An overview on the reproductive toxicity of graphene derivatives: Highlighting the importance
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Nanotechnology Reviews 2022; 11: 1076–1100
Review Article
Nastaran Hadizadeh, Saba Zeidi, Helia Khodabakhsh, Samaneh Zeidi, Aram Rezaei,
Zhuobin Liang, Mojtaba Dashtizad*, and Ehsan Hashemi*
An overview on the reproductive toxicity of
graphene derivatives: Highlighting the
importance
https://doi.org/10.1515/ntrev-2022-0063 term toxic effects. Given the crucial importance of gra-
received July 17, 2021; accepted January 4, 2022 phene’s reproductive toxicity, more attention has been
Abstract: With the glorious discovery of graphene back in recently shifted toward this subject; however, the existing
2004, the field of nanotechnology was faced with a break- literature remains insufficient. Therefore, we have con-
through that soon attracted the attention of many scientists ducted this review with the aim of providing researchers
from all over the world. Owing to its unique bidimensional with assorted information regarding the toxicity of gra-
structure and exquisite physicochemical properties, gra- phene derivatives and their underlying mechanisms, while
phene has successfully managed to cave its way up to the mentioning some of the major challenges and gaps in the
list of the most investigated topics, while being extensively current knowledge to further elucidate the path to exploring
used in various fields of science and technology. However, graphene’s true nature. We hope that our work will effec-
serious concerns have been raised about the safety of gra- tively give insight to researchers who are interested in this
phene, for which numerous studies have been conducted to topic and also aid them in completing the yet unfinished
evaluate the toxicity of graphene derivatives in both in vitro puzzle of graphene toxicity.
and in vivo conditions. The reproductive toxicity of gra- Keywords: graphene, reproductive toxicity, in vivo
phene 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-
1 Introduction
As a unique, two-dimensional nanostructure that is mainly
* Corresponding author: Mojtaba Dashtizad, Department of Animal composed of sp2 hybridized carbon atoms, graphene has
Biotechnology, National Institute of Genetic Engineering and gained tremendous recognition ever since its initial isolation
Biotechnology, Tehran 14965-16, Iran, e-mail: dashtizad@nigeb.ac.ir, in 2004 [1,2]. Characterized by their exceptional physico-
tel: +98-88631298, fax: +98-88220052 chemical properties, high electron mobility, and incredible
* Corresponding author: Ehsan Hashemi, Department of Animal
tensile strength, this inherently honeycomb-shaped family
Biotechnology, National Institute of Genetic Engineering and
Biotechnology, Tehran 14965-16, Iran; Diabetes Research Center,
of nanomaterials are now being extensively exploited in
Endocrinology and Metabolism Clinical Sciences Institute, Tehran various fields of biotechnology (Figure 1) [3], drug delivery
University of Medical Sciences, Tehran, Iran; Institute of Molecular [4], tissue engineering [5], and cancer treatment [6], as well
Physiology, Shenzhen Bay Laboratory, Shenzhen 518132, China, as electronics [7], photo-sensors [8], and solar batteries
e-mail: E_hashemi@nigeb.ac.ir [9–13]. The sp2 orbitals in graphene consist of px, py, and
Nastaran Hadizadeh, Saba Zeidi, Helia Khodabakhsh,
pz orbitals, among which the latter can form π bonds with a
Samaneh Zeidi: Department of Animal Biotechnology, National
Institute of Genetic Engineering and Biotechnology, Tehran 14965- wide range of organic and inorganic materials, while also
16, Iran; Diabetes Research Center, Endocrinology and Metabolism providing the opportunity for surface modifications [14–16].
Clinical Sciences Institute, Tehran University of Medical Sciences, Chemical modification and surface functionalization
Tehran, Iran have actively conferred to this already-distinctive nano-
Aram Rezaei: Nano Drug Delivery Research Center, Health
material the potential to exceed its applicability limita-
Technology Institute, Kermanshah University of Medical Sciences,
Kermanshah, Iran
tions, as well as the capability of being tuned in for use in
Zhuobin Liang: Institute of Molecular Physiology, Shenzhen Bay additional fields of science and technology, and get one
Laboratory, Shenzhen 518132, China step closer to becoming immensely blended in our
Open Access. © 2022 Nastaran Hadizadeh et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0
International License.
Reproductive toxicity of graphene derivatives 1077
Figure 1: Utilization of graphene in different fields of biomedicine.
everyday lives. Therefore, numerous studies have been doses [23–26]. With that in mind, it is also expected
continuously conducted with the aim of investigating for different graphene derivatives to be associated with
this phenomenon and creating new graphene derivatives varying degrees of toxicity [23].
[17]. Graphene oxide (GO), reduced graphene oxide (rGO), Furthermore, targeted organs are yet another critical
and other types of functionally modified graphene deriva- factor that impacts the level of graphene-induced toxi-
tives are other residents of this family that can be pro- city. Metabolism rate, blood circulation, and physiolog-
duced in various forms of 2D nanosheets, nanoflakes ical barriers defenses of an organ are all complicated
and spheres, nanoplatelets, nanofibers, or 3D hydrogels factors that differ from organ to organ, species to species,
and nanocomposites depending on their desired applica- and perhaps even within the same species. Despite the
tions [18,19]. Coming in a wide range of shapes, graphene complexity of all these elements and their possible inter-
derivatives are also available in a variety of sizes ranging connections, it is important to consider their potential
from a few nanometers to several meters, some of which influence on the accumulation and toxicity of graphene.
consist of a limited number of layers (≤5 layers) and mostly A major proportion of studies in this field have investi-
referred to as few-layered graphene (FLG), while some gated the toxicity of graphene in several organs such as
others are produced in more cost-effective bulk forms [20]. lungs [27], liver [28], spleen [29], and kidneys [30], while
Emerging from the extensive utilization of graphene studies regarding graphene toxicity on the reproductive
and our ever-increasing exposure to its derivatives, system remain limited. The multifactorial nature of repro-
serious concerns have been raised regarding its safety ductive studies, sensitivity of embryos, difficulty of long-
toward animals, humans, and the environment. The pos- term few-generation studies, and unpredictable interplay
sibility for the occurrence of undesired and unpredicted between genetic and environmental variables may be
interactions of graphene with different biological com- some of the explanations for the lack of sufficient data
partments has recently led research toward a more toxi- on this subject when compared to other organs. However,
city-based outlook and has also promoted more cautious long-term and short-term reproductive toxicity of gra-
exploitation of graphene as it might induce temporary or phene in the gametes and offspring of those who were
permanent damage to cells, tissues, and organs of dif- in contact with these nanomaterials is an essentially
ferent species [21,22]. Several studies have noted that pivotal matter as they may prove to be harmless to
the toxicity of graphene is in close correlation with a an individual at specific doses and exposure periods,
variety of its characteristics including shape, size, and but have the ability to induce toxicity to the following
state of oxidation, functional groups, methods of synthe- generations and exhibit potential damage in prolonged
sis, as well as administration routes, exposure time, and periods of time due to their accumulation and slow
