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BY 4.0 license Open Access Published by De Gruyter March 4, 2022

An overview on the reproductive toxicity of graphene derivatives: Highlighting the importance

  • Nastaran Hadizadeh , Saba Zeidi , Helia Khodabakhsh , Samaneh Zeidi , Aram Rezaei , Zhuobin Liang , Mojtaba Dashtizad EMAIL logo and Ehsan Hashemi EMAIL logo
From the journal Nanotechnology Reviews

Abstract

With the glorious discovery of graphene back in 2004, the field of nanotechnology was faced with a breakthrough that soon attracted the attention of many scientists from all over the world. Owing to its unique bidimensional structure and exquisite physicochemical properties, graphene has successfully managed to cave its way up to the list of the most investigated topics, while being extensively used in various fields of science and technology. However, serious concerns have been raised about the safety of graphene, for which numerous studies have been conducted to evaluate the toxicity of graphene derivatives in both in vitro and in vivo conditions. The reproductive toxicity of graphene is one of the most important aspects of this subject as it not only affects the individual but can also potentially put the health of one’s offsprings at risk and display long-term toxic effects. Given the crucial importance of graphene’s reproductive toxicity, more attention has been recently shifted toward this subject; however, the existing literature remains insufficient. Therefore, we have conducted this review with the aim of providing researchers with assorted information regarding the toxicity of graphene derivatives and their underlying mechanisms, while mentioning some of the major challenges and gaps in the current knowledge to further elucidate the path to exploring graphene’s true nature. We hope that our work will effectively give insight to researchers who are interested in this topic and also aid them in completing the yet unfinished puzzle of graphene toxicity.

1 Introduction

As a unique, two-dimensional nanostructure that is mainly composed of sp2 hybridized carbon atoms, graphene has gained tremendous recognition ever since its initial isolation in 2004 [1,2]. Characterized by their exceptional physicochemical properties, high electron mobility, and incredible tensile strength, this inherently honeycomb-shaped family of nanomaterials are now being extensively exploited in various fields of biotechnology (Figure 1) [3], drug delivery [4], tissue engineering [5], and cancer treatment [6], as well as electronics [7], photo-sensors [8], and solar batteries [913]. The sp2 orbitals in graphene consist of px, py, and pz orbitals, among which the latter can form π bonds with a wide range of organic and inorganic materials, while also providing the opportunity for surface modifications [1416].

Figure 1 
               Utilization of graphene in different fields of biomedicine.
Figure 1

Utilization of graphene in different fields of biomedicine.

Chemical modification and surface functionalization have actively conferred to this already-distinctive nanomaterial the potential to exceed its applicability limitations, as well as the capability of being tuned in for use in additional fields of science and technology, and get one step closer to becoming immensely blended in our everyday lives. Therefore, numerous studies have been continuously conducted with the aim of investigating this phenomenon and creating new graphene derivatives [17]. Graphene oxide (GO), reduced graphene oxide (rGO), and other types of functionally modified graphene derivatives are other residents of this family that can be produced in various forms of 2D nanosheets, nanoflakes and spheres, nanoplatelets, nanofibers, or 3D hydrogels and nanocomposites depending on their desired applications [18,19]. Coming in a wide range of shapes, graphene derivatives are also available in a variety of sizes ranging from a few nanometers to several meters, some of which consist of a limited number of layers (≤5 layers) and mostly referred to as few-layered graphene (FLG), while some others are produced in more cost-effective bulk forms [20].

Emerging from the extensive utilization of graphene and our ever-increasing exposure to its derivatives, serious concerns have been raised regarding its safety toward animals, humans, and the environment. The possibility for the occurrence of undesired and unpredicted interactions of graphene with different biological compartments has recently led research toward a more toxicity-based outlook and has also promoted more cautious exploitation of graphene as it might induce temporary or permanent damage to cells, tissues, and organs of different species [21,22]. Several studies have noted that the toxicity of graphene is in close correlation with a variety of its characteristics including shape, size, and state of oxidation, functional groups, methods of synthesis, as well as administration routes, exposure time, and doses [2326]. With that in mind, it is also expected for different graphene derivatives to be associated with varying degrees of toxicity [23].

Furthermore, targeted organs are yet another critical factor that impacts the level of graphene-induced toxicity. Metabolism rate, blood circulation, and physiological barriers defenses of an organ are all complicated factors that differ from organ to organ, species to species, and perhaps even within the same species. Despite the complexity of all these elements and their possible interconnections, it is important to consider their potential influence on the accumulation and toxicity of graphene. A major proportion of studies in this field have investigated the toxicity of graphene in several organs such as lungs [27], liver [28], spleen [29], and kidneys [30], while studies regarding graphene toxicity on the reproductive system remain limited. The multifactorial nature of reproductive studies, sensitivity of embryos, difficulty of long-term few-generation studies, and unpredictable interplay between genetic and environmental variables may be some of the explanations for the lack of sufficient data on this subject when compared to other organs. However, long-term and short-term reproductive toxicity of graphene in the gametes and offspring of those who were in contact with these nanomaterials is an essentially pivotal matter as they may prove to be harmless to an individual at specific doses and exposure periods, but have the ability to induce toxicity to the following generations and exhibit potential damage in prolonged periods of time due to their accumulation and slow bio-degradation [31]. Therefore, the safety and toxicity levels of graphene nanomaterials are among important subjects that have to be taken into consideration at the time of investigating this subject.

Despite being investigated for less than a decade, numerous studies now exist in this field, some of which have displayed contradictory results. With a clear understanding of those results and the apparent yet minor inconsistencies in the current literature, this study aims to provide a comprehensive overview of existing data about the reproductive toxicity of graphene nanomaterials and their possible mechanisms while avoiding generalized or biased information to shine light on the path to further exploring the true nature of graphene’s reproductive toxicity [32].

2 Introducing main graphene derivatives

2.1 Pristine graphene

Pristine graphene, as an unoxidized form of graphene consisting of episodic hexagonal carbon structures, owns a remarkably high surface area of ∼2,630 m2/g that declines in parallel with an increase in the number of graphene layers [1416]. Graphene monolayers also benefit from exceptional mechanical resistance as a result of strong C–C sigma bonds with very short interatomic lengths (about 1.42 nm) within the 2D plane of graphene, which subsequently turn them into promising candidates for a variety of constructional and electromechanical applications [3335]. Each carbon atom in pristine graphene consists of four valence electrons that can be shared with other atoms or molecules through covalent bonds. Each of these electrons can be hybridized in sp, sp2, and sp3 forms, among which sp2 contains px and py orbitals as well as a critical pz orbital with a single electron that forms a half-filled weak π bond. Being situated in a perpendicular position above the structural plane of graphene, this half-empty orbital plays a major role in determining the physiochemical properties of graphene and grants it the ability to participate in a broad range of chemical reactions [1416]. Moreover, the half-filled π bonds in graphene create a zero bandgap between the valence and conductive bands that enable electrons to move freely and also result in the formation of weak van der Waals interactions between graphene monolayers that facilitate their gentle movement on each other when subject to weak shear stress [1,36]. Regarding its toxicity, 48 h exposure with pristine graphene (2–3 nm thickness and 500–1,000 nm size) at 5–100 mg/mL concentrations has been associated with Murine RAW 264.7 macrophage cytotoxicity in sequence to increased oxidative stress, apoptosis, and mitochondrial membrane potential damage [37].

2.2 GO

Produced from the oxidation of graphene, GO is an amorphous material with berthollide characteristics such as nonstoichiometric atomic composition [38]. GO might possess a diverse range of functional groups including hydroxyl, carboxyl, and epoxy that vary depending on the employed synthesis methods and oxidation conditions [39,40]. The state of oxidation in GO and the type of functionalization are pivotal factors that impact GO’s physiochemical properties and enhance its potential biomedical applications such as a facilitated bioconjugation with peptides, drugs, and antibodies [41]. However, this advantage is accompanied by its inherent drawback of toxicity, as GO is capable of interacting with natural cellular components such as proteins or DNA and therefore disrupts their normal physiological function [42]. For instance, highly oxidized nano graphene oxide (NGO) is a monolayer graphene sheet that possesses a high number of oxygen-containing functional groups on its edges and basal plane [43]. Due to its significant physicochemical properties such as the ability to absorb aromatic drug molecules as a result of owning two aromatic planes, NGO is considered to be a highly efficient nanomaterial suited for numerous biomedical applications [12]. Despite the advantages that NGO conveys to the field of biomedicine, a previous study has displayed that NGO possesses the highest toxicity among graphene derivatives through the induction of oxidative stress and increasing reactive oxygen species (ROS) generation [44]. For instance, blood cell surface interactions with NGO, even without penetration into RBCs, can lead to the alteration of RBCs’ polarity and permeability, disrupt normal cell membrane functions, and cause RBC hemolysis as a result of excessive electrostatic interactions with NGOs [45].

