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Advances in Pharmacology & Clinical Trials Research Article 31 min read

Self-Assembling Property of Graphene Derivates Chemico-Physical and Toxicological Implication

Luisetto M*
* Corresponding author
ISSN: 2474-9214  10.23880/apct-16000206  Received: September 09, 2022  Published: October 20, 2022
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Keywords
Self-Assembling Graphene Graphene Oxide Chemico-Physicial Property Toxicology Clinical Effect Biopharmaceuticals mRNA Vaccine
Abstract

This work start after seeing an recent open letter for transparency related production an quality control technique of mRNA vaccine first signed by Tarro G, Luisetto M and Monsellato ML and an editorial recognized by IMA Marijnskaya academy: Graphene and Derivate: Physio-Chemical and Toxicology properties in the mRNA Vaccine Manufacturing Strategy, needed specific proof of absence for the regulatory aspects (accepted for publication). Other relevant evidences related to this topic comes from the work of Giovannini, et al. related Dark field microscope assay of the blood of 1086 symptomatic subjects after vaccination with two types of m-RNA vaccine of great interest on this field also the work of P Campra and Young RO, Young Me Lee or Ki-Yeob J. Observing all this recent evidences the aim of this work is to investigate not only the graphene presence (or not) in vials of mRNA Vaccine but also the self-auto assembly properties of graphene and derivate. This is in order to find relationship in some biotechnological application like m-RNA Vaccine’s large scale production. After a review part an experimental hypothesis project will be submitted to the researcher to produce a global conclusion related the topic investigated. The recent evidences published induced the idea to more deeply study these properties for the clinicotoxicological aspects involved. The pro-coagulant properties of the coronavirus covid-19 Spike Protein are well knower by scientific literature as well as the toxicological profile of the graphene derivate. What can happen if this two substantive can act in patients with platelet disorder in the same time?

Introduction

Related various and recent evidence P Campra, Young RO, Young Me Lee, Ki-Yeob J, Giovannini et al. and review works of Luisetto M, Tarro G it is interesting to observe the self-assembling properties of graphene and its derivate and their implication in clinico-oncological and toxicological field. The characteristic pattern of this innovative material used in many of biotechnological application related to their specific chemico-physical properties are reported in various relevant literatures.

As reported in article “Bio-pharmaceutical manufacturing large scale production process: The graphene-derivates role and mRNA vaccine”: Used in many bio-medical and other fields like bio-sensors, in water purifying, to remove heavy metals procedure, in diagnostic field but also in extraction, purifying DNA, RNA and other bio molecule, carrier, adjuvant, antibacterial and other biological and industrial use. In literature it is also possible to see in example, Materials today. New graphene-based material self-assembles into vascular structures 19 March 2020: Self-assembly is the process by which multiple components spontaneously organize into larger, well-defined structures. Biological-systems rely on this process to controllably assemble molecular building blocks into complex and functional-materials exhibiting remarkable properties such as the capacity to grow, replicate and perform robust functions.

There is a relevant great interest to develop materials and fabrication processes that emulate those from nature. The ability to build robust functional materials and devices through the self-assembly of molecular components has until now been limited, said team member Yuan hao Wu, who is also at the Nottingham-Queen M, University, London. This research introduces a new method to integrate proteins with graphene oxide (GO) by self-assembly in a way that can be easily integrated with additive manufacturing to easily fabricate various bio fluidic devices that allow us to replicate key parts of human tissues and organs in the lab (Figures 1-3).

Figure 1: Color online- Schematic depicting 2 un-doped and un-strained freely suspended graphene layers separated by a finite distance (D) [1].
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Figure 1: Color online- Schematic depicting 2 un-doped and un-strained freely suspended graphene layers separated by a finite distance (D) [1].

Reactivity control of the graphene is an important problem because chemical functionalization can modulate graphene unique mechanical, optical, and the electronic properties. Using systematic optical research studies, we demonstrate that van der Waals VDW interaction is the dominant factor for the chemical reactivity of graphene on 2-dimensional (2D) hetero structures. A significant enhancement in chemical stability of graphene is obtained by replacing the common SiO2 substrate with 2D crystals such as an additional graphene layer, WS2, MoS2, or h-BN. Our theoretical/ experimental results show that its origin is a strong van der Waals VDW interaction between graphene layer and the 2D substrate. This results in a high resistive force on the graphene to-ward geometric lattice deformation. We demonstrate that chemical reactivity of the graphene can be controlled by the relative lattice orientation with respect to the substrates and thus can be used for a wide range of applications including hydrogen storage [2].

