• Robert O Young DSc, PhD, Naturopathic Practitioner

Nano and Micro Blood Clots Seen in the Capillary Blood of the VAXXinated & UNVAXXinated! UPDATED!!!

Updated: Aug 25

Dear Family and Friends,


Thank you for your love and support for so many years. I am truly thankful, grateful and humbled by your generosity and your prayers.


The following are just a few examples of the thousands of micrographs I have observed and documented in the last two months viewed under Brightfield, pHase Contrast and Darkfield Microscopy, Scanning Electron Microscopy[SEM} and Transmission Electron Microscopy[TEM].


What Does Healthy Blood Look Like?


1. Healthy capillary human blood from a UNVAXXinated male - appears even in color, even in shape and even in size as observed under Brightfield, pHase Contrast and Darkfield microscopy.


This is What Healthy Blood Looks Like! What Does YOUR Blood Look Like? Keep Reading!


What Does UnHealthy Blood Look Like Full of Graphene, Iron, Aluminum, Parasites, Acid Crystals and Blood Clots?


2. The micrograph below shows a symplast or a micro clot of enlarged red blood cells or a blood clot observed using a compound pHase contrast light microscope at 1500x magnification. These micro clots are now commonly seen in the live unstained blood of humans and animals due to acidic/toxic poison now found in food, water, air, vaccines, nutritional supplements and legend drugs. The hemoglobin, the molecule of the red blood cells adsorbs and absorbs metabolic, dietary, respiratory and environmental acidic poisonous waste in order to protect the delicate pH balance of the vascular fluids at 7.365 pH and -20.5 mV and the interstitial fluids that surround all body cells at a 8.4 pH and -80 mV. The red blood cells NOT the white blood cells are the primary protectors of the body cells that make up all organs, glands and tissues. White blood cell are secondary to red blood cells in providing immunity which is a major oversight in current medical microbiology.



The symptoms of micro clots caused by acidic chemical and radiation poisoning are as follows:


Low energy

Cold hands

Cold feet

Itchy skin

Lightheadedness

Dizziness

Muddle thinking

Forgetfulness

Foggy thinking

Dry cough

Fever

Hypoxia

Hypercapnia

DIC or Pathological Blood Coagulation

Decompensated acidosis of the vascular and interstitial fluids

Death



3. A large symplast of magnetic nano graphene oxide observed in the live unchanged and unstained capillary blood using pHase Contrast Microscopy of a VAXXinated male.


Targeted Red Blood Cell Deficient in Hemoglobin with Large Symplasts of Metallic Graphene Oxide Surrounded by Blood Clots!


4. Ferric oxide with lactic and sulphuric acid crystals observed in the live capillary blood from a VAXXinated male using Brightfield, pHase contrast microscopy and confirmed with UV absorbance and Fluorescence Spectroscopy, Scanning Electron Microscopy, Transmission Electron Microscopy, Energy Dispersive Spectroscopy, X-ray Diffractometer and Nuclear Magnetic Resonance instruments.



5. A large symplast of Nano Graphene oxide particulates observed in the live unchanged and unstained capillary blood of a VAXXinated female with metastatic lung and brain cancer patient after chemotherapy.



6. A large symplast of Nano Graphene Oxide observed in the live unchanged capillary blood under pHase contrast microscopy of a UNVAXXinated female. I call these symplasts of graphene, " Graphene Atomic Bombs"




IMMENSE HARM FROM CRYSTALLIZING BLOOD CELLS & GRAPHENE POISONING POST-VACCINE


https://www.bitchute.com/video/PRBfJ6U9EZvM/

https://www.bitchute.com/video/rdQhuY455VmK/


7. Graphene oxide in the center of what appears as a vulgaris bulgarus parasite at 1500x magnification observed in the live capillary blood under phase contrast microscopy in a VAXXinated female.



8. Pathological blood coagulation combined with a host of Wuchereria bancrofti, Microfilariae and Trypanosoma Cruzi blood parasites found in living capillary blood observed under pHase Contrast Microscopy. These parasites mainly live in the lymphatic vessels, but occasionally they are found in the living capillary blood and can thrive for up to ten years in animal and human blood. These parasites destroy the health of your interstitial and vadcular fluids, causing cellular and tissue acidification, inflammation and degeneration of the cell membranes and genetic matter leading to chronic disease! You pick up these parasites when eating sushi, chicken, beef, shrimp, pork and NOW through VAXXination! It is notable that shrimp, crab and lobster have a countless number of parasites that rot the body from the inside out! Flee from these highly acidic foods and inoculations and NEVER touch them with your hands!!