2.3 rGO

rGO can be produced through the reduction of GO; however, complete elimination of GO’s functional groups (–COOH, –OH, –COH) is rather unlikely, and the final rGO often possesses several remaining functional groups [46,47]. Even though rGO is generally believed to be of lower toxicity than GO [48], many studies have observed rGO-associated toxicity in different cell cultures and animal models [49,50]. Previous investigations have indicated that 24 h exposure of human mesenchymal stem cells (hMSCs) with rGO (11  ±  4 nm thickness and 3.8  ±  0.4 µm lateral diameter) at of 0.01–100 μg/mL concentrations induces DNA fragmentations and genomic aberrations [51]. Moreover, exposure with rGO sheets (∼1.2 nm thickness and ∼2 µm lateral size) at 0.01–100 μg/mL concentrations for 96 h was reported to induce slight cytotoxicity by the disruption of hMSCs membranes, while rGO nanoribbons (thickness of 1 nm thickness, 10 µm length, and 50–200 nm width) under similar conditions damaged hMSCs through DNA fragmentations and chromosome aberrations [52]. While the differences in rGO toxicity may be explained with its correlations with rGO shapes and sizes, further investigations are required for exploring the exact interconnections of these variables. Last but not least, Liu et al. demonstrated that zebrafish embryos incubated with GO and rGO at 1, 5, 10, 50, and 100 µg/mL concentrations for 96 h experienced slight toxicity in hatching speed and larvae length as well some moderate damage to the embryos’ hearts [53]. Interestingly, these results also indicated that rGO was associated with lower toxicity in zebrafish embryos compared to GO (Figure 2).

Figure 2 
                  G(1) Pristine graphene: (a) displays van der Waals bonds between two graphene sheets and (b) displays sp2 carbon atom orbitals. G(2) GO: (c) displays intramolecular bonds between single layers of GO and red dashes are representative of intramolecular hydrogen bonds, G(3) rGO.
Figure 2

G(1) Pristine graphene: (a) displays van der Waals bonds between two graphene sheets and (b) displays sp2 carbon atom orbitals. G(2) GO: (c) displays intramolecular bonds between single layers of GO and red dashes are representative of intramolecular hydrogen bonds, G(3) rGO.

3 Toxicological studies of graphene derivatives in the reproductive system

The ever-growing utilization of graphene in various fields of science and technology has rapidly raised questions regarding the toxicity profile of these extraordinary nanomaterials. The occurrence of molecular interactions between graphene derivatives and cellular compartments is a well-established yet concerning the matter that is capable of causing potential harm in different tissues and organs, including the reproductive system. Even though significant attention has been shifted to the field of graphene toxicity and numerous contributions have been made in recent years, there is still a lack of sufficient understanding regarding how and under what conditions graphene-based materials induce toxicity in the reproductive system of different species. In the following sections, this study aims to discuss recent advancements in the field of graphene toxicity in the reproductive systems of different living species, while explaining possible mechanisms for the induction of toxicity.

3.1 Nematodes

Caenorhabiditis elegans, a free-living nematode with a short life span, is widely used as an animal model for in vivo toxicological studies due to its 40% genomic homology with humans [54,55]. Moreover, its low costs, short life span, ease of handling under the microscope, and significant similarity of its physiological pathways and stress-related responses with mammals are other desirable characteristics that have turned C. elegans to a favorable model for toxicological studies [5658].

Exposure of gravid C. elegans nematodes with 10 mg/L GO for 72 h has been reported to disrupt fatty acid metabolism and spermatogenesis; reduce sperm count, brood size, offspring birth and life span; and alter fat metabolism in sperms. In this study, alleviated expression of beta-oxidation–related genes were indicated to be responsible for disrupted fat metabolism and impaired spermatogenesis in nematodes [59]. Even though the importance of fat metabolism and its critical role in spermatogenesis has been proved earlier [60], the exact impact of GO on them has yet to be fully discovered. However, one of the proposed mechanisms that may explain the detrimental effects of GO in C. elegans worms is considered to be elevated oxidative stress and ROS production in exposed nematodes [61]. These results are consistent with the results from the study by Wu et al., studies in which the toxicity of NGO was investigated on primary (lung, kidney, spleen, and liver) and secondary (the reproductive system and the neurological system) organs of C. elegans nematodes and reported toxic damages to both primary and secondary organs after prolonged exposure to 0.5–100 mg/L GO, mainly via oxidative stress pathways [57,58].

In another study, C. elegans was exposed to GO and rGO, and an integrated systems toxicology approach was used to assess the interactions and underlying mechanisms of GO and rGO’s toxicity. The results of this study indicated a reduction in the worms’ reproductive capabilities, which was more significant followed by GO exposure in comparison with rGO exposure. Unlike rGO, a noncanonical Wnt-MARK signaling cascade (MOM-2 → MOM-5 → MOM-4 → LIT-1 → POP-1 → EGL-5) was proposed to be the underlying mechanism for the induction of reproductive toxicity followed by GO exposure, where GO was responsible for suppressing ELG-5 by the activation of POP-1 and thus resulted in impaired fertilization and egg hatching [62].

A study by Pattammattal et al. reported that no acute toxicity was detected after a 7-day exposure of wild type N2 C. elegans nematodes with graphene at a dose of 50 to 500 μg/mL, while prolonged exposure (10–15 days) with higher doses of graphene (300–500 μg/mL) induced about 95% cytotoxicity in two human cell lines and also reduced C. elegans brood size by 5–10% [63]. Accordingly, the number of eggs produced by high-dose (500 μg/mL) graphene-exposed nematodes had also decreased, which may be explained by the generation of oxidative stress followed by high-dose graphene exposure [64]. Strikingly, this study also displayed that small fragmentations of graphene sheets of less than 200 nm diameters were mainly responsible for the observed toxicity, as larger graphene sheets did not exhibit toxic effects on both human cell lines and nematodes. All in all, 50–100 μg/mL concentrations of graphene revealed desirable safety to worm populations as the nematodes’ survival rate was not decreased after the 10-day duration of this study [63]. These findings are consistent with the results from the previous studies, which proved that the toxicity of pristine graphene in C. elegans follows a dose-dependent pattern [44].

Regarding the role of surface modification, graphene nanoplatelets (GNPs) with different surface modifications (NH2 and COOH) and GO (single and few layers) were evaluated for their toxic effects toward C. elegans reproductive system and Beas2B cell lines. In this study, an order of pristine > NH2 > COOH was obtained for modified GNP toxicity; however, GO exhibited more significant reproductive toxicity than pristine graphene, whereas for BeasB2 cells, pristine graphene induced higher toxicity when compared to GO. This occurrence may be related to different interactions of functional groups with different biological components, thus confirming the pivotal role of varying biological interactions in determining the final toxicity of graphene. GO was revealed to damage cells through the formation of hydrophobic agglomerates that impaired the cellular membrane [65], while the platelet-like structure of pristine GNPs was held accountable for increased toxicity in BeasB2 when compared to GO [66]. On the contrary, decreased bioavailability and biocompatibility of pristine GNPs along with the generation of hydrophobic agglomerates serve as crucial factors that reduce the reproductive toxicity of pristine GNP compared to GO in C. elegans [67]. The oxidation state of GO, increased hydrophilicity and dispersion in exposure mediums, and enhanced biocompatibility also contribute to escalating GO’s toxicity in the reproductive system. Regarding the role of chemical modification, surface functionalization of GNPs with NH2- and COOH- was associated with reduced toxicity, while pristine GNPs displayed excessive agglomeration and superior cell membrane impairment [45,67]. Moreover, while both SLGO and FLGO displayed a clear dose-dependent toxicity pattern, SLGO exhibited more biological interactions owing to its lower stiffness and subsequently higher biological adsorption in comparison with FLGO [68].