Figure 2: Graphene sheets and bond types.
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Figure 2: Graphene sheets and bond types.

Self-assembly is a process-mechanism by which a disordered system of pre-existing components forms an organized structure or pattern like a consequence of specific, local interactions among the components themselves, without external direction. When the constitutive components are molecules, the process is named molecular self-assembly.

Regarding the self-assembly process in Nano science it is possible to see:

Figure 3: Conceptual scheme indicating the main stages of the self-assembly process in Nano science [3].
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Figure 3: Conceptual scheme indicating the main stages of the self-assembly process in Nano science [3].

Material and Methods

Whit an observational point of view various relevant literature and figure are reported and analyzed. After this review part an experimental project hypothesis will be submitted to researcher in order to produce a global conclusion related the topics of this works. All literature comes from bio medical or other scientific or technological involved database.

Results

GO is a unique 2-dimensional (2D) material with interesting physical/chemical properties. GO can be considered as a 2D ampi-philic conjugated polymer, consisting of hydro-philic oxygenated groups and hydro phobic conjugated graphitic-domains. The diverse chemical groups endow GO graphene oxide with high chemical activity to react with other molecules and form new species with graphitic frame work. The ampi-philic properties of GO sheets provide them the abilities to self-assemble into 3-dimensional (3D) structure or reduced-GO (r-GO) gels with porous micro- structures. The pre-condition of these promising properties of GO is its excellent solution-like dispensability in aqueous or non-aqueous media. These liquid media facilitate the exfoliation of GO into single-layer sheets and provide the exfoliated GO sheets with specific chemical environment for functionalization/processing. It is essential to understand the solution-based chemical behavior of GO graphene oxide, which is important for better application of the GO. In this review work, we outline the solution-based chemistry of GO mainly in terms of the molecular structure, dispensability in solvents, solution properties and related processing of GO sheets. This review work aims to systematically present physical/chemical behaviors of GO in solvents including aqueous and non-aqueous solvents, which is helpful for better understanding and application of GO graphene oxide materials [10].

We used GO sheets of 2 different average lateral sizes, including the Larger GO (GO-L) measuring 10.5 ± 4.5 µm and smaller GO (GO-S) of 2.3 ± 0.9 µm, both exhibiting a typical hydro phobic-surface and negatively charged carboxylic- groups on their periphery. We chose ELRs as the protein component because of their modular and disordered nature and the possibility to exhibit different molecular conformations at the different temperatures. The ELK1 sequence is a 51.9 kDa molecule consisting of 24 repeats of single-block made of 4 hydro-phobic penta-peptides (VPGIG) and a positively (+) charged (VPGKG) one. This relatively simple molecular design offers an accessible transition team. (Tt) of 30 °C (at 2% ELK1 in MilliQ water) with clearly different ELR conformations above or below it, as well as medium molecular-weight to enable both cooperative interactions between its charged and hydro-phobic segments as well as with the anionic edge and hydro phobic surface of the G.O . ELRs with similar molecular weight but different levels of charge and hydro-phobicity, as well as a single repeat of an individual block of each of these three ELRs, was used as a controls.

Figure reported 1: Molecular building blocks (and rationale) for When an ELK1 solution at its Tt (30 °C) is immersed in a larger volume of a GO graphene oxide solution, a multilayered membrane of up to 50 µm in thickness develops at the interface around the immersed drop maintaining both solutions separated. This kind of membrane consists of layers made from both GO graphene oxide sheets and ELK1, with GO sheets being present throughout the cross-section of the membrane and ELK1 gradually decreasing in concentration from the inside (ELK1- side) to the outside (GO side) of the membrane. Multi-layered structures are known to emerge from diffusion–reaction mechanisms. We have previously demonstrated that with co-assembling PAs with ELRs, it is possible to trigger a diffusion-reaction mechanism, which generates multi-layered membranes capable of exhibiting dynamic- properties. In Similar way, by touching any surface within the first few seconds of formation, the ELK1-GO membrane adheres, spontaneously and reproducibly opens, and can be manipulated to grow into tubular structures with patio-temporal control. In this case, the underlying ELR-GO mechanism of interaction and supra molecular assembly lead to the growth of a material with remarkably enhanced properties [11] (Figure 7).