9. Graphene Oxide nanowires, threads, dots and bubbles observed in the live unchanged and unstained capillary blood with Brightfield and pHase Contrast microscopy in a UNVAXXinated female.



More Micro and Nanographs of Graphene Nanowires, Threads, Ribbons, Dots and Bubbles of the Vaccinated Observed in LIVE CAPPILARY BLOOD BLOOD CLOTS!


Graphene Based Nanowires are superconductor batteries used for tissue scaffolding inside the human body and for receiving and transmitting data across the Internet of Things.











GRAPHENE NANOWIRES, RIBBONS AND NANO NETWORKS IN THE BLOOD COMBINES WITH EMF 5G RADIATION CAUSES OF INJURY AND DEATH!

https://www.bitchute.com/video/0wrX2Mwfrx4Z/


10. Different morphology of nano graphene oxide as wires, ribbons, tubes, dots and sheets as observed in the live unchanged and unstained capillary blood of a VAXXinated female.



11. Nano Graphene Oxide Nanowires and Threads as observed in the live unchanged and unstained capillary blood under pHase contrast microscopy from a VAXXinated female



12. Nano Graphene Oxide sheets, tubes, nanowires, threads and dots observed in the live unchanged capillary blood under pHase contrast microscopy from a VAXXinated female. Please note the red blood cell clots throughout the smear.



13. A large Nano Graphene Oxide Tubular blood clot observed in the live unchanged and unstained capillary blood under pHase contrast microscopy from a VAXXinated male.



14. A large fibrous graphene oxide micro blood clot observed in the live unchanged and unstained capillary blood of a VAXXinated female under pHase contrast microscopy.



15. A pathological capillary blood clot containing graphene oxide threads and tubes observed in the live unchanged and unstained blood of a VAXXinated female under brightfield microscopy.


Micrographs of Healthy and UnHealthy Blood Under pHase Contrast and Brightfield Microscopy


Graphene in Nano and Micro Blood Clots in the VAXXinated and UNVAXXinated Capillary Blood!

https://www.bitchute.com/video/jnpG3weGmOui/


16. Self-Assembling Graphene Oxide Based Biosensors observed in the unchanged and unstained capillary blood under pHase contrast microscopy of a VAXXinated female.


NEW COVID VACCINE & BLOOD FINDINGS & SELF ASSEMBLY WITH PULSED RADIO & MICROWAVES[5G]!

https://www.bitchute.com/video/qT6iiHrlOGHZ/


17. Graphene, iron and silicon have been found in the Pfizer, Moderna and Johnson and Johnson VAXXines for children 6 months to 5 years old!


https://rumble.com/v197mj7-eua-amendment-request-for-pfizer-biontech-covid-19-vaccine-for-children.html


Figure 1 below reveals the cytotoxic, genotoxic and magnetic toxic spectrum of the Pfizer “vaccine" nano particulates of reduced graphene oxide or graphene hydroxide, magnesium, aluminum, silicon, chloride and calcium identified under an ESEM microscope coupled with an EDS x-ray microprobe. (X axis =KeV, Y axis = Counts)

Figure 2 below reveals the cytotoxic, genotoxic and magnetic toxic spectrum of the Moderna "vaccine" nano particulates of reduced graphene oxide or graphene hydroxide, silicon, copper, titanium, selenium, potassium and cadmium, and lead identified under an ESEM microscope coupled with an EDS x-ray microprobe. (X axis =KeV, Y axis = Counts)

Figure 2 Reveals Nano Particulates Which Are Cytotoxic, Genotoxic and Magnetic Toxic!

These same toxic nondisclosed ingredients of graphene were observed in the VAXXines and the capillary blood of the VAXXinated of young adults and adults. These findings were published in February of 2021.


https://www.bitchute.com/video/Z2sAH0Woz38r/


CLOTS, NANOWIRES, PARASITES, SYMPLASTS OF GRAPHENE IN VAXXINATED and UNVAXXINATED BLOOD of INFANTS, CHILDREN and ADULTS!


https://www.bitchute.com/video/7BM8rMBiQ6f9/


PLUS: A Life of Public Service in Education to Help Heal Humanity!