Consistent with the aforementioned results, Zanni et al. evaluated the toxicity of completely reduced multilayered (3–60 layers) GNPs (with no residual oxygen-containing functional groups) with the lateral size of one to tens of micrometers, thickness of 1–20 nm, and tested concentrations of 100 and 250 μg/mL on adult C. elegans models after 3 h of exposure. Notably, the nematodes’ life span and brood size were not altered, thus displaying the absence of chronic reproductive toxicity [67].

3.2 Aquatic species

Owing to the genomic homology between zebrafish (Daniorerio) and humans along with adequately similar physiological responses during chronic illnesses, zebrafish has become a widely used animal model for studying the toxic effects of nanoparticles in vivo [6971].

John and coworkers [72] studied the adverse effects of GO (0.8–1 nm thickness, 101–258 nm diameters) at 0.01, 0.1, 1, 10, and 100 mg/L concentrations on the embryogenesis of zebrafish and reported that GO had entered and induced hypoxia in the chorion, generated an anoxic space close to the chorion, and also enforced mechanical pressure on its surface area. Given the substantial role of chorion in the development of zebrafish embryos, the aforementioned effects along with the envelopment of chorions by GO resulted in the decreased embryo movement and delayed hatching and development of the embryos. ROS generation, lipid peroxidation (LPO), formation of 8-OHdG (8-hydroxy-2-deoxy-guanosine) adducts, apoptosis, mitochondria activity disruption, and impaired antioxidant enzyme activity are some of the other detected outcomes of GO exposure in zebrafish embryos. Some malformations such as pericardial/yolk sac edema were also observed in different embryonic regions that were resulted from the localization of GO inside the embryos in a dose-dependent and selective manner [73].

A study on Japanese medaka (oryziaslatipes) demonstrated that one-time intraperitoneal injection of (25–200 µg/g body weight) GO to breeding pairs was followed by decreased egg fecundity in early days upon injection in a dose-dependent manner; however, the overall fecundity was not altered significantly. The hatchability of embryos was reported to have undergone drastic reduction when injected with 200 µg/g body weight GO; however, cell morphologies of granulosa and leydig cells remained almost unaltered according to gonad (testis and ovary) histopathological examinations. In spite of the agglomeration of GO in the gonads of Japanese medaka, folliculogenesis in the ovaries and germinal components of the testes experienced almost no deviation. Also, 25,200 g/g GO was not capable of inducing notable reproductive toxicity in Japanese medaka [74]. Results from another study indicated that GO (mean thickness of 1.0 nm and GO flake area of 0.58 µm2) had the ability to partially inhibit the reproduction of Ceriodaphniadubia via increasing ROS production followed by waterborne exposure. GO reduced C. dubia energy levels and thus decreased their reproduction activities. In this study, acute (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/L for 48 h) and chronic exposures (0.05, 0.1, 0.2, 0.4, and 0.8 mg/L for 7 days) were reported among which the latter, especially at higher doses of 0.4 and 0.8 mg/L, was stated to be associated with major reproductive toxicity as it diminished the number of neonates by 12.8 and 44.2%, respectively [75].

3.3 Mammals

3.3.1 Rodents

Three groups of Wistar rats (AS1: 15 days of treatment with seven repeated doses on alternative days; AS2: 30 days of treatment with 15 repeated doses on alternative days; AS3: 30 days of treatment with 15 repeated doses on alternate days and 30 days of recovery) and three subgroups within each group (categorized by the received doses of intraperitoneal NGO as low, mid, and high doses) were evaluated regarding the toxic effects of NGO. Accordingly, the total sperm count of the high-dose subgroup of the AS1, and mid- and high-dose subgroups of the AS2 group experienced a significant reduction. However, these changes were not as prominent in low-dose subgroups. Decreased sperm production in the testes, spermatogonia loss, cell cycle arrest, and occurrence of cell death within the sperm production pathways are potential explanations for this matter. Of note, spermatogonia and spermatid numbers were also reported to have decreased in this study [72]. The high-dose subgroup of the AS2 group also experienced declined sperm motility values, which were restored to normal after a recovery period. On the one hand, SOD, GPx, and GST antioxidant enzymes of the treated rats deteriorated remarkably in a dose-dependent fashion, and ROS generation was increased in parallel with decreased cell proliferation and enhanced cell death [76]. Abnormal morphological alterations were also detected in the high-dose subgroup of the AS2 group. Both such alterations and reduced sperm motility are highly likely to be associated with excessive ROS production and oxidative stress followed by the oxidation of cell membrane lipids that contain a significant amount of polyunsaturated fatty acids. Sperm ATP loss, axonemal damage, and consequent morphological abnormalities and disrupted sperm motility also take place in sequence to accelerate oxidative stress in sperms [77]. Decreased steroidogenesis, germ loss, germ cell apoptosis, and germinal epithelial impairment are some of the other outcomes of oxidative stress that contribute to the induction of reproductive toxicity in Wistar rats [7880]. In spite of the aforementioned toxic effects, reproductive hormone concentrations in the rats’ serum remained unchanged upon NGO treatment, further confirming the results of a study by Liang et al. where serum testosterone levels remained unaltered in adult male mice followed by the injection of 25 mg/kg body weight concentrations of NGO (∼4 nm thickness, ∼238 nm (large GO), and ∼55 nm (small GO) particle size) to tail veins [81]. Weight, sex ratio, and survival rate of the progenies remained normal, and epidermis and testis tissues of the treated mice also displayed no signs of damage. Moreover, intra-abdominal injection of high-dose GO (total 300 mg/kg male mouse, 60 mg/kg every 24 h for 5 days) also exhibited no detectable harm to male mice reproduction, thus indicating little to no toxicity in the reproductive system of male mice. As concluded, blood–testis barrier (BRB) and blood–epididymis barrier (BEB) played a major role in inhibiting the penetration of GO into testis and epididymis, thus impeding the risk of reproductive damage [81].

In contradiction with the previous results, Akhavan et al. [82] discovered that intravenous injection of 4 mg/kg of body weight NGO (∼0.8 nm thick and <100 nm lateral diameters) weekly for 8 weeks to male Balb/C mice exhibited high graphene uptake in testis and subsequently altered spermatozoa viability, morphology, and kinetics and also induced DNA damage and chromosomal aberrations. Doses higher than 200 µg/mL displayed serious toxicity and deteriorated sperm viability by ∼45% at 2,000 µg/mL NGO concentrations. As detected in the semen of treated mice, accelerated ROS generation had the ability to disrupt normal pregnancy, decrease hormone secretion, and also reduce fertility, gestation, and multi-birth ability by ∼44, 35, and 59% in the female mice that were in contact with the NGO-treated male mice, respectively. Upon the birth of the litters, postnatal viability of the offspring also experienced a 15% reduction at 2,000 µg/mL NGO concentration. As proposed in this study, a dose-dependent pattern was unveiled for the toxicity of NGO, as lower doses exhibited lower toxicity while higher NGO concentrations were associated with more significant reproductive toxic effects.

The clear inconsistency observed in the outcomes of the aforementioned studies can raise existing uncertainties regarding the true nature of graphene’s reproductive toxicity. Taking into consideration that the induction of toxic effects in all organs, including the reproductive organs, is under the direct or indirect influence of multiple factors, it can be concluded that graphene characteristics such as size, thickness, and concentrations are some of the critically important factors that impact the eventual toxicity that is observed. For instance, Akhavan et al. [63], synthesized graphene with ∼0.8 nm thickness and <100 nm dimensions and employed a lower dosage of NGO (4 µg/kg to 4 mg/kg body weight) for weekly injection into mice for 8 weeks, while Chatterjee et al. [62] injected mice with much higher concentrations at 25 mg/kg body weight once via tail vein and 60 mg/kg NGO (∼4 nm thickness, ∼238 nm (large GO), and ∼55 nm (small GO) particle size) abdominally for 5 days every 24 h. Despite the higher doses used by Liang and coworkers, no significant reproductive toxicity was detected in mice 30 days after injection, while Akhavan et al. reported prominent toxicity in both treated male mice reproductive system (after 8 weeks from the initial injection) and the viability of delivered offspring (4 days upon birth) followed by the injection of lower NGO doses (4 µg/kg to 4 mg/kg body weight every week for 8 weeks). A possible explanation for the variations in obtained results may be potentially attributed to both specific time-points at which the mice mated and were sacrificed for further investigations. In the study by Akhavan et al., mating took place 1 week after injection and the mice were sacrificed after 8 weeks; however, in the study by Liang et al., mating took place after almost 1 month from injection and also mice were sacrificed at the similar time-point of 1 month after treatment. Akhavan et al.’s study stated the existence of morphological abnormalities in male mice spermatozoa as well as NGO accumulation in the testis 8 weeks upon injection of NGO, while Liang et al.’s results were indicative of the absence of abnormalities in mice spermatozoa, testes, and epididymis after 30 days from the initial injection. Accordingly, the process of spermatogenesis occurs through a ∼8 week period, thus unveiling the importance of evaluation time-points for the detection of toxicity. Therefore, the absence of reproductive toxicity in Liang et al.’s study might be explained by the short duration between injection and evaluation time-frames and thus the lack of completely renewed spermatogenesis before mating. This finding is further confirmed by the same study by Akhavan et al. stating that genotoxicity and chromosomal aberrations in the spermatozoa start to take place ∼6 weeks through the spermatogenesis process. However, variations in NGO size and thickness are also crucial factors that are likely to have affected the final toxicity observed in those studies.