In the present research study, the toxicity of 6 different types of Carbon Nano-particles (CNPs) was investigated using a chicken-embryo model. Fertilized chicken eggs were divided into this following treatment groups: placebo, diamond- NPs, graphite NPs, and pristine graphene, small graphene oxide, large graphene oxide GO, and reduced graphene oxide. Self-assembly of CNPs with albumin amino- acids AA by non-covalent bonds is very efficient, implying that CNPs can be effectively transported into embryos. According to Szmidt et al, lower concentrations (50 and 500 μg/mL) of graphene penetrate the embryo more efficiently than the higher concentrations, due to different NP-dispersion levels. These results were explained by the natural tendency of CNPs to agglomerate when they are coated by albumin- proteins that surround the embryo. In the present Research study work, we also administrated CNPs to egg-albumin, which gets progressively consumed by the embryo during the development process and is ultimately fully absorbed, ensuring that the whole dose was delivered during embryo- genesis [12] (Figure 8).

In blood, non-covalent adsorption occurs through weak van der Waals VDW forces, hydro phobic, electrostatic, and π–π stacking interactions. The sp2 hybridized honey-comb carbon-lattice of rGO and GO is hydro phobic and, interacts with the hydro phobic-regions of proteins, according to the protein geometry. The basal plane of the GO is also enriched with π electrons, making π–π stacking interactions possible. At the same time the oxygen-groups of GO, whose composition is strictly dependent on preparation and storing conditions, allow further hydrogen-bonds and electrostatic bonds. These electrostatic-bonds are strongly influenced by the charge on proteins and by pH-ionic strength of the buffer. Bonding on GO also is mediated by van der Waals VDW-interactions. While the electrostatic- interactions are more pronounced on GO, both van der Waals VDW and electrostatic-interactions play a major role in the adsorption of proteins on RGO due to the increase in the non-functionalized area on the surface. In the following other sections, we will show how functionalization of the GO surface alters protein adsorption and consequently BC -properties.

Figure 7: (a) Table summarizes the key information of the three elastin-like re-combiners (ELRs) used in the study comprising similar molecular- weight but different levels of hydro-phobicity (VPGIG) and positive charge (VPGKG). (b) Illustrations of the molecular-structure of a GO sheet and the supra-molecular organization of ELK1 at its transition temperature (Tt) (30 °C) indicating both the charged (red and green) and hydro phobic (brown) segments. (c) Schematic of the proposed mechanism of formation illustrating the molecular-supra-molecular conformation of the GO and ELK1 before and after the co-assembly at the ELK1’s Tt as well as their interaction for membrane formation.
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Figure 7: (a) Table summarizes the key information of the three elastin-like re-combiners (ELRs) used in the study comprising similar molecular- weight but different levels of hydro-phobicity (VPGIG) and positive charge (VPGKG). (b) Illustrations of the molecular-structure of a GO sheet and the supra-molecular organization of ELK1 at its transition temperature (Tt) (30 °C) indicating both the charged (red and green) and hydro phobic (brown) segments. (c) Schematic of the proposed mechanism of formation illustrating the molecular-supra-molecular conformation of the GO and ELK1 before and after the co-assembly at the ELK1’s Tt as well as their interaction for membrane formation.

Figure 7: (a) Table summarizes the key information of the three elastin-like re-combiners (ELRs) used in the study comprising similar molecular- weight but different levels of hydro-phobicity (VPGIG) and positive charge (VPGKG). (b) Illustrations of the molecular-structure of a GO sheet and the supra-molecular organization of ELK1 at its transition temperature (Tt) (30 °C) indicating both the charged (red and green) and hydro phobic (brown) segments. (c) Schematic of the proposed mechanism of formation illustrating the molecular-supra-molecular conformation of the GO and ELK1 before and after the co-assembly at the ELK1’s Tt as well as their interaction for membrane formation.

