graphene – oxide

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https://www.intechopen.com/books/advances-in-carbon-nanostructures/magnetic-graphene-based-nanocomposites-and-respective-applications

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Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms

Lingling OuBin SongHuimin LiangJia LiuXiaoli FengBin DengTing Sun & Longquan Shao

Particle and Fibre Toxicology volume 13, Article number: 57 (2016) Cite this article

Abstract

Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility and toxicity of GFNs in vivo and in intro. Generally, GFNs may exert different degrees of toxicity in animals or cell models by following with different administration routes and penetrating through physiological barriers, subsequently being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine the toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and corona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha (TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial role in these pathways. In this review, we summarize the available information on regulating factors and the mechanisms of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim of completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and facilitate their wide application.

Background

Graphene, which is isolated from crystalline graphite, is a flat monolayer composed of single-atom-thick, two-dimensional sheets of a hexagonally arranged honeycomb lattice [1]. Because of its unique structural, specific surface area and mechanical characteristics, the functions and applications of graphene have gained considerable attention since the discovery of the material in 2004 [23]. Graphene and its derivatives include monolayer graphene, few-layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO), graphene nanosheets (GNS), and graphene nanoribbons, etc. [47]. GO is one of the most vital chemical graphene derivatives of the graphene-family nanomaterials (GFNs), which attracts increasing attention for its potential biomedical applications. Graphene-based materials usually have sizes ranging from several to hundreds of nanometer and are 1-10 nm thick [89], which is also the definition of ‘nanoparticles’ or ‘nanomaterials’. Due to their exceptional physical and chemical properties, graphene materials have been widely used in various fields, including energy storage; nanoelectronic devices; batteries [1012]; and biomedical applications, such as antibacterials [1314], biosensors [1518], cell imaging [1920], drug delivery [82122], and tissue engineering [2325].

Along with the application and production of GFNs increasing, the risk of unintentional occupational or environmental exposure to GFNs is increasing [26]. And recently, there are some investigation on GFNs exposure in occupational settings and published data showed that the occupational exposure of GFNs had potential toxicity to the workers and researchers [2729]. 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 [229]. Several reviews have outlined the unique properties [3536] and summarized the latest potential biological applications of GFNs for drug delivery, gene delivery, biosensors, tissue engineering, and neurosurgery [3739]; assessed the biocompatibility of GFNs in cells (bacterial, mammalian and plant) [74041] 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. Herein, this review presents a comprehensive summary of the available information on the mechanisms and regulating factors of GFNs toxicity in vitro and in vivo via different experimental methods, with the goals of providing suggestions for further studies of GFNs and completing the toxicology mechanisms to improve the biological safety of GFNs and facilitate their wide application.

Toxicity of GFNs (in vivo and in vitro)

GFNs penetrate through the physiological barriers or cellular structures by different exposure ways or administration routes and entry the body or cells, eventually resulting in toxicity in vivo and in vitro. The varying administration routes and entry paths, different tissue distribution and excretion, even the various cell uptake patterns and locations, may determine the degree of the toxicity of GFNs [4446]. So to make them clear may be helpful to better understand the laws of the occurrence and development of GFNs toxicity.

Administration route

The common administration routes in animal models include airway exposure (intranasal insufflation, intratracheal instillation, and inhalation), oral administration, intravenous injection, intraperitoneal injection and subcutaneous injection. The major exposure route for GFNs in the working environment is airway exposure, thus inhalation and intratracheal instillation are used mostly in mice to simulate human exposure to GFNs. Though the inhalation method provides the most realistic simulation to real life exposure, instillation is more effective and time-saving method, and GFNs was found that causing longer inflammation period using instillation (intratracheal instillation, intrapleural installation and pharyngeal aspiration) than inhalation [24304748]. GFNs were investigated to deposit in the lungs and accumulate to a high level, which retained for more than 3 months in the lungs with slow clearing after intratracheal instillation [49]. Intravenous injection is also widely used to assess the toxicity of graphene nanomaterials, and graphene circulates through the body of mice in 30 min, accumulating at a working concentration in the liver and bladder [325052]. However, GO derivatives had rather finite intestinal adsorption and were rapidly excreted in adult mice via oral administration [3153]. Nano-sized GO (350 nm) caused less mononuclear cells to infiltrate subcutaneous adipose tissue after subcutaneous injection in the neck region compared to micron-sized GO (2 μm) [34]. GO agglomerated near the injection site after intraperitoneal injection, and numerous smaller aggregates settled in the proximity of the liver and spleen serosa [3133]. Experiments on skin contact with or skin permeation of GFNs were not found in the papers reviewed here, and there is insufficient evidence available to conclude that graphene can penetrate intact skin or skin lesions. 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.

GFNs entry paths

GFNs reach various locations through blood circulation or biological barriers after entering the body, which results in varying degrees of retention in different organs. 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.

Blood-air barrier

The lungs are a potential entrance for graphene nanoparticles into the human body through airway. The inhaled GO nanosheets can destroy the ultrastructure and biophysical properties of pulmonary surfactant (PS) film, which is the first line of host defense, and emerge their potential toxicity [54]. The agglomerated or dispersed particles deposit on the inner alveolar surface within the alveoli and then be engulfed by alveolar macrophages (AMs) [55]. Clearance in the lungs is facilitated by the mucociliary escalator, AMs, or epithelial layer [5658]. However, some small, inhaled nanoparticles infiltrate the intact lung epithelial barrier and can then transiently enter the alveolar epithelium or the interstitium [5960]. Intratracheally instilled graphene can redistribute to the liver and spleen by passing through the air-blood barrier [61]. The study of blood-air barrier may draw an intensive attention, since the researchers and workers occupational exposure of GFNs usually through inhalation. To make clear how the blood-air barrier plays a role in the toxicity of GFNs may become a research hot topic.

Blood-brain barrier

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

Blood-testis barrier

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

Blood-placenta barrier

The placental barrier is indispensable in maintaining pregnancy, as it mediates the exchange of nutrients and metabolic waste products, exerts vital metabolic functions and secretes hormones [67]. A recent review suggested that the placenta does not provide a tight barrier against the transfer of nanoparticles to foetuses, specifically against the distribution of carbonaceous nanoparticles to and in the foetus [42]. It was suggested that rGO and gold particles (diameter of 13 nm) are barely present or are absent in the placenta and foetus in late gestation after intravenous injection [4468]. However, other reports showed that transplacental transfer does occur in late gestational stages [6970]. Much attention had been paid to the developmental toxicity of nanomaterials, and reports showed that many nanoparticles did cross the placental barrier and strongly influenced the development of embryos [7175]. But studies of the exposure to graphene materials through the placenta barrier are deficient, and how these particles transfer to embryos should be evaluated in detail in the future.

These four barriers were the most frequently mentioned barriers in the literature, and other barriers have not been evaluated in recent studies, such as skin barriers, which have not been mentioned in any of the hundreds of GFNs toxicity studies searched. Moreover, the mechanism by which GFNs pass through these barriers is not well understood, and more systematic investigations are urgently needed.

Distribution and excretion of GFNs in tissue

The absorption, distribution, and excretion of graphene nanoparticles may be affected by various factors including the administration routes, physicochemical properties, particle agglomeration and surface coating of GFNs.

The different administration routes influence the distribution of GFNs, for example, intratracheally instilled FLG passing through the air-blood barrier mainly accumulated and was retained in the lungs, with 47 % remaining after 4 weeks [61]. Intravenously administered GO entered the body through blood circulation and 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]. Similarly, high accumulation of PEGylated GO derivatives was observed in the reticuloendothelial (RES) system including liver and spleen after intraperitoneal injection. In contrast, GO-PEG and FLG did not show detectable gastrointestinal tract absorption or tissue uptake via oral administration [31].

The different properties of GFNs, such as their size, dose and functional groups, always lead to inconsistent results in the distribution profiles of graphene. For instance, Zhang et al. found that GO was mainly entrapped in mouse lungs [49]; however, Li et al. observed that GO accumulated in mouse liver [76]. Notably, small GO sheets, with diameters of 10–30 nm, were mainly distributed in the liver and spleen, whereas larger GO sheets (10–800 nm) mainly accumulated in the lungs [495277]. If the size of GO is larger than the size of the vessels, GO usually becomes stuck in the arteries and capillaries in the proximity of the injection site. The accumulation of GO in the lungs was shown to increase with an increase in the injected dose and size, but that in the liver significantly decreased [78]. Coating biocompatible polymers onto GO also affects the biodistribution, for instance, the intravenous injection of GO-PEG and GO-dextran (GO-DEX) accumulate in the reticuloendothelial system (RES), including the liver and spleen, without short-term toxicity [3179]. Moreover, the charge of plasma proteins and adsorption of GO by plasma proteins also affects the biodistribution [34].