1078 Nastaran Hadizadeh et al.
bio-degradation [31]. Therefore, the safety and toxicity exposure with pristine graphene (2–3 nm thickness and
levels of graphene nanomaterials are among important 500–1,000 nm size) at 5–100 mg/mL concentrations has
subjects that have to be taken into consideration at the been associated with Murine RAW 264.7 macrophage cyto-
time of investigating this subject. toxicity in sequence to increased oxidative stress, apop-
Despite being investigated for less than a decade, tosis, and mitochondrial membrane potential damage [37].
numerous studies now exist in this field, some of which
have displayed contradictory results. With a clear under-
standing of those results and the apparent yet minor
inconsistencies in the current literature, this study aims 2.2 GO
to provide a comprehensive overview of existing data
about the reproductive toxicity of graphene nanomater- Produced from the oxidation of graphene, GO is an amor-
ials and their possible mechanisms while avoiding gen- phous material with berthollide characteristics such as
eralized or biased information to shine light on the path nonstoichiometric atomic composition [38]. GO might
to further exploring the true nature of graphene’s repro- possess a diverse range of functional groups including
ductive toxicity [32]. hydroxyl, carboxyl, and epoxy that vary depending on
the employed synthesis methods and oxidation condi-
tions [39,40]. The state of oxidation in GO and the type
of functionalization are pivotal factors that impact GO’s
2 Introducing main graphene physiochemical properties and enhance its potential bio-
medical applications such as a facilitated bioconjugation
derivatives with peptides, drugs, and antibodies [41]. However, this
advantage is accompanied by its inherent drawback of
2.1 Pristine graphene toxicity, as GO is capable of interacting with natural cel-
lular components such as proteins or DNA and therefore
Pristine graphene, as an unoxidized form of graphene disrupts their normal physiological function [42]. For
consisting of episodic hexagonal carbon structures, owns instance, highly oxidized nano graphene oxide (NGO) is
a remarkably high surface area of ∼2,630 m2/g that declines a monolayer graphene sheet that possesses a high number
in parallel with an increase in the number of graphene of oxygen-containing functional groups on its edges and
layers [14–16]. Graphene monolayers also benefit from basal plane [43]. Due to its significant physicochemical
exceptional mechanical resistance as a result of strong properties such as the ability to absorb aromatic drug
C–C sigma bonds with very short interatomic lengths (about molecules as a result of owning two aromatic planes,
1.42 nm) within the 2D plane of graphene, which subse- NGO is considered to be a highly efficient nanomaterial
quently turn them into promising candidates for a variety suited for numerous biomedical applications [12]. Despite
of constructional and electromechanical applications [33–35]. the advantages that NGO conveys to the field of biomedi-
Each carbon atom in pristine graphene consists of four cine, a previous study has displayed that NGO possesses
valence electrons that can be shared with other atoms or the highest toxicity among graphene derivatives through
molecules through covalent bonds. Each of these electrons the induction of oxidative stress and increasing reactive
can be hybridized in sp, sp2, and sp3 forms, among which oxygen species (ROS) generation [44]. For instance, blood
sp2 contains px and py orbitals as well as a critical pz orbital cell surface interactions with NGO, even without penetra-
with a single electron that forms a half-filled weak π bond. tion into RBCs, can lead to the alteration of RBCs’ polarity
Being situated in a perpendicular position above the struc- and permeability, disrupt normal cell membrane func-
tural plane of graphene, this half-empty orbital plays a major tions, and cause RBC hemolysis as a result of excessive
role in determining the physiochemical properties of gra- electrostatic interactions with NGOs [45].
phene and grants it the ability to participate in a broad range
of chemical reactions [14–16]. Moreover, the half-filled π
bonds in graphene create a zero bandgap between the
valence and conductive bands that enable electrons to 2.3 rGO
move freely and also result in the formation of weak van
der Waals interactions between graphene monolayers that rGO can be produced through the reduction of GO; how-
facilitate their gentle movement on each other when sub- ever, complete elimination of GO’s functional groups
ject to weak shear stress [1,36]. Regarding its toxicity, 48 h (–COOH, –OH, –COH) is rather unlikely, and the final
Reproductive toxicity of graphene derivatives 1079
rGO often possesses several remaining functional groups hearts [53]. Interestingly, these results also indicated that
[46,47]. Even though rGO is generally believed to be of rGO was associated with lower toxicity in zebrafish embryos
lower toxicity than GO [48], many studies have observed compared to GO (Figure 2).
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 3 Toxicological studies of
diameter) at of 0.01–100 μg/mL concentrations induces
DNA fragmentations and genomic aberrations [51]. More-
graphene derivatives in the
over, exposure with rGO sheets (∼1.2 nm thickness and reproductive system
∼2 µm lateral size) at 0.01–100 μg/mL concentrations for
96 h was reported to induce slight cytotoxicity by the disrup- The ever-growing utilization of graphene in various fields
tion of hMSCs membranes, while rGO nanoribbons (thick- of science and technology has rapidly raised questions
ness of 1 nm thickness, 10 µm length, and 50–200 nm width) regarding the toxicity profile of these extraordinary nano-
under similar conditions damaged hMSCs through DNA materials. The occurrence of molecular interactions between
fragmentations and chromosome aberrations [52]. While graphene derivatives and cellular compartments is a well-
the differences in rGO toxicity may be explained with its established yet concerning the matter that is capable of
correlations with rGO shapes and sizes, further investiga- causing potential harm in different tissues and organs,
tions are required for exploring the exact interconnections including the reproductive system. Even though significant
of these variables. Last but not least, Liu et al. demon- attention has been shifted to the field of graphene toxicity
strated that zebrafish embryos incubated with GO and and numerous contributions have been made in recent
rGO at 1, 5, 10, 50, and 100 µg/mL concentrations for years, there is still a lack of sufficient understanding regarding
96 h experienced slight toxicity in hatching speed and larvae how and under what conditions graphene-based materials
length as well some moderate damage to the embryos’ induce toxicity in the reproductive system of different species.