Figure 8: Blood cell aggregation on incubation of graphene with blood from Blood Compatibility and Bio medical Applications of Graphene [13].
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Figure 8: Blood cell aggregation on incubation of graphene with blood from Blood Compatibility and Bio medical Applications of Graphene [13].

Effects of Bio-Coroneted GO Materials on the Blood Components

BC composition directly influences interactions with the other blood components. The presence of antibodies, complement and clotting factors in the Nano-particle BC may activate clotting and coagulation cascades. The BC coating can promote phagocytosis and elimination from the circulation.

We will first consider data on the GO interaction with the red blood cells RBC, in Table reported. An intravenously IV injected Nano-material is likely to interact first with RBCs rather than other cells, due to their abundance in blood. Hemolysis represents the damage to RBCs that leads to the leakage of hemoglobin into the blood-stream. After hemolysis, the Nano-material may adsorb released hemoglobin HB and/ or adhere to cell debris, which can increase its likelihood of elimination by macro-phages. Although the literature is contradictory regarding the GO effects on RBC, when BC is introduced into the framework the results become clearer. Due to the sharp edges of GO and RGO, hemolytic-effects might be expected In Vivo, possibly caused by Nano-material blades disrupting cell-membranes, as reported for the GO interactions with the bacteria (Figure 9).

Graphene and derivatives are emerging as attractive and interesting materials for the bio medical applications like anti-bacterial, the gene delivery, contrast imaging, and anti- cancer therapy applications. It is of fundamental importance to study the cyto-toxicity and the bio compatibility of these materials as well as how they interact with immune-system.

The present research study was conducted to assess the immuno-toxicity of graphene oxide (GO) and vanillin- functionalized GO (V-rGO) on THP-1 cells, and human acute monocytes leukemia cell-line. The synthesized GO and V-rGO were characterized by using various analytical techniques. Various concentrations of G.O and V-rGO showed toxic effects on THP-1 cells such as the loss of cell viability and proliferation in a dose-dependent manner. Cytotoxicity was further demonstrated as an increased level of lactate dehydrogenase, loss of mitochondrial membrane-potential (MMP), decreased level of ATP content, and the cell death. Increased levels of reactive-oxygen species ROS and lipid- peroxidation caused redox imbalance in THP-1 cells, leading to increased levels of melon-di-aldehyde and decreased levels of anti-oxidants like glutathione, glutathione- peroxidase, super oxide dismutase, and catalase. Increased generation of ROS and reduced MMP with simultaneous increases in the expression of pro-apoptotic genes and down-regulation of anti-apoptotic genes suggest that the mitochondria-mediated pathway is involved in GO graphene oxide and V-rGO induced apoptosis. Apoptosis was induced consistently with the significant DNA damage caused by increased levels of 8-oxo-dG and up-regulation of various key DNA-regulating genes in THP-1 cells; indicating that GO and V-rGO induce cell death through oxidative stress. As a result of these events, GO and V-rGO stimulated the secretion of various cytokines and chemokine’s, indicating that the graphene materials induced potent inflammatory responses to THP-1 cells. The harshness of V-rGO in all assays tested occurred because of better charge transfer, various carbons to oxygen ratios, and chemical compositions in the rGO. These research findings suggest that it is essential to better understand the parameters governing GO and functionalized GO in immuno-toxicity and the inflammation. Rational design of safe GO-based formulations for various applications, including Nano-medicine, may result in the development of risk-management methods for people exposed to graphene and graphene family materials, as these Nano-particles can be used like delivery agents in various bio medical applications [15].

Figure 9: Main results of GO interaction with the blood components are summarized in this illustration of the injection of GO graphene oxide flakes in the blood-stream. The formation of the BC (1) prevents the hemolysis of red blood cells RBC (2a), Thrombosis (2b) and interaction with the complement-proteins (2c) are ascribed to GO. In (2d) some of the possible fates after macro-phage encounters are shown: extra-cellular blocking or intra-cellular uptake. The release of cytokines occurs when macro-phages uptake GO. Aggregates of GO in macro-phage cytoplasm induce the production of the pro-inflammatory cytokines. Dendritic-cells fail to present antigens to lymphocytes when they uptake GO (2e). Lymphocyte activity is not inhibited, and BC protects lymphocytes from apoptosis (2f) [14].
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Figure 9: Main results of GO interaction with the blood components are summarized in this illustration of the injection of GO graphene oxide flakes in the blood-stream. The formation of the BC (1) prevents the hemolysis of red blood cells RBC (2a), Thrombosis (2b) and interaction with the complement-proteins (2c) are ascribed to GO. In (2d) some of the possible fates after macro-phage encounters are shown: extra-cellular blocking or intra-cellular uptake. The release of cytokines occurs when macro-phages uptake GO. Aggregates of GO in macro-phage cytoplasm induce the production of the pro-inflammatory cytokines. Dendritic-cells fail to present antigens to lymphocytes when they uptake GO (2e). Lymphocyte activity is not inhibited, and BC protects lymphocytes from apoptosis (2f) [14].

