the nucleus and out of
hagosome accumulation and the conversion of LC3-I to CT26 cells [205, 211, 212].
LC3-II; inhibit the degradation of the autophagic substrate
p62 protein [207, 208]. Furthermore, GO can simultan- Epigenetic changes
eously trigger TLR4 and TLR9 responses in macrophages Epigenetics involve DNA methylation, genomic imprin-
[34, 192] and colon cancer cells CT26 [206]. The autoph- ting, maternal effects, gene silencing, and RNA editing
agy pathway is linked to phagocytosis by TLR signalling in [213–215]. DNA methylation, which is one of the best-
macrophages [206, 209]. studied epigenetic modifications, includes phosphorylation,
ubiquitination, and ATP-ribosylation and can lead to influence the toxicity and biocompatibility of GFNs,
chromatin remodelling [197, 216, 217]. A recently paper single-factor experimental designs and the exclusion of
reported that SL-GO/FL-GO exposure resulted in global other interfering factors should be considered. Details
DNA hypermethylation through upregulating DNMT3B of the fabrication process should also be provided because
and MBD1 genes; GNP treatment caused hypomethylation the formed oxidative debris could largely alter the surface
by decreasing the expression of DNMT3B and MBD1 structure of graphene and GO during functionalization
genes [216]. GO could activate the miRNA-360 regulation [151]. Importantly, a single, universal method needs to be
pathway to suppress the DNA damage-apoptosis signalling established in graphene technology, which will allow for
cascade by affecting the component of CEP-1 [218]. Taken better comparison of data from different studies or diffe-
together, these data suggest that GFNs could cause subtle rent laboratories.
changes in gene expression programming by modulating Second, different observational criteria, parameters
epigenetic changes. However, studies of GFNs-induced and selection of experimental methods might induce
epigenetic changes are few, and the epigenetic mechanism large inter-laboratory variations [220, 221]. For example,
caused by GFNs exposure is not fully understood. the MTT assay always fails to accurately predict graphene
To conclude, many studies have discussed representa- toxicity because the spontaneous reduction results in a
tive mechanisms of GFNs toxicity involving four signal- false positive signal. Therefore, appropriate alternative
ling pathways: TLRs, TGF-β, TNF-α and MAPKs. assessments should be utilized, such as the water-soluble
These four signalling pathways are correlative and tetrazolium salt reagent (WST-8), ROS assay, and trypan
cross-modulatory, making the inflammatory response, blue exclusion test [106, 222]. Additionally, the comet
autophagy, apoptosis and other mechanisms independent assay often shows higher levels of DNA damage than the
and yet connected to each other. Additionally, oxidative micronucleus assay because the former measures the
stress appears to play the most important role in activa- repairable injury and the latter measures the gene damage
ting these signalling pathways. It has been reported that that remains after cell division [159, 223]. Therefore,
there are intersections of apoptosis, autophagy and necro- caution is required in choosing the most appropriate assay
sis in the studies of other nanomaterials toxicity, they to evaluate the toxicity of graphene materials to avoid
inhibit or promote mutually in some conditions. However, false-positive results.
the signalling pathways of GFNs toxicity investigated in Third, the selection of cell lines is of vital importance
papers to date are only a small part of an intricate web, because cancer cell lines tend to be sensitive or resistant
and the network of signalling pathways needs to be depending upon their genetic background. The same
explored in detail in the future. graphene nanoparticles can cause different reactions
depending on their various cells origins. Suitable cell
lines with good stability must be used to avoid false posi-
Data gaps and future studies tive or negative results. Primary cells derived from
Currently, the literature is insufficient to draw conclu- humans or animals can better simulate the health condi-
sions about the potential hazards of GFNs. Two opposite tions of humans. A large amount of primary cells have
opinions have begun to emerge: some researchers been utilized to test the toxicity of other nanomaterials
suggested that graphene materials are biocompatible in a [224–228], but the culturing of primary cells is extremely
number of studies focused on biomedical applications rare in the experiments with GFNs to date [210, 229].
[119, 154, 162, 219], and other studies reported adverse Various cell experiments combined with primary cells
biological responses and cytotoxicity [32, 118, 135, 138, should be performed to comprehensively evaluate the
192]. These inconsistent results might have been caused physicochemical properties and toxicity of GFNs.
by several factors, including the different research Fourth, the administration route of GFNs plays a very
groups, various cellular or animal models, and varying important role in toxicity studies, and different delivery
physicochemical characterizations of GFNs. When GFNs methods will result in different toxicological reactions
are explored for in vivo applications in the human body [32, 53]. Thus, the route and period of exposure should
or some other biomedical applications, biocompatibility be carefully chosen according to the aim of the study.
must be considered, and more detailed and accurate Nasal drug delivery is often used to study the neurotox-
studies of GFNs toxicity are needed. icity of nanomaterials [230, 231], but this administration
First, detailed physicochemical characterization is im- method has rarely been applied in the testing of GFNs
perative in all future studies of GFNs toxicity. In the toxicity. Toxicological studies of GFNs in the nervous
experiments, feature descriptions of GFNs should in- system are rare, and the mechanism is unclear and needs
clude their size, morphology, surface area, charge, sur- to be studied further in the future. Recent toxicokinetic
face modifications, purity, and agglomeration [88, 141, studies involving the absorption, distribution, metabolism,
148, 162]. Because these physicochemical factors largely accumulation, and excretion of GFNs through different
exposure routes have yielded some results but are far from in the future. Many experiments have shown that GFNs
sufficient to clarify the internal complex mechanisms. For have toxic side effects in many biological applications,
instance, further studies are needed to understand the spe- but the in-depth study of toxicity mechanisms is urgently
cific molecular mechanisms of GFNs passing through the needed. In addition, contrasting results regarding the
physiological barriers and the amount of accumulation or toxicity of GFNs need to be addressed by effective expe-
the excretion period of GFNs in tissues. In addition, given rimental methods and systematic studies. This review
the increased exposure of humans to GFNs, the assess- provides an overview of the toxicity of GFNs by summa-