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.1080 Nastaran Hadizadeh et al.
In the following sections, this study aims to discuss recent of reproductive toxicity followed by GO exposure, where
advancements in the field of graphene toxicity in the repro- GO was responsible for suppressing ELG-5 by the activa-
ductive systems of different living species, while explaining tion of POP-1 and thus resulted in impaired fertilization
possible mechanisms for the induction of toxicity. 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
3.1 Nematodes 500 μg/mL, while prolonged exposure (10–15 days) with
higher doses of graphene (300–500 μg/mL) induced about
Caenorhabiditis elegans, a free-living nematode with a 95% cytotoxicity in two human cell lines and also reduced
short life span, is widely used as an animal model for C. elegans brood size by 5–10% [63]. Accordingly, the
in vivo toxicological studies due to its 40% genomic number of eggs produced by high-dose (500 μg/mL) gra-
homology with humans [54,55]. Moreover, its low costs, phene-exposed nematodes had also decreased, which may
short life span, ease of handling under the microscope, be explained by the generation of oxidative stress followed
and significant similarity of its physiological pathways by high-dose graphene exposure [64]. Strikingly, this study
and stress-related responses with mammals are other also displayed that small fragmentations of graphene sheets
desirable characteristics that have turned C. elegans to of less than 200 nm diameters were mainly responsible for
a favorable model for toxicological studies [56–58]. the observed toxicity, as larger graphene sheets did not
Exposure of gravid C. elegans nematodes with 10 mg/L exhibit toxic effects on both human cell lines and nema-
GO for 72 h has been reported to disrupt fatty acid meta- todes. All in all, 50–100 μg/mL concentrations of graphene
bolism and spermatogenesis; reduce sperm count, brood revealed desirable safety to worm populations as the nema-
size, offspring birth and life span; and alter fat meta- todes’ survival rate was not decreased after the 10-day dura-
bolism in sperms. In this study, alleviated expression tion of this study [63]. These findings are consistent with the
of beta-oxidation–related genes were indicated to be results from the previous studies, which proved that the
responsible for disrupted fat metabolism and impaired toxicity of pristine graphene in C. elegans follows a dose-
spermatogenesis in nematodes [59]. Even though the dependent pattern [44].
importance of fat metabolism and its critical role in sper- Regarding the role of surface modification, graphene
matogenesis has been proved earlier [60], the exact nanoplatelets (GNPs) with different surface modifications
impact of GO on them has yet to be fully discovered. (NH2 and COOH) and GO (single and few layers) were
However, one of the proposed mechanisms that may evaluated for their toxic effects toward C. elegans repro-
explain the detrimental effects of GO in C. elegans worms ductive system and Beas2B cell lines. In this study, an
is considered to be elevated oxidative stress and ROS order of pristine > NH2 > COOH was obtained for modified
production in exposed nematodes [61]. These results GNP toxicity; however, GO exhibited more significant
are consistent with the results from the study by Wu reproductive toxicity than pristine graphene, whereas
et al., studies in which the toxicity of NGO was investi- for BeasB2 cells, pristine graphene induced higher toxicity
gated on primary (lung, kidney, spleen, and liver) and when compared to GO. This occurrence may be related to
secondary (the reproductive system and the neurological different interactions of functional groups with different
system) organs of C. elegans nematodes and reported biological components, thus confirming the pivotal role
toxic damages to both primary and secondary organs of varying biological interactions in determining the final
after prolonged exposure to 0.5–100 mg/L GO, mainly toxicity of graphene. GO was revealed to damage cells
via oxidative stress pathways [57,58]. through the formation of hydrophobic agglomerates that
In another study, C. elegans was exposed to GO and impaired the cellular membrane [65], while the platelet-
rGO, and an integrated systems toxicology approach was like structure of pristine GNPs was held accountable for
used to assess the interactions and underlying mechan- increased toxicity in BeasB2 when compared to GO [66].
isms of GO and rGO’s toxicity. The results of this study On the contrary, decreased bioavailability and biocompat-
indicated a reduction in the worms’ reproductive capabil- ibility of pristine GNPs along with the generation of hydro-
ities, which was more significant followed by GO expo- phobic agglomerates serve as crucial factors that reduce
sure in comparison with rGO exposure. Unlike rGO, a the reproductive toxicity of pristine GNP compared to GO
noncanonical Wnt-MARK signaling cascade (MOM-2 → in C. elegans [67]. The oxidation state of GO, increased
MOM-5 → MOM-4 → LIT-1 → POP-1 → EGL-5) was pro- hydrophilicity and dispersion in exposure mediums, and
posed to be the underlying mechanism for the induction enhanced biocompatibility also contribute to escalatingReproductive toxicity of graphene derivatives 1081
GO’s toxicity in the reproductive system. Regarding the weight) GO to breeding pairs was followed by decreased
role of chemical modification, surface functionalization egg fecundity in early days upon injection in a dose-
of GNPs with NH2- and COOH- was associated with reduced dependent manner; however, the overall fecundity was
toxicity, while pristine GNPs displayed excessive agglom- not altered significantly. The hatchability of embryos was
eration and superior cell membrane impairment [45,67]. reported to have undergone drastic reduction when injected
Moreover, while both SLGO and FLGO displayed a clear with 200 µg/g body weight GO; however, cell morphologies
dose-dependent toxicity pattern, SLGO exhibited more bio- of granulosa and leydig cells remained almost unaltered
logical interactions owing to its lower stiffness and subse- according to gonad (testis and ovary) histopathological
quently higher biological adsorption in comparison with examinations. In spite of the agglomeration of GO in the
FLGO [68]. gonads of Japanese medaka, folliculogenesis in the ovaries
Consistent with the aforementioned results, Zanni and germinal components of the testes experienced almost
et al. evaluated the toxicity of completely reduced multi- no deviation. Also, 25,200 g/g GO was not capable of indu-
layered (3–60 layers) GNPs (with no residual oxygen-con- cing notable reproductive toxicity in Japanese medaka [74].
taining functional groups) with the lateral size of one to Results from another study indicated that GO (mean thick-
tens of micrometers, thickness of 1–20 nm, and tested ness of 1.0 nm and GO flake area of 0.58 µm2) had the ability
concentrations of 100 and 250 μg/mL on adult C. elegans to partially inhibit the reproduction of Ceriodaphniadubia
models after 3 h of exposure. Notably, the nematodes’ life via increasing ROS production followed by waterborne
span and brood size were not altered, thus displaying the exposure. GO reduced C. dubia energy levels and thus
absence of chronic reproductive toxicity [67]. 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
3.2 Aquatic species of 0.4 and 0.8 mg/L, was stated to be associated with major
reproductive toxicity as it diminished the number of neo-
Owing to the genomic homology between zebrafish (Daniorerio) nates by 12.8 and 44.2%, respectively [75].
