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GRAPHENE OXIDE IN C-19 INJECTIONS

https://patentscope.wipo.int/search/en/detail.jsf?docPN=CN112220919 CN112220919 Nano coronavirus recombinant vaccine taking graphene oxide as carrier

(EN) Nano coronavirus recombinant vaccine taking graphene oxide as carrier

(ZH) 以氧化石墨烯为载体的纳米冠状病毒重组疫苗

Abstract (EN) The invention belongs to the field of nano materials and biological medicines, and relates to a vaccine, in particular to development of a 2019-nCoV coronavirus nuclear recombinant nano vaccine. The invention also comprises a preparation method of the vaccine and application of the vaccine in animal experiments. The novel coronavirus vaccine contains graphene oxide, carnosine, CpG and novel coronavirus RBD; The carnosine, the CpG and novel coronavirus RBD are combined on a framework of the graphene oxide; the coding sequence of the CpG is as shown in SEQ ID NO 1; and the novel coronavirus RBDrefers to that a novel coronavirus protein receptor binding region can generate a high-titer specific antibody aiming at the RBD in a mouse body, and strong support is provided for prevention and treatment of the novel coronavirus.

GRAPHENE IS ALSO NEEDED FOR CBDC GRAPHENE BLOCKCHAIN AND FOR INTERNET OF EVERYTHING AND MIND CONTROL AND CAN BE USED IN OTHER SINISTER WAYS

IS TOXIC!

https://patents.google.com/patent/CN112220919A/zh - https://patentimages.storage.googleapis.com/ef/0c/d4/2ed14dae3576f1/CN112220919A.pdf

[0010] Graphene is a two-dimensional carbon nanomaterial consisting of carbon atoms in a hexagonal honeycomb lattice with sp² hybridization orbitals.

The basic structural unit is the most stable benzene six-membered ring among organic materials, and it is the most ideal two-dimensional material at present.

Graphene oxide

Graphene oxide (GO), a derivative of graphene, is an exfoliated form of graphite oxide. Due to its unique SP2 hybridization and perfect two-dimensional structure as well as the high reactivity of the edges make it ideal for the design and development of therapeutic platforms based on it as loading and grafting carriers for nano-drug transport systems, bio-detection, tumor therapy and cell imaging.

[0011] The present invention builds on the above research.

[0012] The present invention is based on graphene oxide material as a backbone for loading CpG molecules and recombinant proteins, and develops a novel method for vaccine development. Based on this technology platform, a new nano-neocrown vaccine was prepared based on the recombinant protein of the RBD region of the Spike protein of SAR-CoV-2. The prepared nano-neocrown vaccine showed strong immunogenicity in mouse test and could produce highly effective antibodies.

[0013] On the one hand, the present invention provides a coronavirus vaccine containing graphene oxide, myostatin, CpG, RBD.

In preferred embodiments of the invention, it is referred to as the GO-Car-Myostatin-CpG-RBD vaccine.

[0014] Graphene oxide (GO, graphene oxide) is an oxide of graphene that has more properties than graphene due to the increase in oxygen-containing functional groups on it after oxidation.

The properties of graphene oxide are more active than those of graphene due to the increase of oxygen-containing functional groups on the oxide. For example, hydroxyl and epoxide groups are randomly distributed on graphene oxide monoliths, while

carboxyl and carbonyl groups are introduced at the edges of the monoliths. Graphene oxide is commonly available commercially in powder, flake, and in solution form, with a brownish-yellow color.

GRAPHENE IS IN MASKS, PCR TESTS, DRUGS AND FOOD.

https://outraged.substack.com/p/graphenenanotechnology-in-masks-and

https://outraged.substack.com/p/can-toxic-substances-be-mandated

https://newatlas.com/graphene-bad-for-environment-toxic-for-humans/31851/

But it’s only been 10 years since graphene was first isolated in the laboratory, and as researchers and industries scramble to bring graphene out of the lab and into a vast range of commercial applications, far less money is being spent examining its potential negative effects.