Another study aimed to assess the cytotoxic and genotoxic effects of rGO (0.1, 1, 10, 100, and 400 µg/mL concentrations) in male mice spermatozoa and indicated that GO induced toxicity in a dose-dependent pattern, in which motility and viability of spermatozoa were decreased after incubation with doses higher than 1 µg/mL for hydrazine-reduced GO and hydrothermally reduced GO and with doses higher than 10 µg/mL for GTP-rGO and GO. Accordingly, N2H4-rGO and HT-rGO promoted the generation of ROS and nitric oxide (NO) and reduced ATP and NAD+/NADH, in contraction with GTP-rGO that deteriorated ROS production and NO owing to GTP’s antioxidant properties [83]. Moreover, less than or equal to three-layered GO and rGO (100 and 400 µg/mL, thickness of ∼0.8 nm for each monolayer)-exposed spermatogonial stem cells of mice (SSCs) were subject to cytotoxicity and genotoxicity through apoptosis, membrane damage, and morphological alterations (such as cell shrinkage and chromatin condensation) after 24 h of incubation. Accordingly, these graphene derivatives have the capability of reducing SSCs viability, disrupting normal functions and genomic material of SSCs, and henceforth inducing reproductive toxicity by impairing the genetic material passed on to the offspring [84].

Surprisingly, the addition of 0.5 µg/mL GO to a mice sperm suspension before performing in vitro fertility (IVF) has been reported to increase the number of fertilized oocytes as well as born pups in a more efficient manner compared to the gold-standard methyl-β-cyclodextrin agent for IVF promotion [85]. To this day, exact underlying mechanisms for explaining this occurrence remain unknown, but it is predicted that different graphene derivatives containing varying diameters, morphological shapes, doses, and chemical functionalizations display a diverse range of outcomes in different experimental settings [86]. However, further investigations are still acquired to reach a well-established consensus regarding how graphene materials can be modified before exploitation to minimize the potential risks to one’s reproductive system as well as offspring health.

3.3.2 Wild pigs

In vitro exposure of boar spermatozoa with 0.5, 1, 5, 10, and 50 μg/mL concentrations of GO (size interval 600–900 nm) has previously shown that sperm capacitation and fertility had enhanced in sequence to exposure with 0.5 and 1 μg/mL doses of GO, while 5, 10, and 50 μg/mL doses were associated with induced cytotoxicity in spermatozoa [87]. It has been proved that GO at high doses is capable of interfering with spermatozoa plasma membrane and consequently impairs their fertilization via causing alterations in cholesterol extraction from the membranes. There is also further evidence supporting this fact, for example, Zhang et al. reported in a computational experiment that graphene has the ability to remove cholesterol from a bilayer membrane and absorb the hydrophilic compartment of cholesterol and henceforth prevents spermatozoa from entering the membrane bilayers [88]. It is possible that this mechanism plays an important role in the dose-dependent toxicity of GO in spermatozoa; however, more experiments are required for exploring the precise correlation between them.

An in vivo study [89] further investigated cholesterol extraction from swine spermatozoa membrane and reported that no toxicity was observed; however, certain physiochemical alterations were detected in the spermatozoa membrane and were associated with promoting sperm fertility, functions, signaling pathways, and increasing overall fertility potential of sperms without negatively influencing sperm interactions with the female environment. Moreover, it is also crucial to mention that the homeostasis of the Ca2+ ion plays an essential role in controlling various functions of the sperm, such as its motility [90], cytoskeleton assembly [91,92], and even apoptosis [93]. According to these results, low concentrations of GO managed to successfully increase Ca2+ ions, all the while leaving the sperms’ membrane potential unaltered, thus proving that GO has the ability to improve sperm capacitation via calcium signaling pathways. Furthermore, the underlying mechanisms that reveal the reasons behind increased sperm fertility have yet to be known; however, it is estimated that membrane glycocalyx is potentially involved in this process as it is the primary component that comes into contact with GO and other external molecules [94].

3.3.3 Humans

Graphene toxicological studies on humans remain rather limited due to ethical reasons; however, an in vitro study demonstrated that incubation of human sperms with 1, 5, and 25 μg/mL GO (1–5 μm) for 0.5–3 h resulted in no detectable toxicity or alterations in the viability of sperms and did not trigger accelerated generation of ROS even at the highest concentration of 25 μg/mL [95]. However, 3 h incubation of sperms with high doses of GO (5 and 25 μg/mL) deteriorated sperm motility.

4 Important factors affecting graphene’s toxicity

4.1 Administration routes and exposure periods

Oral administration, intravenous injection, intraperitoneal injection, subcutaneous injection, intratracheal instillation, intrapleural installation, and pharyngeal aspiration are the main routes of graphene administration that might ultimately result in varying toxic effects [96,97]. While toxicological reactions should be assessed via different administration routes, it is of crucial importance to first choose the most suitable route depending on the target organs of study. For instance, intranasal exposure is often utilized in nanoparticle neurotoxicity studies due to its proper relevance with the nervous system [98,99], while intravenous (via tail vein) or intra-abdominal injection methods are the most common routes for reproductive toxicity studies [82,100]. In spite of these findings, the current literature still suffers from a lack of strong evidence regarding the exact impact of administration routes on reproductive toxicity [23].

Numerous investigations have evaluated the correlation between graphene exposure periods and its eventual toxicity. For instance, an in vitro study that investigated graphene nanoribbons (10–400 mg/mL) toxicity on HeLa cells, NIH-3T3 cells, and breast cancer cells (SKBR3, MCF7) demonstrated a dose- and time-dependent fashion for toxicity, where the longest exposure periods (48 h) and highest concentrations (400 mg/mL) are responsible for dramatically decreased cell viability [101].

Even though the exact correlation of exposure periods with graphene nanomaterials’ eventual reproductive toxicity remains somehow unclear, several studies have confirmed the pivotal role of graphene exposure periods on different species’ reproductive capacities. For instance, 25 μg/mL concentrations of GO had the ability to reduce sperm motility after 3 h of incubation, while shorter incubation periods were not associated with such alterations [95].

While toxicity is generally believed to be directly related to the time span of exposure, several studies have indicated that apoptosis-mediated cytotoxicity, unlike necrosis-mediated cytotoxicity, is independent of the duration of exposure [65,102]. Therefore, it can be concluded that underlying toxicity mechanisms may play a potentially influential role in the determination of time–toxicity correlation for graphene nanomaterials.

4.2 Physiological barriers

Graphene nanoparticles face several biological barriers in their way inside the body, which drastically affect their retention levels in different organs. BRB and blood–placenta barrier (BPB) are two of these physiological walls that influence the entry of different nanoparticles to both the reproductive system and fetus [23].