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 [69–71]. 3.3 Mammals
John and coworkers [72] studied the adverse effects of
GO (0.8–1 nm thickness, 101–258 nm diameters) at 0.01, 3.3.1 Rodents
0.1, 1, 10, and 100 mg/L concentrations on the embryo-
genesis of zebrafish and reported that GO had entered Three groups of Wistar rats (AS1: 15 days of treatment
and induced hypoxia in the chorion, generated an anoxic with seven repeated doses on alternative days; AS2: 30
space close to the chorion, and also enforced mechanical days of treatment with 15 repeated doses on alternative
pressure on its surface area. Given the substantial role of days; AS3: 30 days of treatment with 15 repeated doses on
chorion in the development of zebrafish embryos, the alternate days and 30 days of recovery) and three sub-
aforementioned effects along with the envelopment of groups within each group (categorized by the received
chorions by GO resulted in the decreased embryo move- doses of intraperitoneal NGO as low, mid, and high
ment and delayed hatching and development of the doses) were evaluated regarding the toxic effects of
embryos. ROS generation, lipid peroxidation (LPO), forma- NGO. Accordingly, the total sperm count of the high-
tion of 8-OHdG (8-hydroxy-2-deoxy-guanosine) adducts, dose subgroup of the AS1, and mid- and high-dose
apoptosis, mitochondria activity disruption, and impaired subgroups of the AS2 group experienced a significant
antioxidant enzyme activity are some of the other detected reduction. However, these changes were not as prominent
outcomes of GO exposure in zebrafish embryos. Some mal- in low-dose subgroups. Decreased sperm production in the
formations such as pericardial/yolk sac edema were also testes, spermatogonia loss, cell cycle arrest, and occur-
observed in different embryonic regions that were resulted rence of cell death within the sperm production pathways
from the localization of GO inside the embryos in a dose- are potential explanations for this matter. Of note, sper-
dependent and selective manner [73]. matogonia and spermatid numbers were also reported to
A study on Japanese medaka (oryziaslatipes) demonstrated have decreased in this study [72]. The high-dose subgroup
that one-time intraperitoneal injection of (25–200 µg/g body of the AS2 group also experienced declined sperm motility1082 Nastaran Hadizadeh et al. values, which were restored to normal after a recovery were in contact with the NGO-treated male mice, respec- period. On the one hand, SOD, GPx, and GST antioxidant tively. Upon the birth of the litters, postnatal viability of the enzymes of the treated rats deteriorated remarkably in a offspring also experienced a 15% reduction at 2,000 µg/mL dose-dependent fashion, and ROS generation was increased NGO concentration. As proposed in this study, a dose-depen- in parallel with decreased cell proliferation and enhanced dent pattern was unveiled for the toxicity of NGO, as lower cell death [76]. Abnormal morphological alterations were doses exhibited lower toxicity while higher NGO concentra- also detected in the high-dose subgroup of the AS2 group. tions were associated with more significant reproductive Both such alterations and reduced sperm motility are highly toxic effects. likely to be associated with excessive ROS production and The clear inconsistency observed in the outcomes of oxidative stress followed by the oxidation of cell membrane the aforementioned studies can raise existing uncertain- lipids that contain a significant amount of polyunsaturated ties regarding the true nature of graphene’s reproductive fatty acids. Sperm ATP loss, axonemal damage, and conse- toxicity. Taking into consideration that the induction of quent morphological abnormalities and disrupted sperm toxic effects in all organs, including the reproductive motility also take place in sequence to accelerate oxidative organs, is under the direct or indirect influence of mul- stress in sperms [77]. Decreased steroidogenesis, germ loss, tiple factors, it can be concluded that graphene charac- germ cell apoptosis, and germinal epithelial impairment teristics such as size, thickness, and concentrations are are some of the other outcomes of oxidative stress that some of the critically important factors that impact the contribute to the induction of reproductive toxicity in Wistar eventual toxicity that is observed. For instance, Akhavan rats [78–80]. In spite of the aforementioned toxic effects, et al. [63], synthesized graphene with ∼0.8 nm thickness reproductive hormone concentrations in the rats’ serum and
Reproductive toxicity of graphene derivatives 1083
reproductive toxicity in Liang et al.’s study might be 3.3.2 Wild pigs
explained by the short duration between injection and
evaluation time-frames and thus the lack of completely In vitro exposure of boar spermatozoa with 0.5, 1, 5,
renewed spermatogenesis before mating. This finding is 10, and 50 μg/mL concentrations of GO (size interval
further confirmed by the same study by Akhavan et al. 600–900 nm) has previously shown that sperm capacita-
stating that genotoxicity and chromosomal aberrations tion and fertility had enhanced in sequence to exposure
in the spermatozoa start to take place ∼6 weeks through with 0.5 and 1 μg/mL doses of GO, while 5, 10, and 50 μg/mL
the spermatogenesis process. However, variations in doses were associated with induced cytotoxicity in sperma-
NGO size and thickness are also crucial factors that tozoa [87]. It has been proved that GO at high doses is
are likely to have affected the final toxicity observed capable of interfering with spermatozoa plasma membrane
in those studies. and consequently impairs their fertilization via causing
Another study aimed to assess the cytotoxic and gen- alterations in cholesterol extraction from the membranes.
otoxic effects of rGO (0.1, 1, 10, 100, and 400 µg/mL con- There is also further evidence supporting this fact, for
centrations) in male mice spermatozoa and indicated that example, Zhang et al. reported in a computational experi-
GO induced toxicity in a dose-dependent pattern, in ment that graphene has the ability to remove cholesterol
which motility and viability of spermatozoa were decreased from a bilayer membrane and absorb the hydrophilic
after incubation with doses higher than 1 µg/mL for hydra- compartment of cholesterol and henceforth prevents sper-
zine-reduced GO and hydrothermally reduced GO and matozoa from entering the membrane bilayers [88]. It is
with doses higher than 10 µg/mL for GTP-rGO and GO. possible that this mechanism plays an important role in
Accordingly, N2H4-rGO and HT-rGO promoted the genera- the dose-dependent toxicity of GO in spermatozoa; how-
tion of ROS and nitric oxide (NO) and reduced ATP and ever, more experiments are required for exploring the pre-
NAD+/NADH, in contraction with GTP-rGO that deterio- cise correlation between them.