The excretion and clearance of GFNs vary in different organs. In the lungs, observations indicated that NGO is drawn into and cleared by AMs, which might be eliminated from the sputum through mucociliary clearance or other ways [57], and 46.2 % of the intratracheally instilled FLG was excreted through the faeces 28 d after exposure [61]. In the liver, nanoparticles can be eliminated thorough the hepato-biliary pathway following the biliary duct into the duodenum [80]. In addition, PEGylated GNS that mainly accumulates in the liver and spleen can be gradually cleared, likely by both renal and faecal excretion. As recently reviewed, GO sheets larger than 200 nm are trapped by splenic physical filtration, but small sizes (approximately 8 nm) can penetrate the renal tubules into the urine and be rapidly removed without obvious toxicity [81]. The excretion paths of GFNs have not yet been clearly explained, but renal and faecal routes appear to be the main elimination routes for graphene.

Recently, the distribution and excretion/toxicity strategy has become an important part of nano-toxicological studies. To date, several controversial results regarding the distribution and excretion of graphene in vivo have been reported in several papers, and a systematic evaluation of the toxicokinetics of GFNs is still needed. The metabolism and excretion of nanomaterials are long-period processes, however, the recent studies of GFNs had been limited to short-term toxicological assessments, and the long-term accumulation and toxicity of GFNs on different tissues remain unknown. Therefore, long-term studies on the deposition and excretion of GFNs need to be performed using different cells and animals to ensure the materials’ biosafety before utilization in human biomedical applications.

Uptake and location of GFNs in cells

The uptake and location of GFNs have also been observed to exert different effects in different cell lines. Graphene is taken up into cells via various routes [8283]. Basically, the physicochemical parameters such as the size, shape, coating, charge, hydrodynamic diameter, isoelectric point, and pH gradient are important to allow GO to pass through the cell membrane [84]. As stated previously, nanoparticles with diameters <100 nm can enter cells, and those with diameters <40 nm can enter the nucleus [85]. For example, GQDs possibly penetrate cell membranes directly, rather than through energy-dependent pathways [8687]. Larger protein-coated graphene oxide nanoparticles (PCGO) (~1 μm) enter cells mainly through phagocytosis, and smaller PCGO nanoparticles (~500 nm) enter cells primarily through clathrin-mediated endocytosis [88]. GO sheets could adhere and wrap around the cell membrane, insert in the lipid bilayer or be internalized into the cell as a consequence of interactions with cells [89]. Similarly, PEGylated reduced graphene oxide (PrGO) and rGO were shown to adhere onto the lipid bilayer cell membrane prominently due to the interaction of hydrophobic, unmodified graphitic domains with the cell membrane [9091]. Consequently, it was suggested that prolonged exposure to or a high concentration of graphene induces physical or biological damage to the cell membrane, along with destabilization of actin filaments and the cytoskeleton [92].

Current data demonstrates that GO sheets interact with the plasma membrane and are phagocytosed by macrophages. Three major receptors on macrophages take part in the phagocytosis of GNS: the Fcg receptor (FcgR), mannose receptor (MR), and complement receptor (CR). Furthermore, FcgR is a key receptor in the mediated phagocytic pathway [909394]. The protein corona of GO promotes the recognition by macrophage receptors, especially the IgG contained within the protein corona. Macrophages were observed to undergo prodigious morphological changes upon contact with GO [34]. After internalization, graphene accumulated in the cell cytoplasm, perinuclear space, and nucleus, which induced cytotoxicity in murine macrophages by increasing intracellular ROS through depletion of the mitochondrial membrane potential and by triggering apoptosis through activation of the mitochondrial pathway [83]. The possible interactions and accumulation sites of GFNs are summarized in Fig. 1.

figure1
Fig. 1

Toxicity of GFNs in organs

The toxicity and biocompatibility of GFNs has been observed and assessed through theoretical and animal model studies. At present, there are a mass of data demonstrating the toxicity of GFNs in different organs or systems in animals, so that it is hard to list all the data in this review. Thus we summarized a certain number literature and chose some in vivo toxicological studies of GFNs listed in Table 1.Table 1 Toxicity of GFNs in organsFull size table

Toxicity in internal organs

GO can result in acute inflammation response and chronic injury by interfering with the normal physiological functions of important organs [3281]. Oral gavage experiments did not show detectable absorption of GO through the gastrointestinal tract [95]. Interesting, a low dose of GO caused serious damage to the gastrointestinal tract after maternal mice drank a GO suspension rather than a high-dose of GO because a low dose of GO without agglomeration can easily attach to the gastrointestinal surface and cause destruction through its abundant sharp edges [53]. GFNs caused inflammation and remained in the lung on day 90 after a single intratracheal instillation, and even translocated to lung lymph nodes by a nose-only inhalation [9697]. A high dose of GO that forms aggregations can block pulmonary blood vessels and result in dyspnea [5098], 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. The use of Tween 80 to disperse FLG or a pluronic surfactant to disperse graphene was suggested to reduce the likelihood of lung fibrosis formation in cells or mice, whereas lung fibrosis was observed when graphene was suspended with bovine serum albumin (BSA) [100]. In addition, radioactive isotopes can be delivered into the lungs, accompanied by a depth distribution of 125I-NGO in the lungs, and the isotopes might deposit there and result in mutations and cancers [30]. However, recent publications claimed no obvious pathological changes in mice exposed to low dosages of GO and functionalized graphene by intravenous injection, including aminated GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and GO-PEG; only GO-PEG and GO-PAA induced less toxicity than pristine GO in vivo [317989]. So the functional groups of GFNs and the working concentration or aggregate state largely influence the toxicity of GFNs. Recently, the ways to modify the functional group of GFNs, decrease the working concentration or change the aggregate condition are usually used to decrease the toxicity of GFNs.

Toxicity in the central nervous system

Graphene has largely benefited neurosurgery with the application of drug/gene delivery for brain tumour treatment, intracranial and spinal biocompatible devices, biosensing and bioimaging techniques. Studies regarding the potentialities or risks of graphene in the brain have emerged. 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 [101]. The recent researches of GFNs in the central nervous system are mostly involved in the application rather than the toxicity. The data of the toxic study on GFNs is underway.

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. Although no obvious malformation or mortality was observed in exposed zebrafish embryos [102], 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].

The GFNs exert different toxicological effects on male or female reproductive system. Data showed that GO exerted very low or nearly no toxic effects on male reproduction even at a high dose via intra-abdominal injection [66]. Additionally, rGO did not change the serum estrogen levels of non-pregnant female mice. The condition is different in the female mouse: mouse dams could give birth to healthy offspring after rGO injection before mating or during early gestation, and only a few abnormal foetuses were present among the rGO-injected dam litters. However, the pregnant mice had abortions at all dose, and most pregnant mice died when the 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]. Though the findings indicate that GFNs are potentially harmful to development, but data on reproductive and developmental toxicity are still deficient. Studies of the influence of GFNs on male and female reproduction and development are still required to elucidate the underlying toxicity mechanism.

Influence of haemocompatibility

GO release into the blood is ineluctable. The haemocompatibility of GO was found to be dependent on the functional coating and the exposure conditions. GO with submicron size resulted in the greatest haemolytic activity, while aggregated graphene induced the lowest haemolytic reaction. Pristine graphene and GO demonstrated haemolytic effect up to 75 μg/mL [104]. GO-polyethylenimine (GO-PEI) exhibited notable toxicity by binding to HSA, even at 1.6 μg/mL [105]. Carboxylated graphene oxide (GO-COOH) showed significant cytotoxicity toward T lymphocytes at concentrations above 50 μg/mL and had good biocompatibility below 25 μg/mL, whereas GO-chitosan nearly inhibited haemolytic activity [106]. Until now, the corresponding risk of haemocompatibility has remained largely unknown.

In conclusion, the lung injury induced by GFNs has been studied in several studies, the results of which 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 [107109]. 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 [110112]. 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]. However, GQDs did not pose significant toxicity to human breast cancer cell lines (at a dose of 50 μg/mL) or human neural stem cells (at a dose of 250 μg/mL) [114115]. 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 [82116].

Graphene can increase cell viability [117] or cause cell death [118] depending on the cell line, type of graphene material and the doseage. GO cytotoxicity was observed in human fibroblasts and lung epithelial cells at concentrations above 20 μg/mL after 24 h, but minimal toxicity was found in A549 cells at concentrations higher than 50 μg/mL [119]. The biological responses induced by GO such as ROS, malondialdehyde (MDA), and LDH increased, whereas superoxide dismutase (SOD) decreased dose-dependently in HeLa cells [120]. However, GO-molecular beacon (GO-MB) showed low cytotoxicity even at 20 μg/mL in HeLa cells [121]. GO decreased the viability of A549 cells, while the same concentration and time of exposure increased the cell viability of CaCo2 colorectal carcinoma cells [122]. Another study reported that GO dramatically enhanced the differentiation of SH-SY5Y, accompanied by increasing neurite length and the expression of neuronal marker MAP2 at low concentrations but that GO suppressed the viability of SH-SY5Y cells at high doses (≥80 mg/mL) [123]. Functionalized coatings on GO, such as GO-PEG [124] and GO-chitosan [125], can profoundly attenuate the particles’ cytotoxicity by inhibiting the interactions between cells.