Dr. Robert - The Beatles - https://youtu.be/Tb9L3iAUhc0


Detailed reporting (second half of video) of new findings of blood clots of different sizes, nano and micro, along with nanowires, apparent parasites, symplasts and jagged shards and coils of graphene in the live blood of vaccinated children and adults taken from capillaries (which means the nano graphene in the COVID vaccines has spread everywhere through the body) revealing that all who have taken the vaccine are in great danger of harm of different kinds including paralysis, strokes, heart attacks, sudden death, unless they wake up and start detoxing at speed.


Graphene Wires and Sheets in the Blood of OUR Children!

If you would like to view more blood and vaccine micrographs of the live unchanged and unstained capillary blood and my VAXXine Research please click on the following links below:


1. Pathological Blood Coagulation - https://www.drrobertyoung.com/blog/search/blood


2. SEM and TEM Reveals Nondisclosed Ingredients in ALL VAXXinies -https://www.drrobertyoung.com/post/transmission-electron-microscopy-reveals-graphene-oxide-in-cov-19-vaccines


https://www.bitchute.com/video/Z2sAH0Woz38r/

Scanning and Tranmission Electron Microscopy in the Identificaiton of the Non-disclosed In
.
Download • 1.00MB

3. Rothschild's Planned CONvid Plandemic - https://www.drrobertyoung.com/post/proof-rothchild-s-planned-the-plandemic


4. Second Thoughts About Viruses, Vaccines and the Viral Theory - https://www.drrobertyoung.com/post/second-thoughts-about-viruses-vaccines-the-viral-infectious-hypothesis


5. Young, RO (2016) Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Test (MOST). Int J Vaccines Vaccin 2(6): 00048. DOI: 10.15406/ijvv.2016.02.00048


With Great Love and Respect,


Robert O Young MSc, DSc, PhD, Naturopathic Practitioner www.drrobertyoung.com

https://www.givesendgo.com/research


You can support the research of Dr. Robert O. Young with your prayers and donations at: https://www.givesendgo.com/research


Maybe The Most Important Video Interview That YOU WILL Ever Watch! It May Change YOUR Life and Save YOUR Life!


5G Radiation Poisoning Combined with Graphene Poisoning of the Blood = Injury & Death!

https://rumble.com/v18ku7h-5g-radiation-poisoning-combined-with-graphene-poisoning-of-the-blood.html


Understand Why Blood Clots Form Inside the Blood Vessels!


Read Dr. Robert O. Young's Peered Review Scientific Research Article Published in the International Journal of Vaccines and Vaccination on Pathological Blood Coagulation! (2016)

Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Testing, Young RO (2016) Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Test (MOST). Int J Vaccines Vaccin 2(6): 00048. DOI: 10.15406/ijvv.2016.02.00048

IJVV-02-00048
.pdf
Download PDF • 2.51MB


interstitial-fluid-lung-disease-IFLD-of-the-interstitium-organ-the-cause-and-self-care-to-
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Download • 1.16MB


What-Causes-Oxygen-Deprivation-of-the-Blood-DIC-and-Then-Lungs-SARS-CoV2and12 (1)
.pdf
Download PDF • 1.32MB


cystic-fibrosis-and-pulmonary-adenocarcinoma-lung
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Download PDF • 336KB

https://www.bitchute.com/video/TWbG4qZKfO5N/


Change and Save a LIFE and Share this article with everyone YOU LOVE and CARE ABOUT!

https://www.drrobertyoung.com/post/nano-and-micro-clots-seen-in-the-blood-of-the-vaxxinated-nonvaxxinated


References


[1] Young RO (2016) Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Test (MOST). Int J Vaccines Vaccin 2(6): 00048. DOI: 10.15406/ijvv.2016.02.00048


[2] Young RO (2016) Second Thoughts about Viruses, Vaccines, and the HIV/AIDS Hypothesis - Part 1. Int J Vaccines Vaccin 2(3): 00032. DOI: 10.15406/ijvv.2016.02.00032