Two recent studies give us a less than rosy angle. In the first, a team of biologists, engineers and material scientists at Brown University examined graphene’s potential toxicity in human cells. They found that the jagged edges of graphene nanoparticles, super sharp and super strong, easily pierced through cell membranes in human lung, skin and immune cells, suggesting the potential to do serious damage in humans and other animals.

The bottom corner of a piece of graphene penetrates a cell membrane - mechanical properties like rough edges and sharp corners can make graphene dangerous to human cells. Scale bar represents two microns. (Image: Kane lab/Brown University)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088662/ Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms

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.

GFNs can be delivered into bodies by intratracheal instillation [30], oral administration [31], intravenous injection [32], intraperitoneal injection [33] and subcutaneous injection [34]. GFNs can induce acute and chronic injuries in tissues by penetrating through the blood-air barrier, blood-testis barrier, blood-brain barrier, and blood-placenta barrier etc. and accumulating in the lung, liver, and spleen etc. For example, some graphene nanomaterials aerosols can be inhaled and substantial deposition in the respiratory tract, and they can easily penetrate through the tracheobronchial airways and then transit down to the lower lung airways, resulting in the subsequent formation of granulomas, lung fibrosis and adverse health effects to exposed persons [2, 29]. (COVID?!!!)

Several reviews have outlined the unique properties [35, 36] and summarized the latest potential biological applications of GFNs for drug delivery, gene delivery, biosensors, tissue engineering, and neurosurgery [37–39]; assessed the biocompatibility of GFNs in cells (bacterial, mammalian and plant) [7, 40, 41] and animals (mice and zebrafish) [42]; collected information on the influence of GFNs in the soil and water environments [43]. Although these reviews discussed the related safety profiles and nanotoxicology of GFNs, the specific conclusions and detailed mechanisms of toxicity were insufficient, and the mechanisms of toxicity were not summarized completely. The toxicological mechanisms of GFNs demonstrated in recent studies mainly contain inflammatory response, DNA damage, apoptosis, autophagy and necrosis etc., and those mechanisms can be collected to further explore the complex signalling pathways network regulating the toxicity of GFNs. It needs to point out that there are several factors which largely influence the toxicity of GFNs, such as the concentration, lateral dimension, surface structure and functionalization etc.

The route of nasal drops, which has been widely used to test the neurotoxicity or brain injury potential of other nanomaterials, was not mentioned in the papers reviewed here.

Due to their nanosize, GFNs can reach deeper organs by passing through the normal physiological barriers, such as the blood-air barrier, blood-testis barrier, blood-brain barrier and blood-placental barrier.

Intravenously administered GO entered the body through blood circulation and was highly retained in the lung, liver, spleen and bone marrow, and inflammatory cell infiltration, granuloma formation and pulmonary edema were observed in the lungs of mice after intravenous injection of 10 mg kg/body weight GO [49].

After internalization, graphene accumulated in the cell cytoplasm, perinuclear space, and nucleus, which induced cytotoxicity in murine macrophages by increasing intracellular ROS through depletion of the mitochondrial membrane potential and by triggering apoptosis through activation of the mitochondrial pathway [83].

Toxicity in internal organs

GO can result in acute inflammation response and chronic injury by interfering with the normal physiological functions of important organs [32, 81].

GFNs caused inflammation and remained in the lung on day 90 after a single intratracheal instillation, and even translocated to lung lymph nodes by a nose-only inhalation [96, 97]. A high dose of GO that forms aggregations can block pulmonary blood vessels and result in dyspnea [50, 98], and platelet thrombi were observed at high concentrations of 1 and 2 mg/kg body weight via intravenous injection [89].

GO reportedly disrupted the alveolar-capillary barrier, allowing inflammatory cells to infiltrate into the lungs and stimulate the release of pro-inflammatory cytokines [99]. Fibrosis and inflammation could be verified by the increased levels of the protein markers collagen1, Gr1, CD68 and CD11b in the lungs.