The toxicity of GFNs in vitro is summarized in Table 2. Data on the cytotoxicity of graphene nanomaterials are contrasting, and varying characteristics influence the results. The mechanisms and influencing factors of toxicity need to be elucidated in detail.Table 2 Toxicity of GFNs in cell modelsFull size table

Origins of GFNs toxicity

Reportedly, the characteristics of graphene, including its concentration, lateral dimension, surface structure, functional groups, purity and protein corona, strongly influence its toxicity in biological systems [27104126129].

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 [130134]. 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. Moreover, an in vitro study reported that 20 μg/mL GO nanosheets exhibited no cytotoxicity in A549 within 2 h of incubation, but a higher concentration (85 μg/mL) decreased the cell viability to 50 % within 24 h [136137]. Lü et al. also demonstrated that GO had no obvious cytotoxicity at low concentrations for 96 h in a human neuroblastoma SH-SY5Y cell line, but the viability of cells sharply decreased to 20 % after treatment with 100 mg/mL GO for 96 h of incubation [123]. The results in HeLa cells, NIH-3 T3 cells, and breast cancer cells (SKBR3, MCF7) treated with graphene nanoribbons also showed a dose- (10–400 mg/ml) and time-dependent (12–48 h) decrease in cell viability [138]. Increasing concentrations of GO entered the lysosomes, mitochondria, endoplasm, and cell nucleus [119]. Several data indicated that rGO caused apoptosis-mediated cell death at a lower dose and early time point but that necrosis was prevalent with the increase in time/dose [110135].

Lateral dimension

Nanoparticles with sizes <100 nm can enter the cell, <40 nm can enter nucleus, and smaller than <35 nm can cross the blood brain barrier [85]. One study showed that GO (588, 556, 148 nm) did not enter A549 cells and had no obvious cytotoxicity [112]. When the diameter of graphene is between 100 ~ 500 nm, the smallest size may cause the most severe toxicity, and when the diameter is below 40 nm, the smallest sizes may be 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. However, rGO sheets with diameters of 3.8 ± 0.4 nm exhibited no notable genotoxicity in hMSCs even at a high dose of 100 mg/mL after 24 h [118].

In an in vivo study, s-GO (100–500 nm) preferentially accumulated in the liver, whereas l-GO (1–5 μm) was mainly located in the lungs because l-GO formed larger GO-protein complexes that were filtered out by the pulmonary capillary vessels after intravenously injection [31]. Given the relative lateral sizes (205.8 nm, 146.8 nm and 33.78 nm) of the three GO nanosheets at the same concentration, smaller GO experiences much greater uptake than larger GO in Hela cells [139]. The high uptake of s-GO changed in the microenvironment of cells and consequently induced the greatest viability loss and most serious oxidative stress among three sizes of GO samples [119]. As a result, one study delineated that GO size-dependently induced the M1 polarization of macrophages and pro-inflammatory responses in vitro and in vivo. Larger GO showed stronger adsorption onto the plasma membrane with less phagocytosis, eliciting robust interactions with TLRs and activating NF-κB pathways, compared to smaller GO sheets, which were more likely taken up by cells [94]. To further uncover the detailed mechanism underlying these effects, more studies are needed to illustrate the vital mechanism of the lateral size of graphene materials.

Surface structure

GFNs possess widely varying surface chemistries. For example, the pristine graphene surface is hydrophobic, GO surface is partially hydrophobic with carboxylate groups [140142], and rGO has intermediate hydrophilicity [143]. GFNs were observed to disrupt the function and structure of cell membranes and proteins probably by exceptionally strong molecular interactions with cells [291]. For instance, rGO bonded to cell membranes, stimulated receptors and activated mitochondrial pathways, inducing apoptosis [110111144]. Limited evidence showed that GO is smaller and less toxic than rGO because of the high oxygen content, smoother edges, and hydrophilic properties of the former species [104145146]. Because of the different surface oxidation states of GO and rGO, GO possessing distinct hydrophilicity might be internalized and taken up by HepG2 cells easily. On the contrary, rGO with evident hydrophobicity, could be adsorbed and aggregated at cell surfaces without (or with lower) uptake [110]. Due to strong π-π stacking interactions, graphene is highly capability of breaking many residues of the protein, particularly the aromatic ones, such as the villin headpiece (HP), F10, W23, and F35. The protein’s secondary and tertiary structures are largely lying on the graphene surface, disrupting the structure and function of the protein [41] (Fig. 2). In addition, GO can insert between the base pairs of double-stranded DNA and disturb the flow of genetic information at the molecular level, which might be one of the main causes of the mutagenic effect of GO [7112146147].

figure2
Fig. 2

Charge

A number of studies have highlighted the importance of the GO surface charge because of its ability to affect the internalization and uptake mechanism of cells [148150]. GO internalization was negligible in non-phagocytes, which was likely due to the strong electrostatic repulsion between the negatively charged GO and the cell surface [34]. However, others have suggested that negatively charged nanoparticles can be internalized into non-phagocytic cells by binding to available cationic sites on the cell surface and be taken up by scavenger receptors [110146150]. GO/GS particles reportedly cause morphological changes and significant lysis, leading to high haemolysis in red blood cells (RBCs). RBC membrane disruption is probably attributed to the strong electrostatic interactions between the negatively charged oxygen groups on the GO/GS surface and positively charged phosphatidylcholine lipids on the RBC outer membrane [106].

Functionalization

Studies confirmed that functionalization with PEG [52], PEGylated poly-L-lysine (PLL) [151], poly(ε-caprolactone) [152], polyvinyl alcohol [3], Pluronic [153], amine [98], carboxyl, and dextran [79] groups largely decreases the toxicity and improves the biocompatibility of graphene. In vivo results revealed that only mild chronic inflammation emerged after the subcutaneous injection of GO-Pluronic hydrogel and no noticeable short-term toxicity was tested after the intravenous injection of GO-DEX [79154]. PEGylated GS did not induce appreciable toxicity in mice exposed to 20 mg/kg for 3 months, as evaluated by blood biochemistry and histological examinations, and showed relatively low retention in the RES [52155]. Coating GO with chitosan almost eliminated the haemolytic activity in blood [39]. Moreover, the PEG coating effectively alleviated GO-induced acute tissue injuries; decreased GO aggregation and retention in the liver, lungs, and spleen; and promoted the clearance of GO [81], GO-DEX [79], and fluorinated graphene oxide (FGO) [156].

In vitro, several cell function assays showed clear evidence that the surface functionalization of pristine graphene or GO was critical for reducing the strong toxicity effects [91]. PEG-GO, PEI-GO and LA-PEG-GO damaged human lung fibroblast cells less than GO [148]. PEG-GO exhibited no cytotoxicity toward several cell cultures, such as glioblastoma cells (U87MG), breast cancer cells (MCF-7), human ovarian carcinoma cells (OVCAR-3), colon cancer cells (HCT-116), and lymphoblastoid cells (RAJI), at concentrations up to 100 μg/mL [119157158]. GQDs-PEG exhibited very low or no toxicity against lung and cervical cancer cells even at very high concentrations (200 μg/mL) [159]. However, as a non-biodegradable material with great potential for cellular internalization, further investigation is needed to assess the possible long-term adverse effects of functionalized graphene.

Aggregations and sedimentation

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

Impurities

Nanomaterial purity is an important consideration because residual, contaminating metals may be responsible for the observed toxicity, rather than the nanomaterial itself, which has resulted in conflicting data on GFNs cytotoxicity [35160]. Traditionally prepared GO often contains high levels of Mn2+ and Fe2+, which are highly mutagenic to cells. The nonspecific release of these ions from traditionally prepared GO might lead to unusually high levels of cytotoxicity and DNA fracturing [39]. In particular, Peng et al. [161] produced high-purity GO containing only 0.025 ppm Mn2+ and 0.13 ppm Fe2+, and Hanene et al. [162] invented a new method to prepare high-purity, single-layer GO sheets with good aqueous dispersibility and colloidal stability. GO produced by these new methods did not induce significant cytotoxic responses (at exposure doses up to 100 μg/mL) in vitro, and no obvious inflammatory response or granuloma formation (exposure doses up to 50 μg/animal) were observed in vivo. Therefore, the purity of GFNs deserves attention and is a vital step towards the determination of GFNs involved in bioapplications.

Protein corona effect

Because of the high free surface charge, nanomaterials can easily form “coronas” with proteins in biological systems [163164]. The protein corona is suggested to affect the circulation, distribution, clearance and toxicity of nanoparticles. Several papers reported that GO forms GO-protein coronas with adsorbed plasma proteins in serum and these GO-protein coronas play an important role in deciding the fate of the GO biokinetic behaviour in vivo. Such GO-protein coronas can regulate the adhesion of GO to endothelial and immune cells through both specific and nonspecific interactions [165]. Basically, immunoglobulin G and complement proteins in the protein corona help to reorganize nanoparticles in immune cells, causing the particles to be engulfed by the RES, and IgG-coated GO was taken up by either specific or nonspecific interactions with cell membrane receptors [31165]. However, another study found that GO could not adhere to mucosal epithelial cells directly in the intestinal tract after the filial mice drank an aqueous GO solution because abundant proteins in the milk had adsorbed on the surface of the GO and thus inhibited their direct interaction with the mucosal epithelial cells [53]. Protein corona mitigated the cytotoxicity of GO by limiting its physical interaction with the cell membrane and reducing the cellular morphological damage in HeLa, THP-1 and A549 cells [166168]. The cytotoxic effect was largely reduced when GO was pre-coated with FBS and incubated with cells; nearly ∼ 90 % survival was observed with 100 μg/mL FBS-coated GO and 100 % survival with 20 μg/mL FBS-coated GO. Similar trends were observed for GO covered by BSA [166169]. Consistently, additional serum could neutralize the toxicity of pristine GO in J774.A1 cells at a dose of 4 μg/mL, which lead to a decrease in cell number of 52.5 % compared to untreated cells [89].