[3] Young RO (2016) Second Thoughts Concerning Viruses, Vaccines and the HIV/AIDS Hypothesis - Part 2. Int J Vaccines Vaccin 2(3): 00034. DOI: 10.15406/ijvv.2016.02.00034


[4] Young RO (2016) Second Thoughts Concerning Viruses, Vaccines and the HIV/AIDS Hypothesis - Part 3 HIV/AIDS

and the Monomorphic Disease Model. Int J Vaccines Vaccin 2(3): 00035. DOI: 10.15406/ijvv.2016.02.00035


[5] Nanowires patent:

https://patents.google.com/patent/US8115190B2/en


[6] Nanowire arrays for neurotechnology and other applications ~ patent

https://patents.google.com/patent/WO2016112315A3/e


[7] Internalization of ferromagnetic nanowires by different living cells (2010)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592113


[8] Hydrophobic copper nanowires for enhancing condensation heat transfer

https://www.sciencedirect.com/science/article/abs/pii/S2211285517300186


[9] Rotational Maneuver of Ferromagnetic Nanowires for Cell Manipulation

https://www.academia.edu/es/43298108/Rotational_Maneuver_of_Ferromagnetic_Nanowires_for_Cell_Manipulation


[10] Internalization of ferromagnetic nanowires by different living cells

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592113


[11] Ultrathin gold nanowires to enhance radiation therapy

https://pubmed.ncbi.nlm.nih.gov/32917209/


Scanning Electron Microscopy (SEM) of Nano Reduced Graphene Hydroxide and Nano Gold Observed as Wires, Threads, Ribbons and Tubes Removed from the Arteries and Veins of a Deceased Cancer Patient Who Received Radiation Therapy

The Beatles Song - Dr. Robert - Drink Your Cup of Greens

https://youtu.be/Tb9L3iAUhc0


​https://organichealthylife.com/dr-robert-young-shares-his-thougts-on-blood-health-science-and-the-world-situation/

https://www.drrobertyoung.com/post/nano-and-micro-clots-seen-in-the-blood-of-the-vaxxinated-nonvaxxinated

https://www.drrobertyoung.com/post/nano-and-micro-clots-seen-in-the-blood-of-the-vaxxinated-nonvaxxinated


NEED MORE PROOF!


Twelve YEARS of Peer-Reviewed Research Articles on Graphene, Graphene Oxide and Reduced Graphene Hydroxide Describing USE in Humans and Animals and the Negative Effects of Graphene-Related Materials on Humans and Animals

Article 1

Graphene oxide disrupted mitochondrial homeostasis through inducing intracellular redox deviation and autophagy-lysosomal network dysfunction in SH-SY5Y cells – ScienceDirect

August 2021


Graphene oxide (GO) nanomaterials have significant advantages for drug delivery and electrode materials in neural science, however, their exposure risks to the central nervous system (CNS) and toxicity concerns are also increased. The current studies of GO-induced neurotoxicity remain still ambiguous, let alone the mechanism of how complicated GO chemistry affects its biological behavior with neural cells. In this study, we characterized the commercially available GO in detail and investigated its biological adverse effects using cultured SH-SY5Y cells. We found that ultrasonic processing in medium changed the oxidation status and surface reactivity on the planar surface of GO due to its hydration activity, causing lipid peroxidation and cell membrane damage. Subsequently, ROS-disrupted mitochondrial homeostasis, resulting from the activation of NOX2 signaling, was observed following GO internalization. The autophagy-lysosomal network was initiated as a defensive reaction to obliterate oxidative damaged mitochondria and foreign nanomaterials, which was ineffective due to reduced lysosomal degradation capacity. These sequential cellular responses exacerbated mitochondrial stress, leading to apoptotic cell death. These data highlight the importance of the structure-related activity of GO on its biological properties and provide an in-depth understanding of how GO-derived cellular redox signaling induces mitochondrion-related cascades that modulate cell functionality and survival.