rated ROS production and NO owing to GTP’s antioxidant An in vivo study [89] further investigated cholesterol
properties [83]. Moreover, less than or equal to three- extraction from swine spermatozoa membrane and reported
layered GO and rGO (100 and 400 µg/mL, thickness of that no toxicity was observed; however, certain physio-
∼0.8 nm for each monolayer)-exposed spermatogonial stem chemical alterations were detected in the spermatozoa
cells of mice (SSCs) were subject to cytotoxicity and geno- membrane and were associated with promoting sperm
toxicity through apoptosis, membrane damage, and morpho- fertility, functions, signaling pathways, and increasing
logical alterations (such as cell shrinkage and chromatin overall fertility potential of sperms without negatively
condensation) after 24 h of incubation. Accordingly, these influencing sperm interactions with the female environ-
graphene derivatives have the capability of reducing SSCs ment. Moreover, it is also crucial to mention that the
viability, disrupting normal functions and genomic material homeostasis of the Ca2+ ion plays an essential role in con-
of SSCs, and henceforth inducing reproductive toxicity trolling various functions of the sperm, such as its motility
by impairing the genetic material passed on to the off- [90], cytoskeleton assembly [91,92], and even apoptosis
spring [84]. [93]. According to these results, low concentrations of
Surprisingly, the addition of 0.5 µg/mL GO to a mice GO managed to successfully increase Ca2+ ions, all the
sperm suspension before performing in vitro fertility (IVF) while leaving the sperms’ membrane potential unaltered,
has been reported to increase the number of fertilized thus proving that GO has the ability to improve sperm
oocytes as well as born pups in a more efficient manner capacitation via calcium signaling pathways. Furthermore,
compared to the gold-standard methyl-β-cyclodextrin the underlying mechanisms that reveal the reasons behind
agent for IVF promotion [85]. To this day, exact under- increased sperm fertility have yet to be known; however,
lying mechanisms for explaining this occurrence remain it is estimated that membrane glycocalyx is potentially
unknown, but it is predicted that different graphene deri- involved in this process as it is the primary component
vatives containing varying diameters, morphological shapes, that comes into contact with GO and other external mole-
doses, and chemical functionalizations display a diverse cules [94].
range of outcomes in different experimental settings [86].
However, further investigations are still acquired to reach
a well-established consensus regarding how graphene 3.3.3 Humans
materials can be modified before exploitation to minimize
the potential risks to one’s reproductive system as well as Graphene toxicological studies on humans remain rather
offspring health. limited due to ethical reasons; however, an in vitro study1084 Nastaran Hadizadeh et al.
demonstrated that incubation of human sperms with 1, 5, While toxicity is generally believed to be directly
and 25 μg/mL GO (1–5 μm) for 0.5–3 h resulted in no related to the time span of exposure, several studies
detectable toxicity or alterations in the viability of sperms have indicated that apoptosis-mediated cytotoxicity, unlike
and did not trigger accelerated generation of ROS even at necrosis-mediated cytotoxicity, is independent of the dura-
the highest concentration of 25 μg/mL [95]. However, 3 h tion of exposure [65,102]. Therefore, it can be concluded
incubation of sperms with high doses of GO (5 and 25 μg/mL) that underlying toxicity mechanisms may play a potentially
deteriorated sperm motility. influential role in the determination of time–toxicity corre-
lation for graphene nanomaterials.
4 Important factors affecting
graphene’s toxicity 4.2 Physiological barriers
Graphene nanoparticles face several biological barriers in
4.1 Administration routes and exposure their way inside the body, which drastically affect their
periods retention levels in different organs. BRB and blood–pla-
centa barrier (BPB) are two of these physiological walls
Oral administration, intravenous injection, intraperitoneal that influence the entry of different nanoparticles to both
injection, subcutaneous injection, intratracheal instilla- the reproductive system and fetus [23].
tion, intrapleural installation, and pharyngeal aspiration
are the main routes of graphene administration that might
ultimately result in varying toxic effects [96,97]. While 4.2.1 BTB and BEB
toxicological reactions should be assessed via different
administration routes, it is of crucial importance to first BTB, formed by the tight junctions between sertoli cells
choose the most suitable route depending on the target [103], and BEB are two of the tightest physiological bar-
organs of study. For instance, intranasal exposure is often riers that provide crucial protection to male reproductive
utilized in nanoparticle neurotoxicity studies due to its organs and obstruct the entry of various foreign particles
proper relevance with the nervous system [98,99], while [104]. Most germ cells reside within the closed section of
intravenous (via tail vein) or intra-abdominal injection the BTB and are rather safely protected against external
methods are the most common routes for reproductive materials; however, differentiating spermatogonia and
toxicity studies [82,100]. In spite of these findings, the stem cells are situated in the open part of the BTB and
current literature still suffers from a lack of strong evidence are thus more exposed to exterior materials [105]. Owing
regarding the exact impact of administration routes on to their nano-scale dimensions, some nanoparticles pos-
reproductive toxicity [23]. sess the ability to penetrate through BTB and BEB; reside
Numerous investigations have evaluated the correla- inside the testes, epididymis, or neighboring tissues; and
tion between graphene exposure periods and its eventual alter sperm morphology and impair the spermatogenesis
toxicity. For instance, an in vitro study that investigated process upon entry via the circulation system [106–109].