After reviewing many studies, it can be concluded that the toxicity of graphene is influenced by multiple factors. Those factors combined to largely change the toxicity of GFNs in many cases. Scientific studies often need the clear identification of cause and effect, which should keep only one factor different at a time, so that the effect of that single factor can be determined. But in some papers, several factors influencing GFNs toxicity were studied at the same time, which led to confused results.

Possible toxicity mechanisms of GFNs

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

figure3
Fig. 3

Physical destruction

Graphene is a unique nanomaterial compared with other spherical or one-dimensional nanoparticles due to its two-dimensional structure with sp2-carbons. The physical interaction of graphene nanoparticles with cell membranes is one of the major causes of graphene cytotoxicity [7170171]. Graphene has high capability to bind with the α-helical structures of peptides because of its favourable surface curvature [172]. At concentration above 75 μg/mL, pristine graphene largely adhered to the surfaces of RAW 264.7 cells and resulted in abnormal stretching of the cell membrane [104]. The strong hydrophobic interactions of GFNs with the cell membrane lead to the morphological extension of F-actin filopodial and cytoskeletal dysfunction. Furthermore, the sharpened edges of GNS may act as ‘blades’, inserting and cutting through bacterial cell membranes [173]. Moreover, GO also damaged the outer membrane of E. coli bacteria directly, resulting in the release of intracellular components [173]. However, TEM imaging revealed that pre-coating GO with FBS eliminated the destruction of cell membranes [166].

ROS production leading to oxidative stress

Oxidative stress arises when increasing levels of ROS overwhelm the activity of antioxidant enzymes, including catalase, SOD, or glutathione peroxidase (GSH-PX) [174]. ROS act as second messengers in many intracellular signalling cascades and lead to cellular macromolecular damage, such as membrane lipid breakdown, DNA fragmentation, protein denaturation and mitochondrial dysfunction, which greatly influence cell metabolism and signalling [175177]. The interactions of GO with cells can lead to excessive ROS generation, which is the first step in the mechanisms of carcinogenesis, ageing, and mutagenesis [83122]. Oxidative stress had a significant role in GO-induced acute lung injury [30], and the inflammatory responses caused by oxidative stress often emerged upon exposure to GFNs [133177178]. The activity of SOD and GSH-PX decreased after exposed to GO in a time- and dosage-dependent manner [82106119]. Similarly, oxidative stress was the key cause of apoptosis and DNA damage after HLF cells were exposed to GO [148]. Both the mitogen-activated protein kinase (MAPK) (JNK, ERK and p38) and TGF-beta-related signalling pathways were triggered by ROS generation in pristine graphene-treated cells, accompanied by the activation of Bim and Bax, which are two pro-apoptotic members of the Bcl-2 protein family. As a result, caspase-3 and its downstream effector proteins such as PARP were activated, and apoptosis was initiated [83179]. Detailed information regarding the MAPK-, TGF-β- and TNF-α-related signalling pathways, which induce inflammation, apoptosis and necrosis, are summarized in Fig. 4.

figure4
Fig. 4

Mitochondrial damage

Mitochondria are energy production centres involved in various signalling pathways in cells and are also a key point of apoptotic regulation [83]. After exposure to GO and carboxyl graphene (GXYG), the mitochondrial membrane was depolarized, and the amount of mitochondria decreased in HepG2 cells [180]. Exposure to GFNs resulted in significantly increased coupled and uncoupled mitochondrial oxygen consumption, dissipation of the mitochondrial membrane potential, and eventual triggering of apoptosis by activating the mitochondrial pathway [181]. For instance, GO increased the activity of mitochondrial electron transport complexes I/III and the supply of electrons to site I/II of the electron transport chain, accelerating the generation of ROS during mitochondrial respiration in MHS cells [99]. The formation of •OH mediated by GO and the cytochrome-c/H2O2 electron-transfer system could enhance oxidative and thermal stress to impair the mitochondrial respiration system and eventually result in dramatic toxicity [151]. Additionally, the oxygen moieties on GO might accept electrons from cellular redox proteins, supporting the redox cycling of cytochrome c and electron transport proteins, and cytochromes MtrA, MtrB, and MtrC/OmcA might be involved in transferring electrons to GO [182]. Therefore, except for the plasma membrane damage and oxidative stress induction, GFNs can cause apoptosis and/or cell necrosis by direct influencing cell mitochondrial activity [183184].

DNA damage

Due to its small size, high surface area and surface charge, GO may possess significant genotoxic properties and cause severe DNA damage, for example, chromosomal fragmentation, DNA strand breakages, point mutations, and oxidative DNA adducts and alterations [87122185186]. Mutagenesis was observed in mice after intravenous injection of GO at a dose of 20 mg/kg compared with cyclophosphamide (50 mg/kg), a classic mutagen [112]. Even if GO cannot enter into the nucleus of a cell, it may still interact with DNA during mitosis when the nuclear membrane breaks down, which increases the opportunity for DNA aberrations [87147187188]. The π stacking interaction between the graphene carbon rings and the hydrophobic DNA base pairs can make a DNA segment ‘stand up’ or ‘lay on’ the surface of graphene with its helical axis perpendicular or parallel, respectively. The intermolecular forces severely deform the end base pairs of DNA, which potentially increases the genotoxicity [189]. GO may also induce chromosomal fragmentation, DNA adducts and point mutations by promoting oxidative stress or triggering inflammation through the activation of intracellular signalling pathways such as MAPK, TGF-β and NF-κB [110112146]. Graphene and rGO can also elevate the expression of p53, Rad51, and MOGG1-1, which reflect chromosomal damage, and decrease the expression of CDK2 and CDK4 by arresting the cell cycle transition from the G1 to the S phase in various cell lines [112]. DNA damage can not only initiate cancer development but also possibly threaten the health of the next generation if the mutagenic potential of GO arises in reproductive cells, which impacts fertility and the health of offspring [112190].

Inflammatory response

GFNs can cause a significant inflammatory response including inflammatory cell infiltration, pulmonary edema and granuloma formation at high doses via intratracheally instillation or intravenous administration [3049]. Platelets are the important components in clot formation to attack pathogens and particulate matter during the inflammatory response, and GO could directly activate platelet-rich thrombi formation to occlude lung vessels after intravenous injection [98191]. A strong inflammatory response was induced by subcutaneously injection with GO for 21 days, along with the secretion of key cytokines, including IL-6, IL-12, TNF-α, MCP-1, and IFN-g [34192]. GFNs can trigger an inflammatory response and tissue injury by releasing cytokines and chemokines that lead to the recruitment of circulating monocytes and stimulating the secretion of Th1/Th2 cytokines and chemokines [124193]. Additionally, pristine graphene [193] and rGO [110] evoke an inflammatory response by binding to toll-like receptors (TLRs) and activating the NF-κB signalling pathway in cells. The NF-κB signalling cascade is triggered by TLRs and pro-inflammatory cytokines such as IL-1 and TNF-α. Upon activation, NF-κB shifts from the cytoplasm to the nucleus, facilitating the binding of degrading IκB and acting as a transcription factor to synthesize numerous pro-inflammatory cytokines [194]. A schematic of the signalling pathway of TLR4 and TLR9 activated by GFNs is shown in Fig. 5.

figure5
Fig. 5

Apoptosis

Apoptosis is defined as the self-destruction of a cell regulated by genes through complicated programmes [83195]. GO and rGO caused apoptosis and inflammation in mice lungs after inhalation [99], and GFNs also had pro-apoptotic effects in cells [111113124196]. Additionally, graphene and GO physically damaged cell membranes [166], increased the permeabilization of the outer mitochondrial membrane and changed the mitochondrial membrane potential; the increased ROS triggered the MAPK and TGF-β signalling pathways and activated caspase-3 via mitochondrial-dependent apoptotic cascades, prompting the execution of apoptosis [8399]. Similarly, rGO caused apoptosis at a low dose and an early time point, triggered by the death-receptor and canonical mitochondrial pathway [110]. Another study showed three different apoptosis pathways by GFNs: GO led to ROS-dependent apoptosis through direct interaction with protein receptors and subsequent activation of the B-cell lymphoma-2 (Bcl-2) pathway; GO-COOH transmitted a passive apoptosis signal to nuclear DNA by binding to protein receptors and activating a ROS-independent pathway; However, GO-PEI severely damaged the membranes of T lymphocytes to trigger apoptosis [105197].