Article 2

Biodistribution and pulmonary toxicity of intratracheally instilled graphene oxide in mice | NPG Asia Materials (nature.com) April 2013


Graphene and its derivatives (for example, nanoscale graphene oxide (NGO)) have emerged as extremely attractive nanomaterials for a wide range of applications, including diagnostics and therapeutics. In this work, we present a systematic study on the in vivo distribution and pulmonary toxicity of NGO for up to 3 months after exposure. Radioisotope tracing and morphological observation demonstrated that intratracheally instilled NGO was mainly retained in the lung. NGO could result in acute lung injury (ALI) and chronic pulmonary fibrosis. Such NGO-induced ALI was related to oxidative stress and could effectively be relieved with dexamethasone treatment. In addition, we found that the biodistribution of 125I-NGO varied greatly from that of 125I ions, hence it is possible that nanoparticulates could deliver radioactive isotopes deep into the lung, which might settle in numerous ‘hot spots’ that could result in mutations and cancers, raising environmental concerns about the large-scale production of graphene oxide.


Article 3

A review of toxicity studies on graphene-based nanomaterials in laboratory animals – ScienceDirect

April 2017


We summarized the findings of toxicity studies on graphene-based nanomaterials (GNMs) in laboratory mammals. The inhalation of graphene (GP) and graphene oxide (GO) induced only minimal pulmonary toxicity. Bolus airway exposure to GP and GO caused acute and subacute pulmonary inflammation. Large-sized GO (L-GO) was more toxic than small-sized GO (S-GO). Intratracheally administered GP passed through the air-blood barrier into the blood and intravenous GO distributed mainly in the lungs, liver, and spleen. S-GO and L-GO mainly accumulated in the liver and lungs, respectively. Limited information showed the potential behavioral, reproductive, and developmental toxicity and genotoxicity of GNMs. There are indications that oxidative stress and inflammation may be involved in the toxicity of GNMs. The surface reactivity, size, and dispersion status of GNMs play an important role in the induction of toxicity and biodistribution of GNMs. Although this review paper provides initial information on the potential toxicity of GNMs, data are still very limited, especially when taking into account the many different types of GNMs and their potential modifications. To fill the data gap, further studies should be performed using laboratory mammals exposed using the route and dose anticipated for human exposure scenarios.

Article 4

Dose ranging, expanded acute toxicity and safety pharmacology studies for intravenously administered functionalized graphene nanoparticle formulations. – Abstract – Europe PMC

May 2014


Graphene nanoparticle dispersions show immense potential as multifunctional agents for in vivo biomedical applications. Herein, we follow regulatory guidelines for pharmaceuticals that recommend safety pharmacology assessment at least 10-100 times higher than the projected therapeutic dose, and present comprehensive single dose response, expanded acute toxicology, toxicokinetics, and respiratory/cardiovascular safety pharmacology results for intravenously administered dextran-coated graphene oxide nanoplatelet (GNP-Dex) formulations to rats at doses between 1 and 500 mg/kg. Our results indicate that the maximum tolerable dose (MTD) of GNP-Dex is between 50 mg/kg ≤ MTD < 125 mg/kg, blood half-life < 30 min, and majority of nanoparticles excreted within 24 h through feces. Histopathology changes were noted at ≥250 mg/kg in the heart, liver, lung, spleen, and kidney; we found no changes in the brain and no GNP-Dex related effects in the cardiovascular parameters or hematological factors (blood, lipid, and metabolic panels) at doses < 125 mg/kg. The results open avenues for pivotal preclinical single and repeat dose safety studies following good laboratory practices (GLP) as required by regulatory agencies for investigational new drug (IND) application


Article 5

Synthesis and Toxicity of Graphene Oxide Nanoparticles: A Literature Review of In Vitro and In Vivo Studies (hindawi.com)

July 2021


Nanomaterials have been widely used in many fields in the last decades, including electronics, biomedicine, cosmetics, food processing, buildings, and aeronautics. The application of these nanomaterials in the medical field could improve diagnosis, treatment, and prevention techniques. Graphene oxide (GO), an oxidized derivative of graphene, is currently used in biotechnology and medicine for cancer treatment, drug delivery, and cellular imaging. Also, GO is characterized by various physicochemical properties, including nanoscale size, high surface area, and electrical charge. However, the toxic effect of GO on living cells and organs is a limiting factor that limits its use in the medical field. Recently, numerous studies have evaluated the biocompatibility and toxicity of GO in vivo and in vitro. In general, the severity of this nanomaterial’s toxic effects varies according to the administration route, the dose to be administered, the method of GO synthesis, and its physicochemical properties. This review brings together studies on the method of synthesis and structure of GO, characterization techniques, and physicochemical properties. Also, we rely on the toxicity of GO in cellular models and biological systems. Moreover, we mention the general mechanism of its toxicity.