Figure 9: Main results of GO interaction with the blood components are summarized in this illustration of the injection of GO graphene oxide flakes in the blood-stream. The formation of the BC (1) prevents the hemolysis of red blood cells RBC (2a), Thrombosis (2b) and interaction with the complement-proteins (2c) are ascribed to GO. In (2d) some of the possible fates after macro-phage encounters are shown: extra-cellular blocking or intra-cellular uptake. The release of cytokines occurs when macro-phages uptake GO. Aggregates of GO in macro-phage cytoplasm induce the production of the pro-inflammatory cytokines. Dendritic-cells fail to present antigens to lymphocytes when they uptake GO (2e). Lymphocyte activity is not inhibited, and BC protects lymphocytes from apoptosis (2f) [14].

Nano medicines are being developed to treat various diverse diseases; inadvertent or un-intended health effects have to be considered, especially for those targeting cancers. For the cancers, occurrence of metastasis hints an advanced phase of cancer progression, and Nano-medicines per se should be evaluated for their effects on existing metastatic tumors and triggering the metastases.

Graphene-based 2D Nano-materials, such as (GO), due to its unique characteristics, have been extensively studied for bio medical applications including the cancer therapy. The potential effect of GO on metastasis has not been determined yet. We found that low-dose GO could induce significant morphological and structural changes of the cellular membrane within the cancer cells, suggesting an epithelial- mesenchyme transition, with enhanced invasion/migration and the alterations of representative EMT indicators in GO-treated cells. These changes resulted in enhanced lung- metastasis of cancer cells in various kinds of metastasis models.

The mechanistic investigations unveiled that GO graphene oxide increased the protein levels of the TGF-β receptor, leading to a constitutively activated TGF-β-Smad2/3 signaling path-way that drives the EMT. Our findings enhance the understanding of the un-intended side and detrimental effects of GO Nano-sheets in increasing the progression of metastatic-tumors. So, the likely-hood of pro-EMT effects upon low-dose GO exposure should be considered when developing GO Nano medicines [16].

A high dose of GO that forms aggregations can block the pulmonary blood-vessels and result in dyspnea and platelet PTL thrombi were observed at high concentrations of 1 and 2mg/kg body weight via intravenous IV injection [17].

GO has abundant surfaces oxygen-containing groups like epoxide, hydroxyl, and carboxylic groups; it can be prepared through the oxidative intercalation and exfoliation of graphite on a mass scale. Owing to the enriched surface functionalities, the GO is water-soluble and chemically versatile. The surface functional-groups can also provide plenty of reaction sites for linking the Nano-particles, proteins, enzymes, peptides, bacteria, cells, nucleic acids through the covalent and non-covalent binding. GO graphene oxide has been used as a matrix for protein immobilization in different bio technological applications such as fluorescence or electro chemical-based sensors, labeling and imaging, therapy, and targeted delivery.

Non Covalent-interaction (Physical -adsorption)

Non-covalent protein adsorption into solid supports represents the most simple and desirable strategy of physical immobilization. The mechanisms of proteins adsorption on GO graphene oxide is a kind of non-covalent self-assembly including weak Van der Waals VDW forces, hydro-phobic, electrostatic, and π-π stacking interaction. These types of attractions between the proteins and graphene oxide GO involve solution phase incubation, or direct sonication, followed by a washing step to remove the un-bound proteins. The non-covalent bonds responsible for the interaction between GO graphene oxide and proteins vary depending on the surface properties of graphene oxide, such as morphology and hydro-phobicity [18].