Autophagy

Autophagy is the process of self-degradation of cellular components and recently recognized as non-apoptotic cell death [198200]. Autophagy activation requires autophagosome formation containing Beclin 1, multiple autophagy-related proteins (ATG), microtubule-associated protein light chain 3 (LC3) and p62 [201]. Autophagosome accumulation is associated with exposure to various nanoparticles [202205], and autophagy can remove extracellular organisms and destruct the organisms in the cytosol [206]. GO and GQDs was shown to induce autophagosome accumulation and the conversion of LC3-I to LC3-II; inhibit the degradation of the autophagic substrate p62 protein [207208]. Furthermore, GO can simultaneously trigger TLR4 and TLR9 responses in macrophages [34192] and colon cancer cells CT26 [206]. The autophagy pathway is linked to phagocytosis by TLR signalling in macrophages [206209].

Necrosis

Necrosis is an alternate form of cell death induced by inflammatory responses or cellular injury. The exposure of cells to pristine graphene causes apoptosis and necrosis at high doses (50 mg/mL) [83]. Reportedly, LDH leakage and the opening of the mitochondrial permeability transition pore, induced by elevated level of cytoplasmic Ca2+, lead to apoptosis/necrosis [210]. GO treatment was revealed to induce macrophagic necrosis by activating TLR4 signalling and subsequently partly triggering autocrine TNF-α production [93]. GO combined with CDDP (GO/CDDP) triggered necrosis by decreasing RIP1 and increasing RIP3 proteins, accompanied with the release of high mobility group B1 (HMGB1) into the cytosol from the nucleus and out of CT26 cells [205211212].

Epigenetic changes

Epigenetics involve DNA methylation, genomic imprinting, maternal effects, gene silencing, and RNA editing [213215]. DNA methylation, which is one of the best-studied epigenetic modifications, includes phosphorylation, ubiquitination, and ATP-ribosylation and can lead to chromatin remodelling [197216217]. A recently paper reported that SL-GO/FL-GO exposure resulted in global DNA hypermethylation through upregulating DNMT3B and MBD1 genes; GNP treatment caused hypomethylation by decreasing the expression of DNMT3B and MBD1 genes [216]. GO could activate the miRNA-360 regulation pathway to suppress the DNA damage-apoptosis signalling cascade by affecting the component of CEP-1 [218]. Taken together, these data suggest that GFNs could cause subtle changes in gene expression programming by modulating epigenetic changes. However, studies of GFNs-induced epigenetic changes are few, and the epigenetic mechanism caused by GFNs exposure is not fully understood.

To conclude, many studies have discussed representative mechanisms of GFNs toxicity involving four signalling pathways: TLRs, TGF-β, TNF-α and MAPKs. These four signalling pathways are correlative and cross-modulatory, making the inflammatory response, autophagy, apoptosis and other mechanisms independent and yet connected to each other. Additionally, oxidative stress appears to play the most important role in activating these signalling pathways. It has been reported that there are intersections of apoptosis, autophagy and necrosis in the studies of other nanomaterials toxicity, they inhibit or promote mutually in some conditions. However, the signalling pathways of GFNs toxicity investigated in papers to date are only a small part of an intricate web, and the network of signalling pathways needs to be explored in detail in the future.

Data gaps and future studies

Currently, the literature is insufficient to draw conclusions about the potential hazards of GFNs. Two opposite opinions have begun to emerge: some researchers suggested that graphene materials are biocompatible in a number of studies focused on biomedical applications [119154162219], and other studies reported adverse biological responses and cytotoxicity [32118135138192]. These inconsistent results might have been caused by several factors, including the different research groups, various cellular or animal models, and varying physicochemical characterizations of GFNs. When GFNs are explored for in vivo applications in the human body or some other biomedical applications, biocompatibility must be considered, and more detailed and accurate studies of GFNs toxicity are needed.

First, detailed physicochemical characterization is imperative in all future studies of GFNs toxicity. In the experiments, feature descriptions of GFNs should include their size, morphology, surface area, charge, surface modifications, purity, and agglomeration [88141148162]. Because these physicochemical factors largely influence the toxicity and biocompatibility of GFNs, single-factor experimental designs and the exclusion of other interfering factors should be considered. Details of the fabrication process should also be provided because the formed oxidative debris could largely alter the surface structure of graphene and GO during functionalization [151]. Importantly, a single, universal method needs to be established in graphene technology, which will allow for better comparison of data from different studies or different laboratories.

Second, different observational criteria, parameters and selection of experimental methods might induce large inter-laboratory variations [220221]. For example, the MTT assay always fails to accurately predict graphene toxicity because the spontaneous reduction results in a false positive signal. Therefore, appropriate alternative assessments should be utilized, such as the water-soluble tetrazolium salt reagent (WST-8), ROS assay, and trypan blue exclusion test [106222]. Additionally, the comet assay often shows higher levels of DNA damage than the micronucleus assay because the former measures the repairable injury and the latter measures the gene damage that remains after cell division [159223]. Therefore, caution is required in choosing the most appropriate assay to evaluate the toxicity of graphene materials to avoid false-positive results.

Third, the selection of cell lines is of vital importance because cancer cell lines tend to be sensitive or resistant depending upon their genetic background. The same graphene nanoparticles can cause different reactions depending on their various cells origins. Suitable cell lines with good stability must be used to avoid false positive or negative results. Primary cells derived from humans or animals can better simulate the health conditions of humans. A large amount of primary cells have been utilized to test the toxicity of other nanomaterials [224228], but the culturing of primary cells is extremely rare in the experiments with GFNs to date [210229]. Various cell experiments combined with primary cells should be performed to comprehensively evaluate the physicochemical properties and toxicity of GFNs.

Fourth, the administration route of GFNs plays a very important role in toxicity studies, and different delivery methods will result in different toxicological reactions [3253]. Thus, the route and period of exposure should be carefully chosen according to the aim of the study. Nasal drug delivery is often used to study the neurotoxicity of nanomaterials [230231], but this administration method has rarely been applied in the testing of GFNs toxicity. Toxicological studies of GFNs in the nervous system are rare, and the mechanism is unclear and needs to be studied further in the future. Recent toxicokinetic studies involving the absorption, distribution, metabolism, accumulation, and excretion of GFNs through different exposure routes have yielded some results but are far from sufficient to clarify the internal complex mechanisms. For instance, further studies are needed to understand the specific molecular mechanisms of GFNs passing through the physiological barriers and the amount of accumulation or the excretion period of GFNs in tissues. In addition, given the increased exposure of humans to GFNs, the assessment of systemic toxicity in the human body is indispensable in future studies.

Fifth, another important issue requiring attention is the long-term fate of GFNs after entering the body or being taken up by cells. Most recent studies have consisted of short-term toxicity assessments [89232], and long-term toxic injury has not received much attention since the widespread application of GFNs in 2008. Moreover, a functionalized graphene surface can improve its biocompatibility, but the long-term stability of the surface coatings should be considered [233]. If the surface coatings eventually break down, their toxicity may be significantly different from the short-term exposure results. Extended studies are needed to determine if longer treatment times influence the nanotoxic potential of GFNs.

Sixth, more specific signalling pathways in the mechanism of GFNs toxicity need to be discovered and elucidated. Currently, several typical toxicity mechanisms of GFNs have been illustrated and widely accepted, such as oxidative stress, apoptosis, and autophagy. However, these mechanisms have only been described in general terms, and the specific signalling pathways within these mechanisms need to be investigated in detail. The signalling pathways involved in the toxicity of other nanomaterials may also be relevant to the study of GFNs. Therefore, more signalling pathways should be detected in future research. For instance, nano-epigenetics has been considered in numerous studies of nanomaterials, which is also helpful in assessing the limited toxicity and side effects of GFNs. Recent studies have shown that GFNs could cause epigenetic and genomic changes that might stimulate physical toxicity and carcinogenicity [234]. GFNs have high surface areas, smooth continuous surfaces and bio-persistence, similar to the properties of tumorigenic solid-state implants. It is unknown whether GFNs have the potential to induce foreign body sarcomas, and definitive studies of tumour potentialities or risks of graphene should therefore be conducted as soon as possible.

Conclusions

In the past few years, GFNs have been widely utilized in a wide range of technological and biomedical fields. Currently, most experiments have focused on the toxicity of GFNs in the lungs and livers. Therefore, studies of brain injury or neurotoxicity deserve more attention in the future. Many experiments have shown that GFNs have toxic side effects in many biological applications, but the in-depth study of toxicity mechanisms is urgently needed. In addition, contrasting results regarding the toxicity of GFNs need to be addressed by effective experimental methods and systematic studies. This review provides an overview of the toxicity of GFNs by summarizing the toxicokinetics, toxicity mechanisms and influencing factors and aimed to provide information to facilitate thorough research on the in vitro and in vivo haemo- and biocompatibility of GFNs in the future. This review will help address safety concerns before the clinical and therapeutic applications of GFNs, which will be important for further development of GFNs in biological applications.