4.2.1 BTB and BEB

BTB, formed by the tight junctions between sertoli cells [103], and BEB are two of the tightest physiological barriers that provide crucial protection to male reproductive organs and obstruct the entry of various foreign particles [104]. Most germ cells reside within the closed section of the BTB and are rather safely protected against external materials; however, differentiating spermatogonia and stem cells are situated in the open part of the BTB and are thus more exposed to exterior materials [105]. Owing to their nano-scale dimensions, some nanoparticles possess the ability to penetrate through BTB and BEB; reside inside the testes, epididymis, or neighboring tissues; and alter sperm morphology and impair the spermatogenesis process upon entry via the circulation system [106109]. Accumulation of nanoparticles inside the testes or epididymis not only affects the quality of sperms and the individual’s fertile capacities but also holds notable potential to induce genotoxic effects in the stem cells and sperms and hence increases the risk of fatal defects and malfunctions as well as hereditary mutations and disorders in the offspring [110,111]. The current literature offers insightful yet somewhat conflicting data regarding the penetration of different nanoparticles through the aforementioned barriers. For instance, intramuscular injection of TiO2 nanoparticles (2.5, 5, and 10 mg/kg BW) for 90 days was followed by decreased sperm quality, hormonal alterations, and reproductive toxicity in sequence to BTB penetration and testicular accumulation [112]. However, IV administration of TiO2 nanoparticles (0.1, 1, 2, and 10 mg/kg BW, weekly for 4 weeks) displayed no detectable Ti accumulation in the testes of mice, in contrast to their livers [113,114]. Evidence has shown that BTB penetration can also be followed by reproductive toxicity via altering the body or organ’s weight [115]; however, these data too suffer from a lack of consistency in reported results, which can be explained with the variations between exposure routes and duration, types of nanoparticles, concentrations, and the interplay of those factors with host genomic profile, epigenetic factors, and molecular interactions between the nanoparticles and cellular compartments upon entry. IV injection of Ag nanoparticles (5 and 10 mg/kg BW) with diameters of 20 nm to male Wistar rats did not change their testes weight nor impaired their reproductive capacity [116]. Further confirming these results, when administered orally, Ag nanoparticles (15 and 50 µg/kg BW) with a size of 60 nm also showed no sign of altered body or organ weight in male Wistar rats [117]. Conversely, another study reported the occurrence of male rat reproductive toxicity through decreased testes and epididymis weight as well as body weight upon 7 and 28 days of sub-dermal exposure with 50 mg/kg BW Ag nanoparticles, respectively [118]. Concluded from these results, the inarguable impact of administration route, dose, and size of nanoparticles on the obtained results is evident. Very limited studies have been conducted regarding the passage of graphene derivatives through BTB and BEB; however, penetration of BTB and BEB has been reported to be of major difficulty for GO particles of 54.9 ± 23.1 nm diameters after intra-abdominal injection. Consequently, even high concentrations of GO (300 mg/kg) were observed to be incapable of altering the sperm quality in mice [81].

4.2.2 BPB

In addition to the toxicity of graphene materials on different species’ reproductive health, graphene may also disrupt fetal development by crossing through the BPB and passing from the maternal circulation system to the fetus. The importance of this barrier lies upon its crucial role in the exchange of nutrients, metabolic wastes, and hormones between the fetus and the mother [119], as well as serving as a protective barrier to the fetus that prevents the entry of numerous external particles. However, recent evidence has claimed that the protection provided by the placental barrier toward the entry of carbonaceous nanoparticles is not as strong as the previously mentioned barriers [120]. Particle properties and chemistry are two critical factors that to some extent control this phenomenon, as hydrophilic molecules and particles with more than 1 kDa mass are incapable of crossing this barrier [121,122]. In general, regardless of the wide range of nanoparticles that are capable of transferring through this barrier [123125], some of them might not accumulate inside the fetus [94], while some others have the ability to induce teratogenic effects and developmental toxicity via accumulation inside the fetus’ organs [123]. For instance, IV administration of 6.25 and 12.5 mg/kg concentrations of rGO nanosheets (20–150 nm or 200–1,500 nm, single layered or 3–5 layered) to pregnant mice that was ∼20 days into gestation was followed by dramatically increased miscarriage compared to treated dams in earlier phases of gestation (∼6 days). On the other hand, IV injection of 25 mg/kg rGO to pregnant dams that was 20 days into gestation led to the death of nearly all of them, but exhibited almost no significant toxicity in pregnant mice in early gestation phases, except for a few fetal deformities. This study also claimed that IV administration of rGO nanosheets to female mice ∼30–35 days before cohabitation did not alter the reproductive health of female rats. Moreover, histopathological examinations of the mice placenta exhibited very slight placental damage, thus indicating that only a few rGO nanosheets managed to penetrate through this barrier and the occurrence of dose-dependent toxicity was mostly executed to the embryos through the disruption of their mothers’ health [126].

Even though particle size [127129], exposure route and duration, chemical functionalization of nanoparticles [124,125,130], and maternal physiological and pathological conditions [131] contribute greatly in the determination of whether a nanoparticle, for example, graphene, may or may not cross through the BPB [121,132134], more investigations are required to evaluate their precise relationship with the translocation of graphene into this barrier and their eventual impact on graphene derivatives’ reproductive toxicity.

4.3 Surface functionalization

Being directly influenced by surface chemical properties, biomedical applications of graphene derivatives remain largely dependent on the existing functional groups on its surface that, to a notable extent, determine their eventual interactions with biological components as well as recognition processes [135]. Pristine graphene, as a pure form of graphene that solely composed of sp2-hybridized carbons, suffers from inherently insufficient water solubility and chemical reactivity. GO, on the other hand, is one of the most popular functionalized derivatives of pristine graphene that offers extensive applications (such as DNA or drug bioconjugation for gene and drug delivery purposes) due to the existence of carboxyl, epoxy, and hydroxyl groups on its periphery and basal planes that grant this unique nanomaterial the advantage of colloidal stability, water dispersibility, and hydrophilicity, as well as promoted covalent bond formation and facilitated chemical reactions [135138]. The importance of altering graphene nanomaterials surface chemistry and hydrophilicity also lies upon the consequent biological effects and reactions as a result of varying ionization degrees and dispersibility in physiological fluids, which not only impact graphene’s uses but also shape the state of graphene’s toxicity in different mediums [136,138]. All in all, surface chemical modification is often carried out with the aims of enhancing biocompatibility and stability, leading applications toward desired outcomes, accelerated therapeutic capabilities, target-binding efficacy, and attenuating toxicity [136].

Surface functionalization of pristine graphene and GO has been shown to decrease detrimental toxicity to a desirable extent in vitro [139]. PEG [140] polyvinyl alcohol, PEGylated poly-l-lysine (PLL) [141], and dextran [142] are some of the commonly exploited macromolecules for functionalizing graphene’s structure that enhances its biocompatibility and reduce its potential toxicity. For instance, PEGylated GO with doses of up to 100 μg/mL was reported to be safe toward glioblastoma cells (U87MG), breast cancer cells (MCF-7), human ovarian carcinoma cells (OVCAR-3), colon cancer cells (HCT-116), and lymphoblastoid cells (RAJI) [143145], and induced less cytotoxicity in human lung fibroblast cells in comparison with GO (at a concentration range of 1–100 μg/mL) [146]. PEG coating of GO was shown to significantly reduce acute tissue impairment by reducing the aggregation and retention of GO in lungs, liver, and spleen and also accelerated its elimination from the body [147]. Exposure of human liver cell lines (HL-7702), human lung fibroblast cell line (MRC-5), and human macrophage cell line (U937) with 200 µg/mL PEGylated GO was also suggested to protect the cell growth from inhibition by 30% and reduce DNA toxicity by 40% [148]. Dextran, a branched glucan utilized in diverse biotherapeutic applications, remarkably decreased the inhibition of Hela cell proliferation when conjugated to GO, in comparison with unfunctionalized GO [142]. Moreover, modification of GO with chitosan was also reported to reduce RBC lysis and hemotoxicity [149]. The root of reduced toxicity of macromolecule-conjugated graphene materials may be potentially linked with graphene’s inherent phospholipid extraction/insertion ability caused by notable surface dispersion interactions with cellular membrane lipids, and salient coverage of its surface by large macromolecules that inhibit such dispersion interactions. Besides, the surface coverage of graphene derivatives helps to enhance their cellular recognition, uptake, and clearance [142] and also decreases graphene’s capability to induce cytotoxicity through extracting membrane cholesterols and disrupting calcium hemostasis or neurotransmission potentiation [150]. According to the results from an in vitro study on monkey renal cells, pristine graphene was associated with promoted apoptosis as it exhibited accumulation on the cellular membrane due to the formation of hydrophobic interactions with the cell membrane lipids, whereas carboxylated GO was mainly internalized into cells due to its higher hydrophilicity, did disrupt the cellular membrane, and therefore induced no toxicity at high concentrations (300 µg/mL) [139]. As demonstrated by Teo et al., exposure of A549 cells with halogen functionalized (GO–Cl, GO–Br, GO–I, and GO–F) graphene at 0–200 µg/mL concentrations was followed by a dose-dependent increase in cytotoxicity accompanied with increased halogenation [151]. Interestingly, halogenated graphene at 0–400 µg/mL concentrations displayed superior toxicity compared to GO. An explanation proposed for this result was related to the selective adsorption of crucial micronutrients on the hydrophobic surface of halogenated GO and insufficient nutrient availability to A548 cells due to halogenated GO’s more significant hydrophilicity [152]. Furthermore, different functionalized pristine graphene derivatives (G–COOH, G–NH2, and G–OH) with 0.1 mg/L concentrations were evaluated for their neurotoxic effects on SK–N–SH cells post 24 h exposure [153]. Obtained toxicity results were indicative of a G–OH ≈ G–COOH > rGO > G–NH2 ranking that deteriorated over longer exposure durations. However, G–NH2 possessed higher toxicity persistence in long term as a result of its increased potential for disrupting lipid and carbohydrate metabolism compared to other functionalized forms of pristine graphene. This finding further emphasizes the importance of long-term toxicity assessment of graphene derivatives, as they might be associated with insignificant short-term toxic effects but display persistent long-term toxicity due to slow biodegradation and internal accumulation [138]. Regarding the reproductive system, graphene toxicity has been evaluated and compared among pristine graphene, graphene-NH2, graphene-COOH, and also GO in C. elegans. Pertaining to the results, GO induced higher reproductive toxicity in comparison with other derivatives, among which there was a pristine graphene > NH2 > COOH order for the reported toxicity. It has also been previously suggested that covalent functionalizations (such as –COOH) are capable of reducing the toxicity of graphene nanoparticles through increasing their hydrophilicity and bio-clearance from the body [26,44,68].