Although information on the In Vitro and In Vivo Nano- toxicity of graphene Nano-materials has been increasingly published in the last several years, a complete picture on the bio-compatibility of graphene Nano-materials has not been established. The successful applications of graphene Nano-materials in Nano bio-technology and medicine as well as their effective translation into real clinical utility hinge significantly on a thorough understanding of their Nano toxicological profile. Of all aspects of bio compatibility, the hem-compatibility of graphene Nano materials with the different blood constituents in circulatory system is one of the most important elements that need to be well elucidated. Once administered into the biological systems, graphene Nano-materials may inevitably come into contact with the surrounding plasma proteins PP and blood-cells. Crucially, the interactions between these kinds of hematological entities and graphene Nano-materials will influence the overall efficacy of their bio medical applications. As such, a comprehensive understanding of hemo-toxicity of the graphene Nano materials is critically important. he in vitro evaluations of the potential cytotoxic effects of graphene Nano materials have been actively conducted on different human cell-lines, such as human fibroblasts, human umbilical vein endothelial-cells, normal human lung-cells (BEAS-2B), human lung cancer cells (A549), human hepato-carcinoma cells, HeLa cells, and the human breast cancer cells MCF-7. A majority of these investigations have demonstrated the time and dose dependent cytotoxicity of graphene Nano-materials. Vacuous, In Vitro experimental and theoretical investigations have attributed the cyto-toxicity of both the graphene and its oxygenated derivative GO on the mammalian cells and bacteria to cellular membrane penetration, followed by phospho-lipid molecule extraction from the lipid-bilayer [19, 20].

GO has been demonstrated to possess a high loading capacity for albumin ALB and fibrinogen FIBR in a recent work. [19, 20] While numerous studies have reported observations on graphene Nano material-induced a protein conformational change, the under-lying mechanisms are still poorly understood GO have a surface area of 25nm 2 and randomly decorated hydroxyl and epoxy-groups on its surface. A carboxyl group was attached to the GO edges. While having the same surface area, in comparison to GO graphene oxide, the rGO model possesses fewer oxygenated functional groups GO Nano-sheets have been reported to possess a strong thrombus-inducing potential and considerable thrombo-genecity. They could trigger the activation of platelets PTL and their strong aggregator’s response similar to that evoked by thrombin, an active physiological platelet agonist. The platelet activation by GO was suggested to be extensively dependent on the surface charge distribution of GO graphene oxide as it was revealed that, in contrast to GO, rGO with reduced surface charge density was less capable in activating and aggregating platelets PTL. The pro-thrombotic characteristic of GO Nano-sheets was further verified through the occurrence of significant pulmonary thrombo embolism after their intravenous IV administration in mice [20] (Figure 10).

Figure 10: Nano-bio interactions of graphene Nano-materials with various blood plasma proteins and cells.
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Figure 10: Nano-bio interactions of graphene Nano-materials with various blood plasma proteins and cells.

Figure10: Nano-bio interactions of graphene Nano-materials with various blood plasma proteins and cells.

This critical review work aims at giving insights in the spontaneous tendency of the proteins and their constitutive parts to adsorb on graphitic Nano-materials (GNMs) through non-covalent interactions occurring in their interfaces [21].

This review work collects studies on the toxic effects of GFNs in various organs and cell models. We also point out that various factors determine the toxicity of GFNs including lateral size, surface structure, functionalization, charge, the impurities, aggregations, corona effect. Various typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and the necrosis. In these kind of mechanisms, (toll-like receptors) TLR, TGF-β and TNF-α dependent pathways are involved in the signaling pathway network, and oxidative stress plays a crucial role in these kind of path ways [22].

Toxicity of graphene family Nano-particles the dose, shape, surface-chemistry, exposure route, and purity play important roles in differential toxicity of GFNs. Different various authors have used various toxicity tests to evaluate the toxicity of GFNs. Studies have been conducted to find out the toxicity of GFNs on different cellular/animal models, including stem cells, HeLa cells, HepG2 cells, bacteria, Drosophila melanogaster, Zebra-fish, marine organisms, rats, mice, and mammalian cells. Cytotoxicity tests indicated that the RGO can damage cells with direct contact. In this part of the paper, an attempt has been made to compile the recent and up-to-date research studies related to toxicological aspects of GFNs to different models [23].