In addition, radioactive isotopes can be delivered into the lungs, accompanied by a depth distribution of 125I-NGO in the lungs, and the isotopes might deposit there and result in mutations and cancers [30].

Toxicity in the central nervous system

In the chicken embryo model, pristine graphene flakes decreased the ribonucleic acid level and the rate of deoxyribonucleic acid synthesis, leading to harmful effects on brain tissue development and the atypical ultrastructure was observed in the brain.

Toxicity in reproduction and development system

Pristine graphene reduced the vascularization of the heart and the density of branched vessels after injection into fertilized chicken eggs followed by incubation for 19 d [101]. GO and rGO damage zebrafish embryos by influencing the embryo hatching rate and body length in a concentration-dependent manner. GO adhered to and was wrapped in the chorion of the zebrafish embryos, causing remarkable hypoxia and hatching delay. GO aggregates were retained in many organelles, such as the eyes, heart, yolk sac, and tail of the embryos, and apoptosis and reactive oxygen species (ROS) generation were observed in these regions [103].

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]. Notably, the development of offspring in the high dosage group was delayed during the lactation period. The high dose of GO decreased the maternal mice’s water consumption by oral exposure, which reduced milk production and thus postponed the growth of offspring [53].

In conclusion, the lung injury induced by GFNs has been studied in several studies, the results of which have demonstrated inflammatory cell infiltration, pulmonary edema and granuloma formation in the lungs. However, only a few specific studies have evaluated in other organs, such as the liver, spleen, and kidney, and the injury symptoms, damage index and level of damage to these internal organs were not fully investigated. Moreover, studies on the neurotoxicity of GFNs are quite rare; no data has revealed which nerves or brain areas experience damage, nor have the related behavioural manifestations been studied. The developmental toxicity of GFNs may induce structural abnormalities, growth retardation, behavioural and functional abnormalities, and even death. A study on the reproductive and developmental toxicity of GFNs will be extremely significant and gain extensive attention in the future. Almost all the GFNs toxicity studies were short-period experiments, and no studies have investigated long-term chronic toxic injury. However, based on studies of other nanomaterials toxicity, long-term GFNs exposure may be an important factor harming health [107–109]. Therefore, the long-term study of GFNs is necessary.

Toxicity of GFNs in cell models

The cytotoxicity of GFNs in vitro has been verified in various cells to change the cell viability and morphology, destroy the membrane integrity, and induce DNA damage [110–112]. GO or rGO decrease cell adhesion; induce cell apoptosis; and enter lysosomes, mitochondria, cell nuclei, and endoplasm [113]. GQDs entered cells and induced DNA damage by the increased expression of p53, Rad 51, and OGG1 proteins in NIH-3 T3 cells [87].

GO derivatives dramatically decreased the expression of differential genes that are responsible for the structure and function of the cell membrane, such as regulation of the actin cytoskeleton, focal adhesion and endocytosis [89]. In rat pheochromocytoma cells (PC12 cells), graphene and rGO caused cytotoxic effects and mitochondrial injury, such as the release of lactate dehydrogenase (LDH), an increase in the activation of caspase-3, and the generation of ROS [82, 116].

Graphene can increase cell viability [117] or cause cell death [118] depending on the cell line, type of graphene material and the dosage.

Concentration

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

Lateral dimension

When the diameter of graphene is between 100 ~ 500 nm, the smallest size may cause the most severe toxicity, and when the diameter is below 40 nm, the smallest sizes may be the safest. For instance, rGO with a diameter of 11 ± 4 nm could enter into the nucleus of the hMSCs and cause chromosomal aberrations and DNA fragmentation at very low concentrations of 0.1 and 1.0 mg/mL in 1 h.

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

Mitochondrial damage / DNA damage / Inflammatory response / Apoptosis / Autophagy / Necrosis / Epigenetic changes

Functionalized graphene oxide serves as a novel vaccine nano-adjuvant for robust stimulation of cellular immunity: https://pubs.rsc.org/en/content/articlelanding/2016/nr/c5nr09208f/unauth

https://www.sciencedirect.com/science/article/pii/S1742706120303305