Driven from these results, the essential role of graphene surface functionalization in altering their toxicity is emphasized; however, the lack of sufficient data regarding the role of surface functionalization in long-term reproductive studies highlights the urgent need for further experiments in this field to unravel the true nature of these exquisite materials’ toxicity.

4.4 Oxidation state

The oxidation state of graphene, to some extent, determines its chemical structure, cellular interactions, and cytotoxicity by affecting the carbon radical density on GO’s surface. The existence of unpaired electrons grants carbon radicals with higher reactivity compared to other chemical functional groups and therefore promotes their ability to generate more superoxide radicals, which can ultimately oxidize unsaturated lipids and thiol groups on proteins or glutathione (GSH) [154]. Henceforth, GO species with higher surface carbon radical density are considered to be associated with higher pro-oxidant activity, GSH depletion, and membrane LPO [155]. LPO is a process in which phospholipids and low-density lipoproteins undergo oxidation and result in the loss of cellular membrane integrity, membrane lysis, cell death, and consequently enhanced cytotoxicity [156]. Spermatozoa, polyunsaturated fatty acid (PUFA)-containing cells, are highly susceptible to LPO and spermatogenesis disruption as well as eventual reproductive dysfunction [156158]. The main underlying mechanism for the induction of LPO is considered to be oxidative stress, which disrupts the normal physiological functions of cells through promoting ROS generation. Excessive formation of ROS is inherently linked with decreased sperm count, abnormal shape, and overall reduced fertility and impaired oocyte penetration [159,160]. For instance, a study showed that increased LPO and oxidative stress are responsible for male boar infertility, but can be compensated with enhanced antioxidant enzyme activities. Accordingly, the epididymis head and testes were more prone to the acceleration of ROS formation and LPO. The spermatogenesis process consists of several stages including proliferation, maturation, and spermatozoa storage in epididymis, which are all subject to LPO in different phases [161]. Even though oxidative stress and LPO play an evidently crucial role in reproductive infertility, the specific correlation between the toxicity of graphene derivatives and alteration of LPO levels has not been studied in detail. However, it is important to note that the inherent toxic effects of LPO are not limited to the reproductive organs and may also negatively impact the functions of other cells and organs. For instance, in a study by Xia et al. [25], pristine GO, rGO, and hydrated GO (hGO) were evaluated for their LPO-mediated cytotoxic effects on THP-1 and BEAS-2B cell lines. Carbon radical densities of the utilized derivatives were ranked as hGO > GO > rGO, as hydration of GO was shown to be associated with an increase in ˙C density and C–OH groups and a decline in C–O–C groups, and reduction of GO resulted in a significant decrease in ˙C density. hGO contained the highest carbon radical density and therefore resulted in the most significant cytotoxicity and cell death through LPO and lysis of the cell membrane, whereas rGO exhibited the slightest cytotoxic effects in this study. Accordingly, LPO was measured for these materials and reported as 37, 17, and 5% for hGO, GO, and rGO, respectively.

These data notably reveal a distinct correlation between LPO-induced cytotoxicity of GO derivatives with surface oxidation, hydroxyl, carboxyl, and carbonyl groups, and carbon radical density. However, due to the intrinsic complexity that lies within the interplay of LPO and other cellular mechanisms and molecular pathways, the precise extent to which LPO influences the ultimate toxicity remains largely unknown.

4.5 Number of layers

In spite of their enhanced thickness, multilayered graphene sheets contain smaller volume-specific surface area than single-layered sheets and therefore exhibit varying colloidal attributes, different biological interactions at cell membrane interfaces, and different levels of cellular uptake [138]. Parallel alignment of GO sheets on cell membranes is associated with accelerated GO uptake by cells [68]; however, cellular uptake is highly likely to undergo remarkable deterioration with the increasing number of graphene layers [44]. Few layer graphene (FLG) is often described as graphene with 2–10 layers [162]. SLGO and FLGO have been reported to induce toxicity in a dose-dependent fashion; however, SLGO displayed more prominent dose dependency in comparison with FLGO. Evidence suggests that SLGO is more likely to form interactions with biological components than FLGO, probably because of the higher number of layers in FLGO that accounts for enhanced stiffness and subsequently reduced biological adsorption [68]. In contrast to these results, an in vitro study that investigated the genotoxic effects of graphene derivatives at 10 and 50 mg/L concentrations after 24 h of exposure with human bronchial epithelial cells revealed that FLGO with ∼4–8 layers induces more significant genotoxicity than SLGO owing to increased DNA methylation [163].

4.6 Lateral dimension

Ranging from 10 nm to almost 10 µm, the size of graphene sheets plays a critical role in determining the fate of these unique nanomaterials in physiological mediums. Despite the controversy regarding varying cellular responses after internalization of graphene derivatives with different lateral dimensions, it is well established that cells struggle to uptake large nanoparticles and internalize them via different pathways based on their sizes [164166]. For instance, small graphene sheets can penetrate into cells directly, while large graphene sheets enter cells via the formation of hemispherical lipid vesicles [166]. Moreover, smaller GO sheets were observed to be capable of being internalized by human-derived macrophages a lot more than larger sheets [167]. Another study indicated that upon IV administration of graphene in vivo, a notable amount of larger FLG sheets (330–630 nm) were degraded in the liver of mice by Kupffer cells after 180 days, whereas the smaller sheets (20–40 nm lateral size) that entered and accumulated in the liver had not decreased due to their slower biodegradability [168]. As opposed to these results, the accumulation of large graphene sheets with ∼500 nm lateral dimensions in zebrafish was demonstrated to be significantly higher than smaller graphene sheets with ∼30 nm diameters [169]; however, the majority of smaller sheets were accumulated in zebrafish liver and gut, while larger sheets were mainly detected only in the gut. Furthermore, a study by Heo et al. showed that human dermal fibroblast cells effectively internalized needle-like graphene with 1 mm length in spite of their large dimensions [170]. In striking contrast with previous results, despite their highly diverse sizes, GO with 2 µm (4.05 nm average height) and 350 nm (3.9 nm average height) lateral diameters and 1 nm thickness have displayed similar amounts of uptake by macrophages through antibody opsonization and phagocytosis, thus following a size-independent pattern for macrophage uptake. However, micro-sized GO revealed slighter biocompatibility and hence initiated stronger inflammatory responses compared to nano-sized GO [164].