Abbreviations

AMs:

Alveolar macrophagesBBB:

Blood-brain barrierBEB:

Blood-epididymis barriersBTB:

Blood-testis barrierCR:

Complement receptorFcgR:

Fcg receptorFLG:

Few-layer grapheneGFNs:

Graphene family nanomaterialsGNS:

Graphene nanosheetsGO:

Graphene oxideGO-COOH:

Carboxylated graphene oxideGO-DEX:

GO-dextranGO-MB:

GO-molecular beaconGO-NH2:

Aminated GOGO-PAA:

Poly(acrylic acid)-functionalized GOGO-PAM:

Poly(acrylamide)-functionalized GOGO-PEG:

PEGylated GO derivativesGO-PEI:

GO-polyethylenimineGQDs:

Graphene quantum dotsGSH-PX:

Glutathione peroxidaseGXVG:

Carboxyl grapheneLDH:

Lactate and dehydrogenaseMALDI:

Matrix-assisted laser desorption/ionizationMAPKs:

Mitogen-activated protein kinaseMDA:

MalondialdehydeMØ:

MacrophageMR:

Mannose receptorMSI:

Mass spectrometry imagingPC12 cells:

Rat pheochromocytoma cellsPCGO:

Protein-coated graphene oxide nanoparticlesPrGO:

PEGylated reduced graphene oxideRES:

Reticuloendothelial systemrGO:

Reduced graphene oxideROS:

Reactive oxygen speciesSOD:

Superoxide dismutaseTLRs:

Toll-like receptor

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Acknowledgements

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Funding

This review was supported by the National Natural Science Foundation of China (81550011, 51172283, 81400557), Natural Science Foundation of Guangdong Province (2015A030313299) and Guangdong Provincial Medical Research Foundation (A2016360).

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All authors contributed to the design and concept of this article. LO drafted the manuscript. BS and JL critically revised the manuscript. All authors read and approved the final manuscript.

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  1. Nanfang Hospital, Southern Medical University, Guangzhou, 510515, ChinaBin Song, Huimin Liang, Jia Liu, Xiaoli Feng & Longquan Shao
  2. The First Affiliated Hospital of Jinan University, Guangzhou, ChinaLingling Ou & Ting Sun
  3. The General Hospital of People’s Liberation Army, Beijing, ChinaBin Deng

Corresponding author

Correspondence to Longquan Shao.

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Ou, L., Song, B., Liang, H. et al. Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms. Part Fibre Toxicol 13, 57 (2016). https://doi.org/10.1186/s12989-016-0168-y

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Keywords

  • Graphene-family nanomaterials
  • Toxicity
  • Toxicokinetics
  • Mechanisms
  • Physicochemical properties
  • Future prospects

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https://particleandfibretoxicology.biomedcentral.com/articles/10.1186/s12989-016-0168-y

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Graphene oxide-incorporated hydrogels for biomedical applications. https://www.nature.com/articles/s41428-020-0350-9

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frequency, frequency, frequency…

Like: Location, location, location… (I looked up the etymology and the definition has what might be the first indication of the idea of a standing wave in it…. subtle, but there)

Frequency is a note, a wave amplitude – size, which predicates its speed or space between the waves…

This is worth a reprint in the light of what everybody is now calling Trans-medium qualities, propulsion or ability. With a very slight, out of harmonic change of frequency, what’s the odds that something will not be perceived and become ‘invisible’? Or, that it would be able to move through various mediums without friction because it, it’s self, or a field of energy being generated around it was slightly off the harmonic it was surrounded by, or in? And how would this apply to ‘flying an ET ship’, telepathy and entanglement? To me, it all rolls back to frequency, regardless of which magnitude you look at.

____________________

Frequency Soup

We convert energy into reality. 

We take all orders of magnitude and somehow in our body translate (assign meaning to) all the jiggling and make sense information of it. Then we take that information and do stuff with it – we make ideas about it, we have feelings about it, we create something with it. 

We take/use energy and create reality with it.

I was recently shoved back into the 5G mess of information, so I began to try to see if I could understand the resonant frequencies of the smaller human structures, like DNA and cell walls, etc. (because, John Burroughs) I ran into the ‘Orders of Magnitude’ charts – which are basically big to small chart of all types of energy that we can measure, and it occurred to me that what I was looking at were frequencies stated in different octaves, layers and realms – dimensions. Or sheets if you like. In Harmonies

Which, brought me back around to consciousness and perception – and you know I’ve done some thinking about that lately in my articles – because it would be nice to figure out the base quanta (not only frequency but which order to measure) of telepathy to understand entanglement. And well, I came to the conclusion that we see reality the way we do because we are at the most basic level, putting it together that way out of the frequencies that we do perceive, by giving it meaning and then codifying the information with symbols (converting it into another frequency) that can be shared amongst ourselves.

Everything is a frequency a vibration. We arise out of that soup, and we are all immersed in it. Telepathy must be some frequency range that is similar enough to all of us that we are all stewing in simultaneously. ( See Cliff High’s nanobots: the precog of the universal subconscious mind of man) Now, whether we are aware of this frequency is another thing. The potential for being aware of it is there within each of us because we are similar enough to have a harmonic resonance due to our structure. And, because the idea wouldn’t already exist in our frequency soup if it didn’t have a universal harmonic.

Which, of course, led me to this current “We-are-so-close to something breaking wide open.” thought that has energized the UFO/disclosure community recently and the tipping point theory. That’s the theory that says if you get enough people sharing a reality, or realization, then the mass of that energy will push the rest of humanity over into it and one day everybody will just be aware of an idea like it was theirs all alone for ever – they will not even remember how they learned it because it will seem like a piece of the fabric of reality. (which actually at that point it is, because the frequency of the idea has been added to the communal soup we all float in al la hundredth monkey theory)

So: this UFO/Disclosure paradigm that we are all playing with, IMO, IS the veil we are trying to push through. Therefore, we can’t really, really, discover what is going on until we can stand OUTSIDE that paradigm and see what it is. – Which may be hard, because it’s part of the frequency soup. (Uhm, somebody put it there, more like a lot of somebodies) However, because we have this feeling of expectancy about it I am more than willing to entertain that more than a few people have figured it out, or we wouldn’t feel the expectancy. 

I also would be willing to bet its so far out of the norm, that the perception of it, like telepathy, may take some real practice and work, but is not impossible – or we wouldn’t be feeling it. Maybe it’s like riding a bike – weird to learn, but once you do you never forget.

However, perception implies an impingement upon the sensory capacities of the body. Perception is like noticing. So if you are noticing/perceiving something you aren’t actually touching, it would imply a certain level of entanglement of one or more energetic subsystem of the body. Those have electromagnetic resonances.

In physics, resonance is the tendency of a system to oscillate with greater amplitude at some frequencies than at others. Frequencies at which the response amplitude is a relative maximum are known as the system’s resonant frequencies, or resonance frequencies. At these frequencies, even small periodic driving forces can produce large amplitude oscillations, because the system stores vibrational energy.

Resonance occurs when a system is able to store and easily transfer energy between two or more different storage modes (such as kinetic energy and potential energy in the case of a pendulum). However, there are some losses from cycle to cycle, called damping. When damping is small, the resonant frequency is approximately equal to the natural frequency of the system, which is a frequency of unforced vibrations. Some systems have multiple, distinct, resonant frequencies.

Resonance phenomena occur with all types of vibrations or waves: there is mechanical resonance, acoustic resonance, electromagnetic resonance, nuclear magnetic resonance (NMR), electron spin resonance (ESR) and resonance of quantum wave functions. Resonant systems can be used to generate vibrations of a specific frequency (e.g., musical instruments), or pick out specific frequencies from a complex vibration containing many frequencies (e.g., filters).

By studying the way that waves interact with other waves, researchers have found that even low-powered oscillations can have enormous effects on standing waves, physical structures, and even the human brain. The principle which describes this particular wavelength interaction is known as resonance.

When you resonate with something, you are emitting a wave signature which is “in sync” with it. By applying a constant resonant frequency to a standing wave, you can intensify, reinforce, and prolong the standing frequency of that wave. Researchers posit that by applying these concepts of resonance to waves emitted by the brain, it is possible to induce altered brain states.

So what is actually entangling are wave frequencies. That would, IMO, mean perception IS conscious entanglement.

Which sent me to this gem: Biocentrism Theory

Biocentrism was first proposed in a 2007 article by Robert Lanza that appeared in “The American Scholar,” where the goal was to show how biology could build upon quantum physics. Two years later, Lanza published a book with astronomer and author Bob Berman entitled “Biocentrism: How Life and Consciousness Are the Keys to Understanding the True Nature of the Universe”, which expanded upon the ideas that Lanza wrote about in his essay for the “Scholar”.

Biocentrism argues that the primacy of consciousness features in the work of René Descartes, Immanuel Kant, Gottfried Leibniz, George Berkeley, Arthur Schopenhauer, and Henri Bergson. He sees this as supporting the central claim that what we call space and time are forms of animal sense perception, rather than external physical objects.

Robert Lanza argues that biocentrism offers insight into several major puzzles of science, including Heisenberg’s uncertainty principle, the double-slit experiment, and the fine tuning of the forces, constants, and laws that shape the universe as we perceive it. According to Robert Lanza and Bob Berman, “biocentrism offers a more promising way to bring together all of physics, as scientists have been trying to do since Einstein’s unsuccessful unified field theories of eight decades ago.”