Article 6

The Puzzling Potential of Carbon Nanomaterials: General Properties, Application, and Toxicity (nih.gov)

July 2020


Being a member of the nanofamily, carbon nanomaterials exhibit specific properties that mostly arise from their small size. They have proved to be very promising for application in the technical and biomedical field. A wide spectrum of use implies the inevitable presence of carbon nanomaterials in the environment, thus potentially endangering their whole nature. Although scientists worldwide have conducted research investigating the impact of these materials, it is evident that there are still significant gaps concerning the knowledge of their mechanisms, as well as the prolonged and chronic exposure and effects. This manuscript summarizes the most prominent representatives of carbon nanomaterial groups, giving a brief review of their general physico-chemical properties, the most common use, and toxicity profiles. Toxicity was presented through genotoxicity and the activation of the cell signaling pathways, both including in vitro and in vivo models, mechanisms, and the consequential outcomes. Moreover, the acute toxicity of fullerenol, as one of the most commonly investigated members, was briefly presented in the final part of this review. Thinking small can greatly help us improve our lives, but also obliges us to deeply and comprehensively investigate all the possible consequences that could arise from our pure-hearted scientific ambitions and work.


Article 7

Toxicity Evaluation of Graphene Oxide in Kidneys of Sprague-Dawley Rats – PubMed (nih.gov)

March 2016


Recently, graphene and graphene-related materials have attracted a great deal of attention due their unique physical, chemical, and biocompatibility properties and to their applications in biotechnology and medicine. However, the reports on the potential toxicity of graphene oxide (GO) in biological systems are very few. The present study investigated the response of kidneys in male Sprague-Dawley rats following exposure to 0, 10, 20 and 40 mg/Kg GO for five days. The results showed that administration of GOs significantly increased the activities of superoxide dismutase, catalase and glutathione peroxidase in a dose-dependent manner in the kidneys compared with control group. Serum creatinine and blood urea nitrogen levels were also significantly increased in rats intoxicated with GO compared with the control group. There was a significant elevation in the levels of hydrogen peroxide and lipid hydro peroxide in GOs-treated rats compared to control animals. Histopathological evaluation showed significant morphological alterations of kidneys in GO-treated rats compared to controls. Taken together, the results of this study demonstrate that GO is nephrotoxic and its toxicity may be mediated through oxidative stress. In the present work, however, we only provided preliminary information on toxicity of GO in rats; further experimental verification and mechanistic elucidation are required before GO widely used for biomedical applications.


Article 8

Interactions of graphene with mammalian cells: Molecular mechanisms and biomedical insights – ScienceDirect

October 2016


Carbon-based functional nanomaterials have attracted immense scientific interest from many disciplines and, due to their extraordinary properties, have offered tremendous potential in a diverse range of applications. Among the different carbon nanomaterials, graphene is one of the newest and is considered the most important. Graphene, a monolayer material composed of sp2-hybridized carbon atoms hexagonally arranged in a two-dimensional structure, can be easily functionalized by chemical modification. Functionalized graphene and its derivatives have been used in diverse nano-biotechnological applications, such as in environmental engineering, biomedicine, and biotechnology. However, the prospective use of graphene-related materials in a biological context requires a detailed comprehension of these materials, which is essential for expanding their biomedical applications in the future. In recent years, the number of biological studies involving graphene-related nanomaterials has rapidly increased. These studies have documented the effects of the biological interactions between graphene-related materials and different organizational levels of living systems, ranging from biomolecules to animals. In the present review, we will summarize the recent progress in understanding mainly the interactions between graphene and cells. The impact of graphene on intracellular components, and especially the uptake and transport of graphene by cells, will be discussed in detail.