The peculiar features of these cases were the availability of macroscopic and micro-scope autopsy findings. The main macro-scope finding was that venous-thrombosis was much more widespread and catastrophic than diagnosed by imaging during the life. Microscopic findings were showed vascular thrombotic occlusions occurring in the micro- circulation of multiple organs and increased inflammatory infiltrates [24].

Post mortem investigations of fatalities after COVID-19 vaccination are particularly relevant with regard to the detection of anaphylaxis, VITT, and myocarditis. Vaccine- induced immune thrombotic thrombo-cytopenia (VITT) is characterized by thrombo-cytopenia, combined with thrombosis in most cases. Thrombosis can occur in the both the arterial and, more common, venous system. A distinctive feature of VITT is thrombosis in un-usual locations. These include CVT, as well as splanchnic-venous thrombosis [25].

In this research work, for the first time, we studied the In Vitro and In Vivo interactions of a relatively new derivative of graphene, graphene –Nano-pores (GNPs) in the mammalian systems, to systematically elucidate the possible mechanism of their toxicity over time. Heart tissue showed chemo-dectoma, toxic myocarditis, reddish brown atrophy; yellowish-brown pigments suggesting lipo-fuscin granules as remnants of the cell organelles and cytoplasmic- material [26] (Figures 11-14).

Figure 11: Graphene nanopores toxicology [26].
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Figure 11: Graphene nanopores toxicology [26].
Figure 12: The effect of GO, rGO, and rGO-PEG on the platelet activation: Graphene Nano derivatives were co-incubated with platelet PTL rich plasma at 50µg/ml. Platelet activation was induced by the addition of 2µmol/ml of Adenosine Di-Phosphate (ADP). The Platelet PTL aggregates are pointed by the blue arrows. One representative picture reflects the results of 3 independent experiments. GO; rGO reduced graphene oxide; rGO-PEG, PEGylated reduced graphene oxide; ADP, adenosine-di- phosphate [27].
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Figure 12: The effect of GO, rGO, and rGO-PEG on the platelet activation: Graphene Nano derivatives were co-incubated with platelet PTL rich plasma at 50µg/ml. Platelet activation was induced by the addition of 2µmol/ml of Adenosine Di-Phosphate (ADP). The Platelet PTL aggregates are pointed by the blue arrows. One representative picture reflects the results of 3 independent experiments. GO; rGO reduced graphene oxide; rGO-PEG, PEGylated reduced graphene oxide; ADP, adenosine-di- phosphate [27].

Figure 12: The effect of GO, rGO, and rGO-PEG on the platelet activation: Graphene Nano derivatives were co-incubated with platelet PTL rich plasma at 50µg/ml. Platelet activation was induced by the addition of 2µmol/ml of Adenosine Di-Phosphate (ADP). The Platelet PTL aggregates are pointed by the blue arrows. One representative picture reflects the results of 3 independent experiments. GO; rGO reduced graphene oxide; rGO-PEG, PEGylated reduced graphene oxide; ADP, adenosine-di- phosphate [27].

Figure 13: The schematics of the representative carbon-based nanomaterials [28].
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Figure 13: The schematics of the representative carbon-based nanomaterials [28].
Figure 14: Scanning electron microscopy image shows platelet PTL activation by multi-walled CNTs (M60). Platelets were incubated with 100µg/ml of M60 at 37°C for 15 minutes under gentle agitation. The images are representative of at least three individual experiments with platelets PTL from different donors. (Image: Jan Simak, FDA).
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Figure 14: Scanning electron microscopy image shows platelet PTL activation by multi-walled CNTs (M60). Platelets were incubated with 100µg/ml of M60 at 37°C for 15 minutes under gentle agitation. The images are representative of at least three individual experiments with platelets PTL from different donors. (Image: Jan Simak, FDA).

RBCs were exposed to 3 different forms of GQDs (non- functionalized, hydroxyl-ated, and carboxyl-ated GQDs) at various concentrations (0, 500, 750, and 1000μg/mL) and incubation times (0, 1, 2, 3, or 4 h). The rheological characteristics of the RBCs were measured using micro fluidic-laser diffractometry and aggregometry. The hemolysis rate and rheological alterations of the RBCs were insignificant at a concentration less than 500μg/ml. Carboxylated GQDs were observed to have more substantial hemolytic activity and caused abrupt changes in deformability and aggregation of the RBCs than the non-functionalized or hydroxylated GQDs at concentration 750μg/ml. Our findings indicate that hemo- rheological assessments could be utilized to estimate the degree of toxicity to the cells and to obtain useful information on safety sheets for the Nano-materials [29, 30, 31, 32, 33, 34].