4.7 Cellular uptake, interactions, and accumulation

As previously mentioned, physiochemical properties of graphene derivatives including hydrophilicity, size, and carbon radical density are among the factors that play a major role in their uptake and interactions with biological compartments and influence their final toxicity [171]. Being closely interconnected with particle internalization inside cells, the size of GO flakes can adversely affect their uptake by cells, as larger GO flakes have been observed to limit cell uptake, while smaller GO flakes were associated with facilitated internalization by cells [172]. Cellular accumulation is yet another factor that affects the cytotoxicity caused by graphene nanomaterials. Accordingly, graphene has an amphiphilic nature with hydrophilic edges and a hydrophobic planar structure, owing to which GO and hGO were actively accumulated close to THP-1 macrophage membranes without significant cellular uptake [173], while rGO was reported to be internalized and accumulated inside cells due to its reduced hydrophilicity at edges and enhanced overall hydrophobicity. Consistent with these results, another investigation has reported that chemical reduction of GO is associated with promoted cellular uptake as well as clearance at injection sites [174]. Of note, it should be taken into consideration that several studies have reported the localization of pristine GO inside the cell cytoplasm [175177], which could be explained with the difference of exploited cell types; however, other important parameters such as size, surface charge, and surface functionalization should not be overlooked regarding their vital roles in the eventual cellular uptake and accumulation [178].

Uptake of graphene by cells can occur through different routes [37,64]. Nanoparticles with less than 100 nm diameters are capable of entering cells, while nanoparticles with less than 40 nm diameters can also enter the cellular nucleus [179]. Graphene quantum dots are capable of entering cells by direct penetration into the cellular membrane and do not require energy-dependent pathways [180,181], while protein-coated graphene nanoparticles (∼500 nm) enter cells via cathrin-mediated endocytosis and larger protein-coated graphene nanoparticles (∼1 µm) enter cells through phagocytosis [175]. GO sheets can be adhered to cell membranes, enter and localize between the phospholipidic bilayers of the cell membrane, and also be internalized inside cells [182]. Before accumulation inside organs, it is claimed that larger graphene derivatives, such as micro-scale graphene materials (10–30 µm), are carried within the blood circulation system at a milder speed than small graphene quantum dots (3–5 nm) and hence are more likely to induce long-term toxicity upon exposure [183]. In general, any alterations of graphene’s physiochemical characteristics pre- or post-synthesis, such as sharp edges and structural defects, may serve as highly influential factors that can apply changes to the molecular behaviors and interactions of graphene in vitro and in vivo conditions [184]. For example, sharp edges of graphene can act as highly damaging sites that can facilitate membrane penetration through physical impairment of the cellular membrane [185], while the formation of nanoholes on graphene’s planar surface during a reduction process may impact its rigidity, hydrophilicity, and its eventual toxicity [185].

Despite the ever-increasing evidence confirming the substantial role of graphene materials’ physiochemical properties in determining their eventual toxic effects, it is important to consider the difficulty of controlling such characteristics, including particle geometry and oxidation state, in a homogenously distributed manner without altering other properties of the nanomaterial. Characterization heterogeneity, nonuniform functionalization, and inter-lab differences in carrying out synthesis and characterization processes are some of the most crucial factors that might shift experimental results toward unexpectedly varying outcomes. For instance, nonuniform functionalization can often alter particle hydrophilicity and thus affect the cellular uptake, accumulation, and interactions at nano-bio interfaces, which in turn enforces a notable impact on the eventual toxicity of graphene nanomaterials [138] (Figure 3).

Figure 3 
                  Main underlying factors that influence graphene toxicity.
Figure 3

Main underlying factors that influence graphene toxicity.

5 Underlying mechanisms behind graphene toxicity

5.1 Physical impairment of the cell membrane

The physical damage to the plasma membrane upon interaction with graphene derivatives is one of the most crucial mechanisms for induced cytotoxicity by graphene [13,186,187]. As shown in a study by Akhavan and Ghaderi [188], graphene forms strong hydrophobic bonds with the cell membrane, which results in cytoskeletal dysfunction and then cuts through the cell membrane with its sharp edges, which may lead to the release of intracellular materials. Zhou and coworkers [189] further illustrated that pristine and oxidized graphene nanosheets (200–700 nm lateral dimensions and 1 nm thickness) at 10, 50, and 200 μg/mL GO concentrations attenuated A549 and Raw264.7 viability by recruiting membrane phospholipids, reducing cell surface hydrophobicity, and forming water-permeable pores along the surface of cell membranes. By employment of a computer-assisted approach, this investigation confirmed the previously reported results [190] on edge-dependent perforation manner due to the edges’ exceptional sharpness and lipid-extracting ability. However, single sheets of graphene seemed to lack the ability to form holes on the cell membrane, as consistent with previous simulations that disclosed that single-layer graphene sheets could not create holes on membranes to a nonrestorable extent [191,192].

5.2 Oxidative stress

Oxidative stress, as fostered by the overproduction of free radicals and/or depletion of antioxidant enzymes, is generally considered to be both a cause and an outcome of pathogenic mishaps or negatively altered physiological conditions. Due to its crucial contribution in the occurrence or progress of inflammatory [193], auto-immune [194], gastrointestinal [195], and reproductive disorders [196], it is often measured in both in vivo and in vitro toxicological investigations to estimate the induction of toxicity based on enzymatic elevations or ROS production. Graphene and its derivatives are considered to take part in the initiation of accelerated oxidative stress that is not able to be replenished by key ROS-scavenging enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GPx) [13,187,197], which might henceforth damage lipid structures, impair DNA, induce mitochondrial dysfunction and protein denaturation [197200], and also disrupt metabolic pathways [13,199]. Moreover, the activity of different antioxidant enzymes such as SOD and GSH-PX were reported to have decreased after exposure with GO dose dependently [45,64,143]. Interestingly, a group of researchers claimed that even though low GO concentrations of up to 200 μg/mL proved to be capable of escalating oxidative stress levels in human lung carcinoma epithelial cell line A549, they served as adequately safe materials in vitro as no obvious toxicity was spotted in the duration of that experiment [143]. Oxidative stress is also closely related to apoptosis and genotoxicity as previously shown in a study on HLF cells in exposure with GO [146]. An investigation carried out regarding the effects of two key oxidized graphene derivatives (GO and rGO) on HepG2 cells demonstrated that both graphene species elicited oxidative stress. Accordingly, rGO-mediated ROS production was induced by NADPH oxidase alterations and accompanied with enhanced expression of antioxidant enzyme genes (SOD1, SOD2, CAT, GSTA1, and GSTA4), whereas physical interactions played the main role as an underlying factor in GO-initiated oxidative stress [65].

5.3 Mitochondrial impairment

While graphene nanomaterials have the ability to damage cell viability by inducing oxidative stress and damaging cell membranes, they can also directly induce apoptosis or necrosis by altering the normal functions of cell mitochondria [201,202]. Accordingly, graphene nanomaterials are capable of increasing oxygen consumption levels of mitochondria and activating mitochondrial pathways that eventually trigger apoptosis [203]. For example, GO has been reported to increase the electron supply to specific mitochondrial complexes and thus promote the generation of ROS in MHC cells [204]. The increase in the generation of ˙OH radicals after exposure to GO is also capable of inducing oxidative stress as well as thermal stress, which can lead to mitochondrial respiration damage and cause consequent cytotoxicity [141].

5.4 DNA damage

Graphene derivatives may induce DNA damage or genotoxicity, which can develop into different diseases later in life. Not only genotoxicity can trigger carcinogenesis but it can also threaten the reproductive health as well as offspring health if these genotoxic effects take place in the reproductive cells [115,205]. During cell mitosis when the nuclear membrane breaks, GO can interact with DNA without even entering the nucleus. GO may initiate various genotoxic effects such as chromosomal fragmentation, breaking DNA strands, point mutations, and other types of DNA alterations [181,206210]. These DNA alterations can be caused by GO through oxidative stress pathways or inflammatory responses, which are triggered by the activation of several intracellular signaling pathways such as MAPK, TGF-β, and NF-κB [65,211,212]. The molecular interactions between graphene and DNA strands may change the natural form of end bases of the DNA strands and thus result in increased genotoxicity [213]. It is also of significant importance to mention that graphene usually deploy intercalation and cleavage mechanisms to interact with a cell’s genome [211]. Morphological and structural alterations in graphene derivatives can ameliorate their potential to enter cell organelles and also come in physically close contact with the nuclear genome [214]. Furthermore, graphene and rGO can play a pivotal role in promoting the expression of different genes such as p53, Rad51, and MOGG1-1, which impair the DNA [211]. The toxicity of GO flakes with 1.32 μm and 130 nm sizes and concentrations of 10, 50, and 100 μg/mL were further evaluated on a similar cell line after 24 h of incubation. Obtained results were indicative of slightly declined A549 cell viability upon exposure with both sizes of GO. However, 130 nm sized GO flakes appeared to enforce significant genotoxicity on cells even at lower doses [214]. According to recent advances in the field of graphene genotoxicity, it has been revealed that even though both GO and rGO were able to induce harm to single- and double-stranded DNA, rGO did not remarkably affect the expression of DNA repair genes in HepG2 cells [65].