Seven principles form the core of biocentrism: 

The first principle of biocentrism is based on the premise that what we observe is dependent on the observer, and says that what we perceive as reality is “a process that involves our consciousness.” 

The second and third principles state that “our external and internal perceptions are intertwined” and that the behavior of particles “is inextricably linked to the presence of an observer,” respectively. 

The fourth principle suggests that consciousness must exist and that without it “matter dwells in an undetermined state of probability.” 

The fifth principle points to the structure of the universe itself, and that the laws, forces, and constants of the universe appear to be fine-tuned for life. Finally, 

The sixth and seventh principles state that space and time are not objects or things, but rather tools of our animal understanding. Lanza says that we carry space and time around with us “like turtles with shells.”

Robert Lanza claims that biological observers actually create the arrow of time. In his papers on relativity (also published in Annalen der Physik), Einstein showed that time was relative to the observer; in their paper, Podolskiy and Lanza argue that quantum gravitational decoherence is too ineffective to guarantee the emergence of the arrow of time and the “quantum-to-classical” transition to happen at scales of physical interest. They argue that the emergence of the arrow of time is directly related to the way biological observers with memory functions process and remember information. They cite Robert Lanza’s American Scholar paper on biocentrism, stating that the “brainless” observer does not experience time and/or decoherence of any degrees of freedom.   From <http://www.robertlanzabiocentrism.com/biocentrism-wikipedia/>

When a property of an electron suddenly switches from possibility to reality, some physicists say its wave function has collapsed.

What accomplishes this collapse? Messing with it. Hitting it with a bit of light in order to take its picture. Just looking at it does the job. Experiments suggest that mere knowledge in the experimenter’s mind is sufficient to collapse a wave function and convert possibility to reality.    From <http://discovermagazine.com/2009/may/01-the-biocentric-universe-life-creates-time-space-cosmos>

But then I got frustrated and realized it is simpler. I just want to ‘feel’ my way through my own psyche in a less left brain, more right brain way because then I will get it really, instead of giving my left brain indigestion… I wanted to give my right brain wings.  

___________________

So, IMO, the only way to know the taste of a hamburger is eat a hamburger. It’s impossible to describe to someone without any referents. And here we are struggling to understand things in different orders of magnitude from our own experience, and not realizing that to simply detune a thing with an energetic shield makes it…vanish. What is it they say about 6 degrees of separation?

Wild

If we as adults have lost our link to the wild, then imagine how much more our children have lost. The wildness of spirit, of heart, of exploration. This morning I sat writing and the thoughts were about this transitioning of the human race we are all a part of. How our wildness is being bred out of us, trained out of us – especially our children. 

The world is an awful place right now, especially when you finally understand that there are people out there who drive society who are trying to kill the human race. If its apparent to you, it is apparent to the children, and they have far less defense against how that idea will operate in their hearts and minds. As an adult, looking at the patterns, I find it hard not to see that this current mess has been a plan in the works covertly since before WW1, but rather overtly since WW2. Its unmistakable. It is the culmination of generations of planning and strategizing.

When it gets this bad, we all slip into, if we are not careful, poor coping methods. And, let me ask, how are we to know we should be in coping mode when we are told there is nothing going on? We are involved in a sneaky, underhanded stealth war that doesn’t even have a name, that gets you from the inside out, instead of the outside in. 

Our inner sentinels – our dark and wild sides are clamoring for notice, attention and a voice. They are truth telling at a level that nobody wants to hear and, IMO, these inner voices will get louder in an attempt to be heard. We must listen. We must be willing to take up our wildness and stand in our strength and at every juncture where we are caged, boxed and shuttered, push back. If we don’t we will lose our very souls.

The world will be remade after this. The very basis of reality will change. This wonderful green planet, the loveliness I feel and sense and touch may still be here, but our ability to know it’s wildness, may never come back. I was hoping we’d make this looming transition with all that we are intact. But, will we? Will any part of us that is wild and good survive to make it through? Will any part of our wild unity with this earth that makes humans human come with us as we pass through the eye of the needle? STING was right, we are so very fragile. We are not quite aware of what we are sacrificing in this bid for survival. 

In another 100 years will the children even be able to understand those that came before? Those qualities, sensibilities are vanishing as I speak. Humans are vanishing, real wild humans, without any insane modifications, the ones that know unity with nature: the skies above me, the earth below me. Or will this primal awareness vanish as the people are changed from the inside out by those who would remake us in their image and kill our souls? I wonder what it will be like in the future and if the true soul of this world will even be remembered.

Fragile

Listen to me now, believe me later.

When I get stuck in a rut so deep that I can’t see out, sometimes I remember to listen to the wild women’s voices that got me through my first journey of winter. The journey that proves unequivocally whether you will live or die during the first winter of your heart. Whether you will have any say in how you will live the rest of your days and whether you can make it through to “Stand in your own danger”, as Clarissa Pinkola Estes says. 

To stand in your own danger means you have become dangerous to rigid ideas and societal programming and anything that would cage the magnificent honesty of your soul and its creative power. You radiate power and danger to all around you in your potential to upset apple carts and yell the truth by just standing still. To reach this strength you must, in your very first winter, learn to do what a new sapling does in order to survive a real winter. You must harden the outer layer of your heart a bit to protect it from the cold that would kill it to its very center so that in spring the living tissues can once again grow strong and tall. You must harden off the outer cells of your heart wood to survive your winter. And protect that which is nearest and dearest to your soul.

Then and only then can you stand in your own danger, aware of what is true and what is not, who is good and who is not and avoid the ruts ahead of you in your journey. If you are like me, sometimes you walk straight into that rut, but by now can mostly navigate your self out. Old, wise women know this and old wild women do too. They are two halves of the same ripe fruit. 

When you stand in your own danger, you willingly do what you feel called to, what is right and you are honest with a purpose. You can see what the young ones cannot and you tell them, “Listen to me now, believe me later” to hopefully hit that one ear that hears and learns. 

We have all been saying it, warning about it – this coming chaos, this explosion of consequences we all face. But if we all die tomorrow from nuclear spike protein poisoning, then all those words and warnings fall on dead bones, sandblasted clean, glowing in the dark of the newly created desert. Then we will all have to become our wild selves, no metaphor implied. Once again we will have failed miserably like we did the last 5 times. 

If we can just make it through this, clean up our mess, there is still hope for a future that does NOT include 24/7 censorship and mental monitoring along with the death of our souls, or living on a chip somewhere… that is not life. That is not a real and juicy, messy and creative, gloriously wrong and gloriously right, beautiful, soul enwrapped life, it is an empty clanging metal box, not human.  

I quit yelling about covid, when I saw the right people step up and the groups form and the information go out to more and more people – the right people for the job, the doctors who stood in their danger and made life uncomfortable for those asleep and those purposefully in the wrong, with the power of who they were behind the truths they were telling. That is not for me.

The same has occurred in the world with the UFOs to the point that one day that term will be an anachronism – they will be identified. There are the legal warriors, the ones developing science with their intuition, and the ones who have created their own disclosure by telling their stories of contact. This has now moved beyond going back, toothpaste metaphor and all. That is not for me.

Now the wild old crone that howls at the moon needs a new project. Its best left to young hearts and minds how the world goes anyway, and the wild deep forest calls out to me on the silvery light of the moon.

Thank you Clarissa, for the reminder.

The Big Nothing (burger)

Did you really expect that the same people that are raking in tons of money from us by keeping a whole world of things secret, were going to officially let the cat out of the bag?  

Let me reiterate: Do you think the same people who are systematically, genetically killing us with a lab manufactured poisonous spike protein (being forced into us from every available vector) will actually release information on anything but the next thing they want you to believe in their bid to rid the earth of us? Did you believe Covid? Then you go right ahead and believe that they really don’t know what is going on in our skies, or that they have no instrumentation to measure or collect data, or data before 2004. 

Go ahead, sure. But the rest of us will not wait for you because: Liars always lie. They tell the first one because they are cowards and thieves. They tell the next one to cover for the first one – and because they are cowards. They do NOT want to face the consequences of their actions of what ever they lied about in the first place… and on it goes until most times the liars even begin to believe their lies.

However:

You know what you know. We all who have had contact know our experiences are real. We don’t need mommy or daddy or any authority figure to tell us it is so. So why do we so desperately need the government to tell us it is real? WE ALREADY KNOW. Is it so that we can prove to all those who call us names that we are right? Are our egos really that fragile? 

If we still need ‘officials’ to tell us anything at all, we ARE still slaves, and what is worse we are slaves in our own minds – we are NOT even sovereign inside. 

All things are self apparent – in a self governing society, which, we are supposed to be… But we are not and because we are such good little slaves and we don’t govern our selves, we get away with anything we can, waiting to get caught and punished, while most take no responsibility for themselves or their own thoughts. 

Because of the above we are very susceptible to being gas lighted, used by narcissists and psychopaths who laugh at us and see easy marks, lying about everything they do because they know we will never question it, or even notice it. 

Trump isn’t coming back.

The ETs aren’t going to save us.

And yes, Virginia, they DID just try to kill us all off with lab generated Spike Proteins.