graphene nanoribbons (10–400 mg/mL) toxicity on HeLa Accumulation of nanoparticles inside the testes or epidi-
cells, NIH-3T3 cells, and breast cancer cells (SKBR3, dymis not only affects the quality of sperms and the indi-
MCF7) demonstrated a dose- and time-dependent fashion vidual’s fertile capacities but also holds notable potential
for toxicity, where the longest exposure periods (48 h) to induce genotoxic effects in the stem cells and sperms
and highest concentrations (400 mg/mL) are responsible and hence increases the risk of fatal defects and malfunc-
for dramatically decreased cell viability [101]. tions as well as hereditary mutations and disorders in the
Even though the exact correlation of exposure per- offspring [110,111]. The current literature offers insightful
iods with graphene nanomaterials’ eventual reproductive yet somewhat conflicting data regarding the penetration
toxicity remains somehow unclear, several studies have of different nanoparticles through the aforementioned
confirmed the pivotal role of graphene exposure periods barriers. For instance, intramuscular injection of TiO2 nano-
on different species’ reproductive capacities. For instance, particles (2.5, 5, and 10 mg/kg BW) for 90 days was followed
25 μg/mL concentrations of GO had the ability to reduce by decreased sperm quality, hormonal alterations, and
sperm motility after 3 h of incubation, while shorter incuba- reproductive toxicity in sequence to BTB penetration and
tion periods were not associated with such alterations [95]. testicular accumulation [112]. However, IV administration ofReproductive toxicity of graphene derivatives 1085
TiO2 nanoparticles (0.1, 1, 2, and 10 mg/kg BW, weekly for 4 than 1 kDa mass are incapable of crossing this barrier
weeks) displayed no detectable Ti accumulation in the [121,122]. In general, regardless of the wide range of nano-
testes of mice, in contrast to their livers [113,114]. Evidence particles that are capable of transferring through this
has shown that BTB penetration can also be followed by barrier [123–125], some of them might not accumulate
reproductive toxicity via altering the body or organ’s weight inside the fetus [94], while some others have the ability
[115]; however, these data too suffer from a lack of consis- to induce teratogenic effects and developmental toxicity
tency in reported results, which can be explained with the via accumulation inside the fetus’ organs [123]. For instance,
variations between exposure routes and duration, types of IV administration of 6.25 and 12.5 mg/kg concentrations of
nanoparticles, concentrations, and the interplay of those rGO nanosheets (20–150 nm or 200–1,500 nm, single layered
factors with host genomic profile, epigenetic factors, and or 3–5 layered) to pregnant mice that was ∼20 days into
molecular interactions between the nanoparticles and cel- gestation was followed by dramatically increased miscar-
lular compartments upon entry. IV injection of Ag nanopar- riage compared to treated dams in earlier phases of gestation
ticles (5 and 10 mg/kg BW) with diameters of 20 nm to male (∼6 days). On the other hand, IV injection of 25 mg/kg rGO to
Wistar rats did not change their testes weight nor impaired pregnant dams that was 20 days into gestation led to the
their reproductive capacity [116]. Further confirming these death of nearly all of them, but exhibited almost no signifi-
results, when administered orally, Ag nanoparticles (15 and cant toxicity in pregnant mice in early gestation phases,
50 µg/kg BW) with a size of 60 nm also showed no sign of except for a few fetal deformities. This study also claimed
altered body or organ weight in male Wistar rats [117]. Con- that IV administration of rGO nanosheets to female mice
versely, another study reported the occurrence of male rat ∼30–35 days before cohabitation did not alter the reproduc-
reproductive toxicity through decreased testes and epidid- tive health of female rats. Moreover, histopathological exam-
ymis weight as well as body weight upon 7 and 28 days of inations of the mice placenta exhibited very slight placental
sub-dermal exposure with 50 mg/kg BW Ag nanoparticles, damage, thus indicating that only a few rGO nanosheets
respectively [118]. Concluded from these results, the inargu- managed to penetrate through this barrier and the occur-
able impact of administration route, dose, and size of nano- rence of dose-dependent toxicity was mostly executed to
particles on the obtained results is evident. Very limited the embryos through the disruption of their mothers’ health
studies have been conducted regarding the passage of gra- [126].
phene derivatives through BTB and BEB; however, penetra- Even though particle size [127–129], exposure route
tion of BTB and BEB has been reported to be of major diffi- and duration, chemical functionalization of nanoparticles
culty for GO particles of 54.9 ± 23.1 nm diameters after intra- [124,125,130], and maternal physiological and pathological
abdominal injection. Consequently, even high concentra- conditions [131] contribute greatly in the determination of
tions of GO (300 mg/kg) were observed to be incapable of whether a nanoparticle, for example, graphene, may or
altering the sperm quality in mice [81]. may not cross through the BPB [121,132–134], more inves-
tigations are required to evaluate their precise relationship
with the translocation of graphene into this barrier and
4.2.2 BPB their eventual impact on graphene derivatives’ reproduc-
tive toxicity.
In addition to the toxicity of graphene materials on dif-
ferent species’ reproductive health, graphene may also
disrupt fetal development by crossing through the BPB
and passing from the maternal circulation system to the 4.3 Surface functionalization
fetus. The importance of this barrier lies upon its crucial
role in the exchange of nutrients, metabolic wastes, and Being directly influenced by surface chemical properties,
hormones between the fetus and the mother [119], as well biomedical applications of graphene derivatives remain
as serving as a protective barrier to the fetus that prevents largely dependent on the existing functional groups on
the entry of numerous external particles. However, recent its surface that, to a notable extent, determine their even-
evidence has claimed that the protection provided by the tual interactions with biological components as well as
placental barrier toward the entry of carbonaceous nano- recognition processes [135]. Pristine graphene, as a pure
particles is not as strong as the previously mentioned form of graphene that solely composed of sp2-hybridized
barriers [120]. Particle properties and chemistry are two carbons, suffers from inherently insufficient water solu-
critical factors that to some extent control this phenom- bility and chemical reactivity. GO, on the other hand, is
enon, as hydrophilic molecules and particles with more one of the most popular functionalized derivatives of1086 Nastaran Hadizadeh et al.