ment of systemic toxicity in the human body is indispens- rizing the toxicokinetics, toxicity mechanisms and influen-
able in future studies. cing factors and aimed to provide information to facilitate
Fifth, another important issue requiring attention is thorough research on the in vitro and in vivo haemo- and
the long-term fate of GFNs after entering the body or biocompatibility of GFNs in the future. This review will
being taken up by cells. Most recent studies have con- help address safety concerns before the clinical and
sisted of short-term toxicity assessments [89, 232], and therapeutic applications of GFNs, which will be im-
long-term toxic injury has not received much attention portant for further development of GFNs in biological
since the widespread application of GFNs in 2008. More- applications.
over, a functionalized graphene surface can improve its
Abbreviations
biocompatibility, but the long-term stability of the surface AMs: Alveolar macrophages; BBB: Blood-brain barrier; BEB: Blood-epididymis
coatings should be considered [233]. If the surface coat- barriers; BTB: Blood-testis barrier; CR: Complement receptor; FcgR: Fcg
ings eventually break down, their toxicity may be signifi- receptor; FLG: Few-layer graphene; GFNs: Graphene family nanomaterials;
GNS: Graphene nanosheets; GO: Graphene oxide; GO-COOH: Carboxylated
cantly different from the short-term exposure results. graphene oxide; GO-DEX: GO-dextran; GO-MB: GO-molecular beacon; GO-
Extended studies are needed to determine if longer NH2: Aminated GO; GO-PAA: Poly(acrylic acid)-functionalized GO; GO-
treatment times influence the nanotoxic potential of PAM: Poly(acrylamide)-functionalized GO; GO-PEG: PEGylated GO derivatives;
GO-PEI: GO-polyethylenimine; GQDs: Graphene quantum dots; GSH-
GFNs. PX: Glutathione peroxidase; GXVG: Carboxyl graphene; LDH: Lactate and
Sixth, more specific signalling pathways in the mech- dehydrogenase; MALDI: Matrix-assisted laser desorption/ionization;
anism of GFNs toxicity need to be discovered and eluci- MAPKs: Mitogen-activated protein kinase; MDA: Malondialdehyde;
MØ: Macrophage; MR: Mannose receptor; MSI: Mass spectrometry imaging;
dated. Currently, several typical toxicity mechanisms of PC12 cells: Rat pheochromocytoma cells; PCGO: Protein-coated graphene
GFNs have been illustrated and widely accepted, such as oxide nanoparticles; PrGO: PEGylated reduced graphene oxide;
oxidative stress, apoptosis, and autophagy. However, RES: Reticuloendothelial system; rGO: Reduced graphene oxide; ROS: Reactive
oxygen species; SOD: Superoxide dismutase; TLRs: Toll-like receptor
these mechanisms have only been described in general
terms, and the specific signalling pathways within these Acknowledgements
mechanisms need to be investigated in detail. The sig- Not applicable.
nalling pathways involved in the toxicity of other nano- Funding
materials may also be relevant to the study of GFNs. This review was supported by the National Natural Science Foundation of
Therefore, more signalling pathways should be detected China (81550011, 51172283, 81400557), Natural Science Foundation of
Guangdong Province (2015A030313299) and Guangdong Provincial Medical
in future research. For instance, nano-epigenetics has Research Foundation (A2016360).
been considered in numerous studies of nanomaterials,
which is also helpful in assessing the limited toxicity and Availability of data and materials
Databases/repositories and materials is not applicable in this review.
side effects of GFNs. Recent studies have shown that
GFNs could cause epigenetic and genomic changes that Authors’ contributions
might stimulate physical toxicity and carcinogenicity All authors contributed to the design and concept of this article. LO drafted
the manuscript. BS and JL critically revised the manuscript. All authors read
[234]. GFNs have high surface areas, smooth continuous and approved the final manuscript.
surfaces and bio-persistence, similar to the properties of
tumorigenic solid-state implants. It is unknown whether Competing interest
The authors declare that they have no competing interests.
GFNs have the potential to induce foreign body sarco-
mas, and definitive studies of tumour potentialities or Consent for publication
risks of graphene should therefore be conducted as Not applicable.
soon as possible. Ethics approval and consent to participate
Not applicable.
Conclusions
Author details
In the past few years, GFNs have been widely utilized in 1
Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
a wide range of technological and biomedical fields. 2
The First Affiliated Hospital of Jinan University, Guangzhou, China. 3The
Currently, most experiments have focused on the tox- General Hospital of People’s Liberation Army, Beijing, China.
icity of GFNs in the lungs and livers. Therefore, studies Received: 13 June 2016 Accepted: 13 October 2016
of brain injury or neurotoxicity deserve more attention
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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 at
sheets, 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 note
as 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 administered
subcutaneously, 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.
 Dosage: higher concentra ons of GO tend to cause more damage: in one study, a
high 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 have
depends 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 can
reportedly 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 such
coronas and these may regulate the adhesion of GO to certain cells and even
mi 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) – can
penetrate the body’s exis ng protec ve barriers and in"ltrate the brain, placenta and
poten 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 inamma on and injury to vital organs. In the lungs, it was found to
cause inamma on, pulmonary oedema and the forma on of granulomas. There has been
very li@le research into the toxicity of GFNs to other organs such as the liver, spleen and
kidneys.
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 these
materials 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 mice
found that all pregnant females injected with rGO had abor ons, regardless of dose. Most
of 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 to
disease. It also causes apoptosis – a form of programmed cell death – and enters into the
lysosomes, mitochondria, nucleus and endoplasm of cells. GO deriva ves drama cally
decrease the expression of di%eren al genes that are responsible for the structure and
func 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 the
genera 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 inammatory responses and ssue injury. GO has
been found to trigger apoptosis and autophagy – both forms of programmed cell death – as
well 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 the
body. 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 is
unknown as to whether GFNs could induce foreign body sarcomas, so more studies on this
are urgently needed.