They can’t seem to resist the temptation to abuse. They are addicted. It is their illness – it is  the core of their beingness, their raison d’etre.

Absolute power corrupts Absolutely

About all this waking up bs: it doesn’t mean that we all get superpowers, it means we all get to see these people for who they really are, take them out of power or at least take them at their deeds and words – for real. It means we step up and take care of business, get our own shit straight. My father used to tell me there were consequences for every action. There are. 

As an adult you become responsible for those consequences, for yourself and you DO NOT LET children and idiots run your house – the same as (one would hope) you don’t let criminals and psychopaths run your world.

But, here we are amidst idiots and criminals and rats in the middle of a gigantic mess and we must save ourselves. Only we have that skill set. 

Are you gonna eat that Nothing Burger?

Conversations

I’m back in my body and I open my eyes – 4:01am. When that happens it is a signal that communication will happen and there is something to write about. But… my body was going, you’re kidding me? I feel like horse shit. So I pass out again. I promptly open my eyes again at 5:01am. The conversation goes:

“Really? But what is there to say? It’s all bullshit right now.”

“Well, how about that there are indigenous energetic beings who live on the planet.

“I already wrote about that…”

“And the really tall people have had more lifetimes here.”

“I’m short – explains a lot…”

“The super tall people may be regenerated instead of born and have continuous memories of the entire time here, like a 1,000 years or more. Which makes them insane. Need I continue?”

“Ok, as always you got me – now I’m interested”

“(background humor)”

Without fail, fat cat feels my waking mind and comes in the bedroom and meows at the top of his lungs and both dogs go instantaneously from deep sleep to loud barking. I grab my clothing head for the bathroom and the damned cat will not even let me pee in peace. I head down stairs where I open the door for the one dog who followed me, immediately feed the cat before I wind up killing him for all the noise, make my coffee, get my candle and and note book and head out doors to write in my garden. Writing for me is sometimes a form of meditation – I hit the zone there. But it requires silence and not just ‘sound’ silence – it requires psychic silence. Just about the whole world is asleep by 4am – even the night owls in a regular suburban neighborhood like mine. 

However, I can fuck that up by looking at my phone or wrangling to many cats and dogs before I actually make it outside to write. This morning I was rather too awake but I think I got the gist of it. 

  1. To go Interdimensional or Transmedium using technology simply means you create your own field of energy around you or your vehicle that translates you to a place or a time or both instantaneously, with the added benefit of molecular frequency mobility allowing for penetration of solids, liquids and other media out of their native phase, also creating invisibility to normal human sight.
  2. It could be that the souls here are not trapped, but, they are addicted to being 3D.
  3. Somebody or group here MOST definitely has the tech mentioned in #1. They are not willing to share. It would mean financial devastation to them. They do not care that others suffer because of this.
  4. I speculate that the human body was created as an avatar on this planet for other species to use to come and visit. That earth is like a national park for this galaxy, one of many, but the avatars became too conscious for use by others. 
  5. Further, when the avatars got out of hand it was decided to reduce the population, while genetically modifying intelligence to make the avatars more amenable to inhabitation by galactic travelers. (? Another speculation as to why. It is rather obvious we are being systematically culled, however.)
  6. We do have a group of immortals on the planet. They are very good at hiding. Many legends have grown up around them, mostly to obfuscate their existence. ( I’m not the only one who thinks so…) Obviously they would need and want to remain unknown.
  7. I asked if consciousness is information (what you know) or what you sense, or both. Then I speculated that it is both. Sensing increases information, but wrong information can block the recognition of what is right in front of your eyes. In other words you can block any or all of that information. You can be programmed and not even know it. We are all waking up to this right now. It’s interesting. 
  8. Systems resonance (part of #1 at the top) is also a biological function and we know as the earth transits space it changes its frequency and thusly we do too. Because of that resonance we are coming out of sleep all of us – because we resonate in the human field frequency and there is feed back between us and the earth in all of her components. 

These things will bounce around in my head now, (they are not new ideas, but there is a different feeling attached to them) creating new connection points, new ideas and greater awareness – hopefully. My best conversations occur in the semi-dream state which is fleeting, because, loud cats, dogs, a husband that objects to lights on while he sleeps and my immediate need for coffee two seconds after I awake. And yet whom ever is with me has a delightful sense of humor and has stuck with me all the years of my life. Through thick and thin, stupidity and moments of brilliance, through devastation and joy. I have found more moments of wisdom in this writing process than any teacher taking multitudes of years with me could ever teach, because I need not translate the movements of my soul to another to try to explain (that happens when I write) because they are a seamless part of me, and only there when I ask. That is why I return the curtesy of hauling my old, creaky, cranky ass out of bed at 4am. That and because I love this time of communion. It took me close to 60 years to figure out what it was, I still may not have a clue, but I enjoy it muchly.

Fool me once…

What do I think I know?

Lairs always lie. They tell the first one because they are cowards and thieves. They tell the next one to cover for the first one – and because they are cowards. They do NOT want to face the consequences of their actions of what ever they lied about in the first place… and on it goes until most times the liars even begin to believe their lies. 

I think it appears to be that the liars have been given an ultimatum – tell the truth, or ‘We’ will. The ‘We’ being what the first lie was told about. What we are seeing now is a very convoluted plan to squirrel out of most of the blame. And, they are running out of time or they never would have gone this far with it. Plus, they are trying to get the biggest bang for their buck financially while they tell us only partial truths. 

IMO it’s going to back fire in their faces. 

There are many who would let what truth they do know out, but they can’t – not the whole truth. And half truths can be used and spun – twisted to not exactly lie – but lie anyway. Someone coined the phrase ‘truth embargo’. (Richard Dolan, perhaps?) And somewhere in the game, trying to hide the truth with misdirection and talking around it, it tumbled into outright lies, deception and even murder. To make a lie work, you must always tell the next lie. There is never just one lie. Ever. 

IMO, we are fast approaching a deadline of sorts. We all know it, we have all felt it – even not knowing what is coming, we are aware of it. I do believe the gig is up and the game is over. The reveal of what ever it is will occur and the chips will fall where they may. In other words it has been taken out of the secret holder’s hands and they have no recourse but to stand muster and take their consequences. (and probably wish they had told the truth in the first place) 

Never has this earth been a place where all were equal. In the 20th and 21st century the gap between the have and have-nots has gotten so out of hand, the usury so over the top, that it must stop. Now, the financial system that fed the liars is crashing, and the information they used to bank on, will be revealed. Society is already scrambling to reorganize, preparing, mobilizing. The timeline where we wind up in the future as a society that cannot reproduce because we have destroyed our genetics and end up like the greys is being halted, intervened, actually stopped in it’s tracks. 

Intervention is always messy and painful, but with the weight of all of us standing behind it, it will be successful. Now the real work begins. Liars never clean up their messes, those who got hurt do. They quietly begin again and with a greater wisdom and a wary eye on the offenders, rebuild their lives, trusting the liars to always be liars and taking appropriate action. Fool me once, shame on you. Fool me twice, shame on me. 

Aliens exist.

We travel the solar system and beyond.

Technology to greatly alleviate suffering exists.

We are being used, sold, killed off, culled: a commodity without consent.

We have the power to stop this, we just don’t know it.

But we will, very soon.

There would come a time…

I aways knew there would come a time when a man with enough spine, honor and integrity would arrive to handle a corrupt system and see us through to the future. Unflinchingly ethical, principled with a high ideal for the problems ahead in getting and setting a standard we have not seen for a very long time. So watch this. Here he is.

Do You???

Some points to ponder, things I’m wondering about…

  • Why does Lue Elizondo make an issue out of needing to get this done – this whole roll out quickly?
  • It is interesting how we are calling mainstream media lamestream – in light of all the lies and misrepresentations disinformation and outright censorship on everything recently. Now the UFO community is cheering the lamestream for any tidbit of information they ‘report’ on their version of UFO’s? What’s to say the talking heads aren’t feeding us a line of bullshit on this too? (its called spin)
  • This recent flurry of mainstream media reports on supposed UFO’s have over shadowed Jacques Vallee’s and Paola Harris’ new book “Trinity” – that has more truth in it than all the recent lamestream reports put together. 
  • It would seem the way that Elizondo and team are pushing the importance of the UFO community that they might be on a mining expedition for new information from a very rich source, namely us.
  • At the same time people like Danny Sheehan are going full speed ahead to pry information out of the legal dungeon its been hidden in for the last 70 years in an effort to clarify matters.
  • IMO we have two or more parties vying for who’s narrative is going to win and come out on top setting the standard for ET relations for probably the next 500 years. 
  • Which, IMO, leads one to speculate back around to that time limit that Lue mentioned in one of the recent flurry of video interviews he did last week. 
  • Are we looking at some sort of intervention time line that if they do not reveal information to the public about this topic, it will be taken out of their hands???

Trust me what is being bandied about in the media today is simply one snowflake on an iceberg so vast and huge that its almost useless. And here we are havin’ a par-tey over it all, acting like idiots when we know better… 

Do you trust the same people (who in lying to us about CV19 are systematically, genetically killing us) to actually report anything on the UFO topic except what they want you to believe?

Do You???