pristine graphene that offers extensive applications (such salient coverage of its surface by large macromolecules
as DNA or drug bioconjugation for gene and drug delivery that inhibit such dispersion interactions. Besides, the sur-
purposes) due to the existence of carboxyl, epoxy, and face coverage of graphene derivatives helps to enhance
hydroxyl groups on its periphery and basal planes that their cellular recognition, uptake, and clearance [142]
grant this unique nanomaterial the advantage of colloidal and also decreases graphene’s capability to induce cyto-
stability, water dispersibility, and hydrophilicity, as well toxicity through extracting membrane cholesterols and
as promoted covalent bond formation and facilitated disrupting calcium hemostasis or neurotransmission poten-
chemical reactions [135–138]. The importance of altering tiation [150]. According to the results from an in vitro study
graphene nanomaterials surface chemistry and hydro- on monkey renal cells, pristine graphene was associated
philicity also lies upon the consequent biological effects with promoted apoptosis as it exhibited accumulation on
and reactions as a result of varying ionization degrees the cellular membrane due to the formation of hydrophobic
and dispersibility in physiological fluids, which not only interactions with the cell membrane lipids, whereas car-
impact graphene’s uses but also shape the state of gra- boxylated GO was mainly internalized into cells due to its
phene’s toxicity in different mediums [136,138]. All in all, higher hydrophilicity, did disrupt the cellular membrane,
surface chemical modification is often carried out with the and therefore induced no toxicity at high concentrations
aims of enhancing biocompatibility and stability, leading (300 µg/mL) [139]. As demonstrated by Teo et al., exposure
applications toward desired outcomes, accelerated thera- of A549 cells with halogen functionalized (GO–Cl, GO–Br,
peutic capabilities, target-binding efficacy, and attenu- GO–I, and GO–F) graphene at 0–200 µg/mL concentrations
ating toxicity [136]. was followed by a dose-dependent increase in cytotoxicity
Surface functionalization of pristine graphene and accompanied with increased halogenation [151]. Interest-
GO has been shown to decrease detrimental toxicity to ingly, halogenated graphene at 0–400 µg/mL concen-
a desirable extent in vitro [139]. PEG [140] polyvinyl trations displayed superior toxicity compared to GO. An
alcohol, PEGylated poly-L-lysine (PLL) [141], and dextran explanation proposed for this result was related to the selec-
[142] are some of the commonly exploited macromole- tive adsorption of crucial micronutrients on the hydro-
cules for functionalizing graphene’s structure that enhances phobic surface of halogenated GO and insufficient nutrient
its biocompatibility and reduce its potential toxicity. For availability to A548 cells due to halogenated GO’s more
instance, PEGylated GO with doses of up to 100 μg/mL significant hydrophilicity [152]. Furthermore, different func-
was reported to be safe toward glioblastoma cells (U87MG), tionalized pristine graphene derivatives (G–COOH, G–NH2,
breast cancer cells (MCF-7), human ovarian carcinoma cells and G–OH) with 0.1 mg/L concentrations were evaluated for
(OVCAR-3), colon cancer cells (HCT-116), and lymphoblas- their neurotoxic effects on SK–N–SH cells post 24 h expo-
toid cells (RAJI) [143–145], and induced less cytotoxicity in sure [153]. Obtained toxicity results were indicative of a
human lung fibroblast cells in comparison with GO (at a G–OH ≈ G–COOH > rGO > G–NH2 ranking that deteriorated
concentration range of 1–100 μg/mL) [146]. PEG coating of over longer exposure durations. However, G–NH2 possessed
GO was shown to significantly reduce acute tissue impair- higher toxicity persistence in long term as a result of its
ment by reducing the aggregation and retention of GO in increased potential for disrupting lipid and carbohydrate
lungs, liver, and spleen and also accelerated its elimination metabolism compared to other functionalized forms of
from the body [147]. Exposure of human liver cell lines (HL- pristine graphene. This finding further emphasizes the impor-
7702), human lung fibroblast cell line (MRC-5), and human tance of long-term toxicity assessment of graphene derivatives,
macrophage cell line (U937) with 200 µg/mL PEGylated GO as they might be associated with insignificant short-term toxic
was also suggested to protect the cell growth from inhibition effects but display persistent long-term toxicity due to slow
by 30% and reduce DNA toxicity by 40% [148]. Dextran, a biodegradation and internal accumulation [138]. Regarding
branched glucan utilized in diverse biotherapeutic applica- the reproductive system, graphene toxicity has been evaluated
tions, remarkably decreased the inhibition of Hela cell pro- and compared among pristine graphene, graphene-NH2, gra-
liferation when conjugated to GO, in comparison with phene-COOH, and also GO in C. elegans. Pertaining to the
unfunctionalized GO [142]. Moreover, modification of GO results, GO induced higher reproductive toxicity in comparison
with chitosan was also reported to reduce RBC lysis and with other derivatives, among which there was a pristine gra-
hemotoxicity [149]. The root of reduced toxicity of macro- phene > NH2 > COOH order for the reported toxicity. It has also
molecule-conjugated graphene materials may be poten- been previously suggested that covalent functionalizations
tially linked with graphene’s inherent phospholipid (such as –COOH) are capable of reducing the toxicity of gra-
extraction/insertion ability caused by notable surface dis- phene nanoparticles through increasing their hydrophilicity
persion interactions with cellular membrane lipids, and and bio-clearance from the body [26,44,68].Reproductive toxicity of graphene derivatives 1087
Driven from these results, the essential role of gra- the functions of other cells and organs. For instance, in a
phene surface functionalization in altering their toxicity study by Xia et al. [25], pristine GO, rGO, and hydrated GO
is emphasized; however, the lack of sufficient data regarding (hGO) were evaluated for their LPO-mediated cytotoxic
the role of surface functionalization in long-term reproductive effects on THP-1 and BEAS-2B cell lines. Carbon radical
studies highlights the urgent need for further experiments in densities of the utilized derivatives were ranked as hGO >
this field to unravel the true nature of these exquisite mate- GO > rGO, as hydration of GO was shown to be associated
rials’ toxicity. 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
4.4 Oxidation state highest carbon radical density and therefore resulted in
the most significant cytotoxicity and cell death through
The oxidation state of graphene, to some extent, deter- LPO and lysis of the cell membrane, whereas rGO exhibited
mines its chemical structure, cellular interactions, and the slightest cytotoxic effects in this study. Accordingly,
cytotoxicity by affecting the carbon radical density on LPO was measured for these materials and reported as 37,
GO’s surface. The existence of unpaired electrons grants 17, and 5% for hGO, GO, and rGO, respectively.
carbon radicals with higher reactivity compared to other These data notably reveal a distinct correlation between
chemical functional groups and therefore promotes their LPO-induced cytotoxicity of GO derivatives with surface
ability to generate more superoxide radicals, which can oxidation, hydroxyl, carboxyl, and carbonyl groups, and
ultimately oxidize unsaturated lipids and thiol groups on carbon radical density. However, due to the intrinsic com-
proteins or glutathione (GSH) [154]. Henceforth, GO spe- plexity that lies within the interplay of LPO and other cel-
cies with higher surface carbon radical density are con- lular mechanisms and molecular pathways, the precise
sidered to be associated with higher pro-oxidant activity, extent to which LPO influences the ultimate toxicity remains
GSH depletion, and membrane LPO [155]. LPO is a pro- largely unknown.