Article 9

Cellular and molecular mechanistic insight into the DNA-damaging potential of few-layer graphene in human primary endothelial cells – ScienceDirect

July 2016


Despite graphene being proposed for a multitude of biomedical applications, there is a dearth in the fundamental cellular and molecular level understanding of how few-layer graphene (FLG) interacts with human primary cells. Herein, using human primary umbilical vein endothelial cells as model of vascular transport, we investigated the basic mechanism underlying the biological behavior of graphene. Mechanistic toxicity studies using a battery of cell based assays revealed an organized oxidative stress paradigm involving cytosolic reactive oxygen stress, mitochondrial superoxide generation, lipid peroxidation, glutathione oxidation, mitochondrial membrane depolarization, enhanced calcium efflux, all leading to cell death by apoptosis/necrosis. We further investigated the effect of graphene interactions using cDNA microarray analysis and identified potential adverse effects by down regulating key genes involved in DNA damage response and repair mechanisms. Single cell gel electrophoresis assay/Comet assay confirmed the DNA damaging potential of graphene towards human primary cells.


Article 10

Graphene oxide induces apoptotic cell death in endothelial cells by activating autophagy via calcium-dependent phosphorylation of c-Jun N-terminal kinases – ScienceDirect

December 2016


Despite the rapid expansion of the biomedical applications of graphene oxide (GO), safety issues related to GO, particularly with regard to its effects on vascular endothelial cells (ECs), have been poorly evaluated. To explore possible GO-mediated vasculature cytotoxicity and determine lateral GO size relevance, we constructed four types of GO: micrometer-sized GO (MGO; 1089.9 ± 135.3 nm), submicrometer-sized GO (SGO; 390.2 ± 51.4 nm), nanometer-sized GO (NGO; 65.5 ± 16.3 nm), and graphene quantum dots (GQDs). All types but GQD showed a significant decrease in cellular viability in a dose-dependent manner. Notably, SGO or NGO, but not MGO, potently induced apoptosis while causing no detectable necrosis. Subsequently, SGO or NGO markedly induced autophagy through a process dependent on the c-Jun N-terminal kinase (JNK)-mediated phosphorylation of B-cell lymphoma 2 (Bcl-2), leading to the dissociation of Beclin-1 from the Beclin-1–Bcl-2 complex. Autophagy suppression attenuated the SGO- or NGO-induced apoptotic cell death of ECs, suggesting that SGO- or NGO-induced cytotoxicity is associated with autophagy. Moreover, SGO or NGO significantly induced increased intracellular calcium ion (Ca2+) levels. Intracellular Ca2+chelation with BAPTA-AM significantly attenuated microtubule-associated protein 1A/1B-light chain 3-II accumulation and JNK phosphorylation, resulting in reduced autophagy. Furthermore, we found that SGO or NGO induced Ca2+ release from the endoplasmic reticulum through the PLC β3/IP3/IP3R signaling axis. These results elucidate the mechanism underlying the size-dependent cytotoxicity of GOs in the vasculature and may facilitate the development of a safer biomedical application of GOs.


Article 11

Understanding the hemotoxicity of graphene nanomaterials through their interactions with blood proteins and cells | SpringerLink

January 2018


The successful applications of graphene nanomaterials in nanobiotechnology and medicine as well as their effective translation into real clinical utility hinge significantly on a thorough understanding of their nanotoxicological profile. Of all aspects of biocompatibility, the hemocompatibility of graphene nanomaterials with different blood constituents in the circulatory system is one of the most important elements that needs to be well elucidated. Once administered into biological systems, graphene nanomaterials may inevitably come into contact with the surrounding plasma proteins and blood cells. Crucially, the interactions between these hematological entities and graphene nanomaterials will influence the overall efficacy of their biomedical applications. As such, a comprehensive understanding of the hemotoxicity of graphene nanomaterials is critically important. This review presents an up-to-date elucidation of the hemotoxicity of graphene nanomaterials through their interactions with blood proteins and cells, as well as offers some perspectives on the current challenges, opportunities, and future development of this important field.