5.5 Inflammation

Graphene derivatives can trigger the release of various chemokines and cytokines that result in the secretion of Th1/Th2 cytokines and thus trigger the initiation of inflammatory responses [215,216]. Pristine graphene [216] and rGO [65] can also induce inflammatory responses by activating the NF-κB signaling cascade [217]. Graphene-derived nanomaterials have also been indicated to induce inflammatory responses such as pulmonary edema and the formation of granuloma [100,218].

5.6 Apoptosis

Apoptosis, as a crucial gene-regulated destructive function, is another mechanism by which graphene derivatives have the capability of inducing toxicity [37,219]. Besides from the complex interconnections of apoptosis with oxidative stress and physical damage, apoptosis and/or necrosis can take place as the result of DNA damage or disrupted mitochondrial activities [178]. Graphene nanomaterials possess pro-apoptic properties that at certain doses might critically threaten the viability of cells [215,220222]. For instance, GO and rGO were proved to induce inflammation and apoptosis in mouse pulmonary tissue followed by exposure via inhalation [204]. In addition, even low doses of rGO were shown to induce apoptosis via triggering the death receptor as well as the canonical mitochondrial pathway [65]. Chemically functionalized graphene quantum dots (GQD–NH2, GQD–COOH, and GQD–CO–N(CH3)2) at concentrations up to 200 μg/mL were shown to not induce notable cytotoxicity to the A459 cell line through triggering apoptosis and/or necrosis. However, despite their small diameters, GQDs were mostly accumulated in the cytoplasm to have not entered the cell nucleus [65]. Furthermore, rGO and GO were reported to trigger apoptic pathways in HepG2 cells in somewhat different manners. For instance, rGO increased apoptosis at low concentrations and early times during exposure, contrarily to which, GO led to the elevation of principal apoptosis-related genes and elicited apoptosis through a concentration and time-dependent fashion [65]. Results from another study were indicative of rGO’s apoptosis-mediated toxicity in U87 and U118 glioma cell lines, which was notably higher than that of GO, thus proving that GO may have a milder toxic profile toward glioma cells [49].

5.7 Necrosis

Necrosis, a type of cell death often followed by cell damage or inflammation, is another toxicity induction pathway in response to exposure with the graphene nano-family. For example, the combination of GO and CDDP was revealed to cause necrosis, which led to the loss of viability in CT26 cells [223225]. Similarly, GO can partially trigger TNF-α production and result in subsequent macrophagic necrosis and death [226]. In regards with pristine graphene, it has been reported that graphene, by reducing the mitochondrial membrane potential (MMP) and increasing the generation of ROS, activates mitochondrial pathways and induces cytotoxicity in murine macrophage cells [37]. Strikingly, HeLa cells showed no sign of obvious signs of apoptosis or necrosis-mediated toxicity after incubation with 160 μg/mL PEGylated GQDs, and no increased ROS generation was reported. PEG polymer coating and the small dimensions of GQDs (below 5 nm) were addressed as the underlying factors that efficiently reduced toxicity in this study [197]. In contradiction with the previous results, O-GNR-PEG-DSPE (oxidized-graphene nanoribbons with an amphiphilic polymer) with 400 μg/mL concentration reduced HeLa cells viability drastically in parallel with the release of LDH [101]. As stated in the experiments of Jaworski et al., highly malignant human glioma cells (U87 and U118) were subject to 24 h exposure with graphene platelets at 100 µg/mL concentrations. Followed by treatment with graphene, the number of vital cells dropped down by 54 and 58% and apoptosis surged strikingly by 68 and 99% in U87 and U118, all in a respective order. Regarding necrosis, however, a 24% increase was detected only in U87 glioma cells [101].

In summary, different mechanisms can be deployed by the infamous graphene nanofamily to induce toxicity, each of which possesses the ability to independently alter the viability of cells through interconnected cellular pathways. However, it is important to take into deep consideration their correlations and potentially joint activities of these complex mechanisms to elicit toxicity and eventual cell death, as their signaling pathways are often cross-modulatory. For instance, TLRs, MARKs, TGF-β, and TNF-α are among the most common toxicity induction pathways that can be closely interrelated with each other, thus making it difficult for scientists to point to one specific mechanism as the only pathway responsible for the induction of toxicity [23] (Figure 4).

Figure 4 
                  (a) Formation of hydrophobic bonds between graphene and the cell membrane; (b) localization of graphene between phospholipidic layers of the membrane; (c) cutting through the cell membrane with sharp graphene edges; (d) entering of graphene into cell plasma through the formation of endosomes; (e) internalization of graphene inside the plasma and induction of cytotoxicity via different pathways; (f) internalization of graphene inside mitochondria and inducing mitochondria-mediated toxicity; (g) internalization of graphene inside the nucleus and induction of genotoxicity; and (h) internalization of graphene inside exosomes.
Figure 4

(a) Formation of hydrophobic bonds between graphene and the cell membrane; (b) localization of graphene between phospholipidic layers of the membrane; (c) cutting through the cell membrane with sharp graphene edges; (d) entering of graphene into cell plasma through the formation of endosomes; (e) internalization of graphene inside the plasma and induction of cytotoxicity via different pathways; (f) internalization of graphene inside mitochondria and inducing mitochondria-mediated toxicity; (g) internalization of graphene inside the nucleus and induction of genotoxicity; and (h) internalization of graphene inside exosomes.

6 Gaps and insights

The majority of currently available evidence regarding toxicity mechanisms is related to cells or organs other than the reproductive system, the reason for which lies in the insufficiency of experimental data and the difficult nature of related investigations. We believe that a large amount of addressed data can be possibly related to the reproductive system as well, since a lot of intracellular signaling pathways are similar between different cell lines. However, even the slightest of differences between cells should not be overlooked, as they can affect the behaviors of cells and final toxicity in exposure with graphene derivatives. Moreover, differences between the male and female reproductive system call for more thorough experiments, in which physiological conditions and barriers of the reproductive systems in both genders are taken into deep consideration. As discussed in the previous sections, the entry of graphene through physiological barriers of the reproductive system such as blood–testes barrier and BEB are crucially important topics that remain in apparent infancy and require further studies. Similarly, the placenta–blood barrier can also be subject to further evaluations in which its ability to prevent graphene nanomaterials from entering the fetus’ body in different phases of pregnancy is of significant importance.

Last but not least, we believe that to fully explore the nature of graphene and its effects in biological and physiological systems, more attention should be shifted from short-term affects to long-term toxicity of graphene, as they may prove to be safe in a short-term experimental model, but display life-threatening effects to the offspring.

7 Conclusion

With the emergence of graphene as one of the most exquisite nanomaterials that are utilized in various fields of medicine, biotechnology, engineering and environment, serious concerns are rapidly arising regarding graphene’s potential hazards to public health. While graphene derivatives have exhibited desirable safety in certain concentrations, there is still a long debate about the exact doses and conditions under which the graphene nanofamily does not elicit toxicity. Owing to the previous reports regarding graphene toxicity in various cell lines and organs, the path to exploring graphene materials’ harmful impacts in the reproductive system can take a much faster and detailed route. However, due to the existence of numerous substantial challenges (precise controlling graphene’s physiochemical characteristics, sensitivity and error-susceptibility of female reproductive system studies, difficulty of fetus assessment studies, and the complex multifactorial essence of reproductive toxicity studies), this field to this day remains as one of the least-investigated aspects of graphene toxicity. While it is generally of notable difficulty to evaluate the long-term toxicity of graphene, especially in concerns with the toxicity of graphene on the reproductive system and health of next generations, we still believe that it is crucially urgent to further explore this topic. Therefore, we aimed to provide readers with a detailed review of the existing literature related to this subject as well as mention some of the major gaps in the current data to further elucidate the importance of this topic and encourage more investigations to be conducted in this field.


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  1. Funding information: The authors state no funding involved.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Conflict of interest: The authors state no conflict of interest.

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Received: 2021-07-17
Revised: 2021-10-06
Accepted: 2022-01-04
Published Online: 2022-03-04

© 2022 Nastaran Hadizadeh et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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