cess in which phospholipids and low-density lipopro-
teins undergo oxidation and result in the loss of cellular
membrane integrity, membrane lysis, cell death, and
consequently enhanced cytotoxicity [156]. Spermatozoa, 4.5 Number of layers
polyunsaturated fatty acid (PUFA)-containing cells, are
highly susceptible to LPO and spermatogenesis disruption In spite of their enhanced thickness, multilayered gra-
as well as eventual reproductive dysfunction [156–158]. phene sheets contain smaller volume-specific surface
The main underlying mechanism for the induction of area than single-layered sheets and therefore exhibit
LPO is considered to be oxidative stress, which disrupts varying colloidal attributes, different biological interac-
the normal physiological functions of cells through pro- tions at cell membrane interfaces, and different levels of
moting ROS generation. Excessive formation of ROS is cellular uptake [138]. Parallel alignment of GO sheets on
inherently linked with decreased sperm count, abnormal cell membranes is associated with accelerated GO uptake
shape, and overall reduced fertility and impaired oocyte by cells [68]; however, cellular uptake is highly likely to
penetration [159,160]. For instance, a study showed that undergo remarkable deterioration with the increasing
increased LPO and oxidative stress are responsible for male number of graphene layers [44]. Few layer graphene
boar infertility, but can be compensated with enhanced (FLG) is often described as graphene with 2–10 layers
antioxidant enzyme activities. Accordingly, the epididymis [162]. SLGO and FLGO have been reported to induce toxi-
head and testes were more prone to the acceleration of ROS city in a dose-dependent fashion; however, SLGO dis-
formation and LPO. The spermatogenesis process consists played more prominent dose dependency in comparison
of several stages including proliferation, maturation, and with FLGO. Evidence suggests that SLGO is more likely to
spermatozoa storage in epididymis, which are all subject form interactions with biological components than FLGO,
to LPO in different phases [161]. Even though oxidative probably because of the higher number of layers in FLGO
stress and LPO play an evidently crucial role in reproductive that accounts for enhanced stiffness and subsequently
infertility, the specific correlation between the toxicity of reduced biological adsorption [68]. In contrast to these
graphene derivatives and alteration of LPO levels has not results, an in vitro study that investigated the genotoxic
been studied in detail. However, it is important to note effects of graphene derivatives at 10 and 50 mg/L concen-
that the inherent toxic effects of LPO are not limited to trations after 24 h of exposure with human bronchial
the reproductive organs and may also negatively impact epithelial cells revealed that FLGO with ∼4–8 layers1088 Nastaran Hadizadeh et al.
induces more significant genotoxicity than SLGO owing carbon radical density are among the factors that play a
to increased DNA methylation [163]. major role in their uptake and interactions with biological
compartments and influence their final toxicity [171].
Being closely interconnected with particle internalization
4.6 Lateral dimension inside cells, the size of GO flakes can adversely affect
their uptake by cells, as larger GO flakes have been
Ranging from 10 nm to almost 10 µm, the size of graphene observed to limit cell uptake, while smaller GO flakes
sheets plays a critical role in determining the fate of these were associated with facilitated internalization by cells
unique nanomaterials in physiological mediums. Despite [172]. Cellular accumulation is yet another factor that
the controversy regarding varying cellular responses after affects the cytotoxicity caused by graphene nanomater-
internalization of graphene derivatives with different lat- ials. Accordingly, graphene has an amphiphilic nature
eral dimensions, it is well established that cells struggle with hydrophilic edges and a hydrophobic planar struc-
to uptake large nanoparticles and internalize them via ture, owing to which GO and hGO were actively accumu-
different pathways based on their sizes [164–166]. For lated close to THP-1 macrophage membranes without
instance, small graphene sheets can penetrate into cells significant cellular uptake [173], while rGO was reported
directly, while large graphene sheets enter cells via the to be internalized and accumulated inside cells due to its
formation of hemispherical lipid vesicles [166]. Moreover, reduced hydrophilicity at edges and enhanced overall
smaller GO sheets were observed to be capable of being hydrophobicity. Consistent with these results, another
internalized by human-derived macrophages a lot more investigation has reported that chemical reduction of
than larger sheets [167]. Another study indicated that upon GO is associated with promoted cellular uptake as well
IV administration of graphene in vivo, a notable amount of as clearance at injection sites [174]. Of note, it should
larger FLG sheets (330–630 nm) were degraded in the liver be taken into consideration that several studies have
of mice by Kupffer cells after 180 days, whereas the smaller reported the localization of pristine GO inside the cell
sheets (20–40 nm lateral size) that entered and accumu- cytoplasm [175–177], which could be explained with the
lated in the liver had not decreased due to their slower difference of exploited cell types; however, other impor-
biodegradability [168]. As opposed to these results, the tant parameters such as size, surface charge, and surface
accumulation of large graphene sheets with ∼500 nm lat- functionalization should not be overlooked regarding
eral dimensions in zebrafish was demonstrated to be sig- their vital roles in the eventual cellular uptake and accu-
nificantly higher than smaller graphene sheets with ∼30 nm mulation [178].
diameters [169]; however, the majority of smaller sheets Uptake of graphene by cells can occur through dif-
were accumulated in zebrafish liver and gut, while larger ferent routes [37,64]. Nanoparticles with less than 100 nm
sheets were mainly detected only in the gut. Furthermore, a diameters are capable of entering cells, while nanoparti-
study by Heo et al. showed that human dermal fibroblast cles with less than 40 nm diameters can also enter the
cells effectively internalized needle-like graphene with cellular nucleus [179]. Graphene quantum dots are cap-
1 mm length in spite of their large dimensions [170]. In able of entering cells by direct penetration into the cel-
striking contrast with previous results, despite their highly lular membrane and do not require energy-dependent
diverse sizes, GO with 2 µm (4.05 nm average height) and pathways [180,181], while protein-coated graphene nano-
350 nm (3.9 nm average height) lateral diameters and 1 nm particles (∼500 nm) enter cells via cathrin-mediated endo-
thickness have displayed similar amounts of uptake by cytosis and larger protein-coated graphene nanoparticles
macrophages through antibody opsonization and phago- (∼1 µm) enter cells through phagocytosis [175]. GO sheets
cytosis, thus following a size-independent pattern for macro- can be adhered to cell membranes, enter and localize
phage uptake. However, micro-sized GO revealed slighter between the phospholipidic bilayers of the cell membrane,
biocompatibility and hence initiated stronger inflammatory and also be internalized inside cells [182]. Before accumu-
responses compared to nano-sized GO [164]. lation inside organs, it is claimed that larger graphene
derivatives, such as micro-scale graphene materials
(10–30 µm), are carried within the blood circulation
4.7 Cellular uptake, interactions, and system at a milder speed than small graphene quantum
accumulation dots (3–5 nm) and hence are more likely to induce long-
term toxicity upon exposure [183]. In general, any altera-
As previously mentioned, physiochemical properties of tions of graphene’s physiochemical characteristics pre- or
graphene derivatives including hydrophilicity, size, and post-synthesis, such as sharp edges and structural defects,You can also read