Article 12

A systems toxicology approach to the surface functionality control of graphene–cell interactions – ScienceDirect

January 2014


The raised considerable concerns about the possible environmental health and safety impacts of graphene nanomaterials and their derivatives originated from their potential widespread applications. We performed a comprehensive study about biological interaction of grapheme nanomaterials, specifically in regard to its differential surface functionalization (oxidation status), by using OMICS in graphene oxide (GO) and reduced graphene oxide (rGO) treated HepG2 cells. Differential surface chemistry (particularly, oxidation – O/C ratio) modulates hydrophobicity/philicity of GO/rGO which in turn governs their biological interaction potentiality. Similar toxic responses (cytotoxicity, DNA damage, oxidative stress) with differential dose dependency were observed for both GO and rGO but they exhibited distinct mechanism, such as, hydrophilic GO showed cellular uptake, NADPH oxidase dependent ROS formation, high deregulation of antioxidant/DNA repair/apoptosis related genes, conversely, hydrophobic rGO was found to mostly adsorbed at cell surface without internalization, ROS generation by physical interaction, poor gene regulation etc. Global gene expression and pathway analysis displayed that TGFβ1 mediated signaling played the central role in GO induced biological/toxicological effect whereas rGO might elicited host-pathogen (viral) interaction and innate immune response through TLR4–NFkB pathway. In brief, the distinct biological and molecular mechanisms of GO/rGO were attributed to their differential surface oxidation status.


Article 13

A DIFFERENTIAL EFFECT OF GRAPHENE OXIDE ON THE PRODUCTION OF PROINFLAMMATORY CYTOKINES BY MURINE MICROGLIA | Taiwan Veterinary Journal (worldscientific.com)

2015


Graphene oxide (GO) is a promising nanomaterial for application in a variety of biomedical fields, including neuro-oncology, neuroimaging, neuroregeneration and drug delivery. Microglia are the central macrophage-like cells critically involved in neuroimmunity. However, the interaction between GO and microglia remained mostly unknown. The present study investigated the influence of GO on the production of proinflammatory cytokines by microglia. Primary murine microglial cells were treated with GO (1–25 μg/mL) followed by stimulation with lipopolysaccharide (LPS) for 24 h. The cell viability was measured by spectrophotometry using AlamarBlueⓇ. The levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). The IL-1β converting enzyme (ICE) activity was measured using a specific fluorescent substrate. The activity of cathepsin B and the lysosomal permeability and alkalinity were determined by flow cytometry. Treatment with GO did not affect cell viability, but significantly suppressed the production of IL-1β. In contrast, the production of TNF-α was unaltered. In addition, the lysosomal permeability and alkalinity in microglia treated with GO were increased, whereas the activity of cathepsin B and ICE was decreased. Collectively, these results demonstrated that exposure to GO differentially affected the production of proinflammatory cytokines, which is associated with the modulation of the lysosomal pathway of cytokines processing.


Article 14

Graphene toxicity as a double-edged sword of risks and exploitable opportunities: a critical analysis of the most recent trends and developments – IOPscience

January 2017


Increased production volumes and a broadening application spectrum of graphene have raised concerns about its potential adverse effects on human health. Numerous reports demonstrate that graphene irrespective of its particular form exerts its effects on a widest range of living organisms, including prokaryotic bacteria and viruses, plants, micro- and macro-invertebrates, mammalian and human cells and whole animals in vivo. However, the available experimental data is frequently a matter of significant divergence and even controversy. Therefore, we provide here a critical analysis of the most recent (2015–2016) reports accumulated in the graphene-related materials biocompatibility and toxicology field in order to elucidate the cutting edge achievements, emerging trends and future opportunities in the area. Experimental findings from the diverse in vitro and in vivo model systems are analysed in the context of the most likely graphene exposure scenarios, such as respiratory inhalation, ingestion route, parenteral administration and topical exposure through the skin. Key factors influencing the toxicity of graphene and its complex derivatives as well as potential risk mitigation approaches exploiting graphene physicochemical properties, surface modifications and possible degradation pathways are also discussed along with its emerging applications for healthcare, diagnostics and innovative therapeutic approaches.


Article 15

Nanotoxicity of Graphene and Graphene Oxide | Chemical Research in Toxicology (acs.org)

January 2014


Graphene and its derivatives are promising candidates for important biomedical applications because of their versatility. The prospective use of graphene-based materials in a biological context requires a detailed comprehension of the toxicity of these materials. Moreover, due to the expanding applications of nanotechnology, human and environmental exposures to graphene-based nanomaterials are likely to increase in the future. Because of the potential risk factors associated with the manufacture and use of graphene-related materials, the number of nanotoxicological studies of these compounds has been increasing rapidly in the past decade. These studies have researched the effects of the nanostructural/biological interactions on different organizational levels of the living system, from biomolecules to animals. This review discusses recent results based on in vitro and in vi