Experimental Project Hypothesis

In order to verify in vitro the self-assembling property of graphene GO it is necessary to test 100 human blood specimen with added graphene GO as concentration similar to as reported in literature 100 human blood specimen with no added (control-group). These entire samples must to be sent to various certified and individual analytical laboratory and tested using the blind. If possible send some sample also at various university centers. With various methods (micro- scopic cytology, dark field microscope analysis, RAMAN destructive methods, micro-scope Raman et other useful).

Results: The result must be reported as: a) Sample + graphene, b) Control. Object of the search: Self-aggregates of graphene. Time of observation: T 1H after collecting sample and added graphene, at 4 H, after 24 H, the after 1-week (needed to use anti-coagulant that non produce interference with graphene GO). At time after 1h after the graphene addition coagulation test must to be performed (DD, fibrinogen and other as well as emocromocitometrico assay (Platelet, RBC, micro-scope assay).

Results

To verify if there is difference between the group a and b in significant way p < 0,005.

Discussion

  1. In the literature reported it is clear the self-assembling properties of graphene derivate as well as clear is the effect that this products and aggregate produce on blood.
  2. The same it is clear by scientific literature the pro- coagulant effect of spike protein during the pathological process in covid-19 disease.
  3. Because there is now-a-days a public debate about the presence or absence of graphene derivate in some vials of covid-19 new vaccine it is crucial to think at what can happen when this 2 toxic molecule act in the same time (spike protein and graphene-aggregates) in an human body.
  4. Because many biotechnological process in last decades see the introduction of innovative material like graphene derivate this molecule must to be analytically excluded for release of biopharmaceuticals and so for the covid-19 vaccine.
  5. The m RNA large scale purification process use TFF Tangential Flow filtration added to other chromatographic separation and in this last phases are used silica columns.
  6. This column in commerce are characterized with various level of Carbon Load (from 0 to 100%) so the amount of the graphitic substantive can vary from low to high level.
  7. The same in various researches are reported use of magnetic beads graphene coated to increase efficacy in purification of RNA since before pandemic.
  8. Because since today the full productive process of mRNA Vaccine is not clarified and it is not declared the material used it is of great interest to get public official information about this productive strategy even if present patents or industrial secrets.

9. Thrombosis effect can be increased when 2 different stimuli actin simultaneity way like a Synergic effect. 10. And what can be the global effect in an unbalanced blood system like VITT? 11. The clinical effect of this poisonous association must to be deeply more investigated for public safety need.

Conclusion

Because in toxicology are well known, various situation of combined toxic effect by multiple chemical dangerous exposure. It is needed to verify the clinical effect of the self- assembling graphene GO effect added to spike protein using In vitro sample (Animal model and sample from humans specimens: subjects volunteers). The experimental project submitted can help for this scope. It is also of interest to verify if the cumulative effect of this two substantial Graphene GO and SPIKE protein show and added toxic effect (synergic) or this is greater then the single molecule acting alone and the kinetic related.

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@article{luisetto2022,
  title   = {Self-Assembling Property of Graphene Derivates Chemico-Physical and Toxicological Implication},
  author  = {Luisetto M},
  journal = {Advances in Pharmacology & Clinical Trials},
  year    = {2022},
  volume  = {7},
  number  = {4},
  doi     = {10.23880/apct-16000206}
}
Luisetto M (2022). Self-Assembling Property of Graphene Derivates Chemico-Physical and Toxicological Implication. Advances in Pharmacology & Clinical Trials, 7(4). https://doi.org/10.23880/apct-16000206
TY  - JOUR
TI  - Self-Assembling Property of Graphene Derivates Chemico-Physical and Toxicological Implication
AU  - Luisetto M
JO  - Advances in Pharmacology & Clinical Trials
PY  - 2022
VL  - 7
IS  - 4
DO  - 10.23880/apct-16000206
ER  -