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Case Briefing Document

● Gross Criminal Manslaughter

Halt the roll-out of experimental treatments known as “COVID-19

● National Health Service (NHS)

● Medicines & Healthcare products Regulatory Agency (MHRA)

● Joint Committee on Vaccination and Immunisation (JCVI)

● Her Majesty’s Government

Post Vaccination Death & Injury Reports

A full peer reviewed scientific paper is expected to be provided in due course.

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

The following documents are attached in support of this submission:

Laboratory Report Summary

RAMAN Spectroscopy discovered the following particles -

A summary of the findings detailed in the attached toxicology report is as follows:

● Graphene oxide (GO):

a. can damage internal organs

b. damages the reproduction and development system

c. destroys blood health

d. damages and destroys cells

e. can trigger cancer and accelerate ageing

f. damages mitochondria and DNA

g. triggers an inflammatory response and three different kinds of cell death

h. causes changes in gene function

Corporate Manslaughter and Corporate Homicide Act 2007

UK Public General Acts <https://www.legislation.gov.uk/ukpga>2007 c. 19

Gross Criminal Manslaughter

Police and Criminal Evidence Act 1984

UK Public General Acts <https://www.legislation.gov.uk/ukpga>1984 c. 60

Home Office Guidelines on the Reporting and Recording of Crime

The HOCR also promote a victim-oriented approach (PDF document)

1. Incident reporting and recording

The HOCR require:

Qualitative Evaluation of Inclusions

UNIT was commissioned by EbMCsquared CiC under project UNITC-112980 to investigate

undeclared ingredients that may cause bodily harm.

THIS REPORT AND ANY OF ITS ASSOCIATED

Any reproduction and distribution queries should be directed

UNIT takes no responsibility for interpretations drawn by readers of the report.

The manufacturing label had the following information on the vial:

1.2.2. Moderna 02 Labelling

The manufacturing label had the following information on the vial:

1.2.3. AstraZeneca Labelling

The manufacturing label had the following information on the vial:

Covid-19 Vaccine 4 ml, Astrazeneca Injection. Covid-19 Vaccine:

1.2.4. Pfizer Labelling

Figure 1.1. Pfizer vaccine vial

2.2. Process of Sampling and Slide Preparation

Stage 1 - Opening of the vial

Stage 3- Partitioning of sample for clean slide preparation

The identified inclusions were-

3.1.1. Graphene Composites in the form of Nano Ribbons

3.1.3. Graphene Composite Form 2

Figure 3.3. Carbon composite Form 2

Figure 3.4. Microcrystalline Calcite with graphene inclusions.

3.1.5. Graphene Nano Forms

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

3.1.6. Crystalised forms of the solution

Figure 3.7. Filament forms as observed under wet microscopy.

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